Post on 12-Feb-2017
Taller 5 Controles de calidad para muestras bioloacutegicas
Coordina Andreacutes C Garciacutea Montero Banco Nacional de ADN Salamanca
Ponentes Carmen Garciacutea MaciacuteasServicio de Patologiacutea Molecular CIC Salamanca Rosa Pinto Labajo Banco Nacional de ADN SalamancaAnna Bosch Comas Biobanco IDIBAPS BarcelonaAndreacutes C Garciacutea MonteroBanco Nacional de ADN Salamanca
Controles de calidad de aacutecidos nucleicos
Taller 5 Controles de calidad para muestras bioloacutegicas
Dra Rosa Mariacutea Pinto Labajo Aacuterea de Control de Calidad
Banco Nacional de ADN Carlos III- Universidad de Salamanca
CRITERIOS DE CALIDAD DE ADN Y ARN
Concentracioacuten
Pureza
Integridad
Funcionalidad
wwwbancoadnorg
Trazabilidad
- Espectrofotometriacutea
1 unidad de absorbancia en 1cm de trayectoria oacuteptica para ADN de doble cadena = 50 microg ml y para ARN = 40 microg ml
Cada moleacutecula absorbe la energiacutea radiante a una longitud de ondaespeciacutefica a partir de la cual es posible extrapolar la concentracioacuten de unsoluto en una solucioacuten
Ley de Lambert-Beer una relacioacuten lineal entre absorbancia A (DO) y concentracioacuten
A = DO = αlc (1)
α coeficiente de extincioacuten molar (probabilidad de que el fotoacuten sea absorbido por el material a esa λl distancia que recorre la luz para atravesar el materialc concentracioacuten
CONCENTRACIOacuteN
-basa la cuantificacioacuten en la medida de absorbancia 260 nmde todos los nucleoacutetidos
- concentracioacuten y pureza
- Espectrofotometriacutea Nanodrop ND1000
Ventajas
- sencillo raacutepido y econoacutemico
Incovenientes
La capacidad de absorcioacuten a una λ especiacutefca de lasposibles sustancias contaminantes
ADN degradadoADN monocatenariooligonucleoacutetidosARN
-No aporta informacioacuten sobre la integridad
CONCENTRACIOacuteN
-No discrimina entre ADN y ARN
Fundamento determinacioacuten de la concentracioacuten en funcioacuten de la fluorescenciaemitida por un fluoroacuteforo de unioacuten especiacutefica al ADN de doble cadena
Cuantificacioacuten relativa necesario realizar una recta patroacuten con un ADN estaacutendar (iacutentegro) de concentracioacuten conocida
PicoGreenreg SYBR Green reg QuantiFluortrade
Ventajas- concentracioacuten e integridad
Unioacuten exclusiva a ADN de doble cadena
- coste elevado metodologiacutea maacutes compleja (gran precisioacuten)
-No aporta informacioacuten sobre la pureza del ADN
RELACIOacuteN PicoGreenreg Nanodrop ND1000R lt 1
CONCENTRACIOacuteN DE ADN
- Fluorimetriacutea
Inconvenientes
CONCENTRACIOacuteN DE ADN
muestra oacuteptima de ADN R 08gt
Picogreen PromediosPicogreen
MEDIDAS DE CONCENTRACION (ngul))MEDIDAS DE CONCENTRACION (ngul))
ltgt 1 2 3 ltgtA 9829 9678 9740 A 9749B 16827 17030 16198 B 16685C 40833 41898 41583 C 41438D 21674 21724 21649 D 21682E 9611 9224 9088 E 9308
Promedios NanodropMEDIDAS DE CONCENTRACIOacuteN (ngul))ltgtA 11800B 18730C 47950D 25650E 10480
Ratios piconano
A 083B 089C 086D 085E 089
Meacutetodo precipitacioacuten con salesldquosalting outrdquo
A4 B3 B6 B9 B10 C2 C3085 076 074 025 024 055 074
RELACIOacuteN PicoGreenreg Nanodrop
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
A4 B3 B6 B9 B10 C2 C3ng microl 588 658 68 50 455 455 42
A4 B3 B6 B9 B10 C2 C3ng microl 50 50 50 125 11 25 312
concentracioacuten PicoGreenreg
concentracioacuten Nanodrop ND1000
Metodologiacutea RT-PCR a tiempo real
Cuantificacioacuten relativa necesario realizar una recta patroacuten con un ADN estaacutendar (iacutentegro) de concentracioacuten conocida
Ventajas- concentracioacuten e integridad mediante el caacutelculo de los valores de ciclo de umbral o Ct
- coste elevado metodologiacutea maacutes compleja interpretacioacuten de los resultados
CONCENTRACIOacuteN DE ARN
- Fluorimetriacutea
Inconvenientes
Los fluoroacuteforos pueden ser de dos tipos a) fluoroacuteforos con afinidad por el ADN b) sondas especificas para fragmentos del ADN
Fundamento determinacioacuten de la concentracioacuten en funcioacuten de la fluorescencia emitida por un fluoroacuteforo de unioacuten al ADN sintetizado
Hay que tener en cuenta que el tamantildeo de los fragmentos amplificados es limitado
- deteccioacuten de contaminacioacuten por ADN- funcionalidad
- no aporta informacioacuten sobre presencia de contaminantes en la muestra
PUREZA
Capacidad de absorbancia a una λ determinada de las posibles sustancias contaminantes presentes en una solucioacuten de ADN
RELACIOacuteN DE ABSORBANCIAS
A280 proteiacutenas fenol (A270)A230 sales caotroacutepicas fenol hidratos de carbono
wwwbancoadnorg
A260A280Contaminacioacuten c aromaacuteticoslt 16
18 ndash 20 PUREZA OacutePTIMAgt 21 Contaminacioacuten ARN
A260A230 20-22 PUREZA OacutePTIMA
ADN
A260A280 20 - 21
A260A230 ~20
ge 18 Pureza aceptablePUREZA OacutePTIMA
PUREZA OacutePTIMAARN
A260280 187
A260230 246
A260280 204
A260230 168
ADN alta pureza
PUREZA
ARN alta pureza
Precipitacioacuten con etanol y sales
A260280 190
A260230 237
ngmicrol 1400
A260280 168
A260230 125
ngmicrol 2551
Contaminacioacuten con fenol
Relacioacuten A260230
A230sales caotroacutepicas fenolhidratos de carbono
Puede resultar muy variable dependiendo del tampoacuten que utilicemos como blanco en la medida del espectrofotoacutemetro
muestras concentracioacuten A260280 A26023010-3 5618 191 -6610-4 5736 197 -6810-5 577 191 -72510-6 567 187 -63910-8 548 188 -595
muestras concentracioacuten A260280 A26023010-3 5578 19 22510-4 5598 201 23910-5 5541 194 22610-6 5483 201 2310-8 532 203 235
Buffer TE
Buffer kit bolas magneacuteticas
A260280 189A260230 226
ngmicrol 1316
A260280 186A260230 172
ngmicrol 111
dilucioacuten 110
Si diluimos una muestra a la mitad de concentracioacuten en un tampoacuten salino larelacioacuten 260280 se mantiene constante (la muestra tiene la misma pureza) sinembargo el ratio 260230 disminuye por el efecto del incremento proporcional desales respecto al ADN
INTEGRIDAD
ELECTROFORESIS EN GEL DE AGAROSA
28 S18 S
gDNA
agarosa 07 agarosa 1
integridad bandas ribosomales 28S y 18S discretas en proporcioacuten 21 21
integridad una uacutenica banda definidaen la parte superior del gel
pureza deteccioacuten de presencia de ADN en la parte superior del gel
pureza deteccioacuten de presencia de ARN en la parte inferior del gel
ge 50 ng
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
Controles de calidad de aacutecidos nucleicos
Taller 5 Controles de calidad para muestras bioloacutegicas
Dra Rosa Mariacutea Pinto Labajo Aacuterea de Control de Calidad
Banco Nacional de ADN Carlos III- Universidad de Salamanca
CRITERIOS DE CALIDAD DE ADN Y ARN
Concentracioacuten
Pureza
Integridad
Funcionalidad
wwwbancoadnorg
Trazabilidad
- Espectrofotometriacutea
1 unidad de absorbancia en 1cm de trayectoria oacuteptica para ADN de doble cadena = 50 microg ml y para ARN = 40 microg ml
Cada moleacutecula absorbe la energiacutea radiante a una longitud de ondaespeciacutefica a partir de la cual es posible extrapolar la concentracioacuten de unsoluto en una solucioacuten
Ley de Lambert-Beer una relacioacuten lineal entre absorbancia A (DO) y concentracioacuten
A = DO = αlc (1)
α coeficiente de extincioacuten molar (probabilidad de que el fotoacuten sea absorbido por el material a esa λl distancia que recorre la luz para atravesar el materialc concentracioacuten
CONCENTRACIOacuteN
-basa la cuantificacioacuten en la medida de absorbancia 260 nmde todos los nucleoacutetidos
- concentracioacuten y pureza
- Espectrofotometriacutea Nanodrop ND1000
Ventajas
- sencillo raacutepido y econoacutemico
Incovenientes
La capacidad de absorcioacuten a una λ especiacutefca de lasposibles sustancias contaminantes
ADN degradadoADN monocatenariooligonucleoacutetidosARN
-No aporta informacioacuten sobre la integridad
CONCENTRACIOacuteN
-No discrimina entre ADN y ARN
Fundamento determinacioacuten de la concentracioacuten en funcioacuten de la fluorescenciaemitida por un fluoroacuteforo de unioacuten especiacutefica al ADN de doble cadena
Cuantificacioacuten relativa necesario realizar una recta patroacuten con un ADN estaacutendar (iacutentegro) de concentracioacuten conocida
PicoGreenreg SYBR Green reg QuantiFluortrade
Ventajas- concentracioacuten e integridad
Unioacuten exclusiva a ADN de doble cadena
- coste elevado metodologiacutea maacutes compleja (gran precisioacuten)
-No aporta informacioacuten sobre la pureza del ADN
RELACIOacuteN PicoGreenreg Nanodrop ND1000R lt 1
CONCENTRACIOacuteN DE ADN
- Fluorimetriacutea
Inconvenientes
CONCENTRACIOacuteN DE ADN
muestra oacuteptima de ADN R 08gt
Picogreen PromediosPicogreen
MEDIDAS DE CONCENTRACION (ngul))MEDIDAS DE CONCENTRACION (ngul))
ltgt 1 2 3 ltgtA 9829 9678 9740 A 9749B 16827 17030 16198 B 16685C 40833 41898 41583 C 41438D 21674 21724 21649 D 21682E 9611 9224 9088 E 9308
Promedios NanodropMEDIDAS DE CONCENTRACIOacuteN (ngul))ltgtA 11800B 18730C 47950D 25650E 10480
Ratios piconano
A 083B 089C 086D 085E 089
Meacutetodo precipitacioacuten con salesldquosalting outrdquo
A4 B3 B6 B9 B10 C2 C3085 076 074 025 024 055 074
RELACIOacuteN PicoGreenreg Nanodrop
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
A4 B3 B6 B9 B10 C2 C3ng microl 588 658 68 50 455 455 42
A4 B3 B6 B9 B10 C2 C3ng microl 50 50 50 125 11 25 312
concentracioacuten PicoGreenreg
concentracioacuten Nanodrop ND1000
Metodologiacutea RT-PCR a tiempo real
Cuantificacioacuten relativa necesario realizar una recta patroacuten con un ADN estaacutendar (iacutentegro) de concentracioacuten conocida
Ventajas- concentracioacuten e integridad mediante el caacutelculo de los valores de ciclo de umbral o Ct
- coste elevado metodologiacutea maacutes compleja interpretacioacuten de los resultados
CONCENTRACIOacuteN DE ARN
- Fluorimetriacutea
Inconvenientes
Los fluoroacuteforos pueden ser de dos tipos a) fluoroacuteforos con afinidad por el ADN b) sondas especificas para fragmentos del ADN
Fundamento determinacioacuten de la concentracioacuten en funcioacuten de la fluorescencia emitida por un fluoroacuteforo de unioacuten al ADN sintetizado
Hay que tener en cuenta que el tamantildeo de los fragmentos amplificados es limitado
- deteccioacuten de contaminacioacuten por ADN- funcionalidad
- no aporta informacioacuten sobre presencia de contaminantes en la muestra
PUREZA
Capacidad de absorbancia a una λ determinada de las posibles sustancias contaminantes presentes en una solucioacuten de ADN
RELACIOacuteN DE ABSORBANCIAS
A280 proteiacutenas fenol (A270)A230 sales caotroacutepicas fenol hidratos de carbono
wwwbancoadnorg
A260A280Contaminacioacuten c aromaacuteticoslt 16
18 ndash 20 PUREZA OacutePTIMAgt 21 Contaminacioacuten ARN
A260A230 20-22 PUREZA OacutePTIMA
ADN
A260A280 20 - 21
A260A230 ~20
ge 18 Pureza aceptablePUREZA OacutePTIMA
PUREZA OacutePTIMAARN
A260280 187
A260230 246
A260280 204
A260230 168
ADN alta pureza
PUREZA
ARN alta pureza
Precipitacioacuten con etanol y sales
A260280 190
A260230 237
ngmicrol 1400
A260280 168
A260230 125
ngmicrol 2551
Contaminacioacuten con fenol
Relacioacuten A260230
A230sales caotroacutepicas fenolhidratos de carbono
Puede resultar muy variable dependiendo del tampoacuten que utilicemos como blanco en la medida del espectrofotoacutemetro
muestras concentracioacuten A260280 A26023010-3 5618 191 -6610-4 5736 197 -6810-5 577 191 -72510-6 567 187 -63910-8 548 188 -595
muestras concentracioacuten A260280 A26023010-3 5578 19 22510-4 5598 201 23910-5 5541 194 22610-6 5483 201 2310-8 532 203 235
Buffer TE
Buffer kit bolas magneacuteticas
A260280 189A260230 226
ngmicrol 1316
A260280 186A260230 172
ngmicrol 111
dilucioacuten 110
Si diluimos una muestra a la mitad de concentracioacuten en un tampoacuten salino larelacioacuten 260280 se mantiene constante (la muestra tiene la misma pureza) sinembargo el ratio 260230 disminuye por el efecto del incremento proporcional desales respecto al ADN
INTEGRIDAD
ELECTROFORESIS EN GEL DE AGAROSA
28 S18 S
gDNA
agarosa 07 agarosa 1
integridad bandas ribosomales 28S y 18S discretas en proporcioacuten 21 21
integridad una uacutenica banda definidaen la parte superior del gel
pureza deteccioacuten de presencia de ADN en la parte superior del gel
pureza deteccioacuten de presencia de ARN en la parte inferior del gel
ge 50 ng
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
CRITERIOS DE CALIDAD DE ADN Y ARN
Concentracioacuten
Pureza
Integridad
Funcionalidad
wwwbancoadnorg
Trazabilidad
- Espectrofotometriacutea
1 unidad de absorbancia en 1cm de trayectoria oacuteptica para ADN de doble cadena = 50 microg ml y para ARN = 40 microg ml
Cada moleacutecula absorbe la energiacutea radiante a una longitud de ondaespeciacutefica a partir de la cual es posible extrapolar la concentracioacuten de unsoluto en una solucioacuten
Ley de Lambert-Beer una relacioacuten lineal entre absorbancia A (DO) y concentracioacuten
A = DO = αlc (1)
α coeficiente de extincioacuten molar (probabilidad de que el fotoacuten sea absorbido por el material a esa λl distancia que recorre la luz para atravesar el materialc concentracioacuten
CONCENTRACIOacuteN
-basa la cuantificacioacuten en la medida de absorbancia 260 nmde todos los nucleoacutetidos
- concentracioacuten y pureza
- Espectrofotometriacutea Nanodrop ND1000
Ventajas
- sencillo raacutepido y econoacutemico
Incovenientes
La capacidad de absorcioacuten a una λ especiacutefca de lasposibles sustancias contaminantes
ADN degradadoADN monocatenariooligonucleoacutetidosARN
-No aporta informacioacuten sobre la integridad
CONCENTRACIOacuteN
-No discrimina entre ADN y ARN
Fundamento determinacioacuten de la concentracioacuten en funcioacuten de la fluorescenciaemitida por un fluoroacuteforo de unioacuten especiacutefica al ADN de doble cadena
Cuantificacioacuten relativa necesario realizar una recta patroacuten con un ADN estaacutendar (iacutentegro) de concentracioacuten conocida
PicoGreenreg SYBR Green reg QuantiFluortrade
Ventajas- concentracioacuten e integridad
Unioacuten exclusiva a ADN de doble cadena
- coste elevado metodologiacutea maacutes compleja (gran precisioacuten)
-No aporta informacioacuten sobre la pureza del ADN
RELACIOacuteN PicoGreenreg Nanodrop ND1000R lt 1
CONCENTRACIOacuteN DE ADN
- Fluorimetriacutea
Inconvenientes
CONCENTRACIOacuteN DE ADN
muestra oacuteptima de ADN R 08gt
Picogreen PromediosPicogreen
MEDIDAS DE CONCENTRACION (ngul))MEDIDAS DE CONCENTRACION (ngul))
ltgt 1 2 3 ltgtA 9829 9678 9740 A 9749B 16827 17030 16198 B 16685C 40833 41898 41583 C 41438D 21674 21724 21649 D 21682E 9611 9224 9088 E 9308
Promedios NanodropMEDIDAS DE CONCENTRACIOacuteN (ngul))ltgtA 11800B 18730C 47950D 25650E 10480
Ratios piconano
A 083B 089C 086D 085E 089
Meacutetodo precipitacioacuten con salesldquosalting outrdquo
A4 B3 B6 B9 B10 C2 C3085 076 074 025 024 055 074
RELACIOacuteN PicoGreenreg Nanodrop
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
A4 B3 B6 B9 B10 C2 C3ng microl 588 658 68 50 455 455 42
A4 B3 B6 B9 B10 C2 C3ng microl 50 50 50 125 11 25 312
concentracioacuten PicoGreenreg
concentracioacuten Nanodrop ND1000
Metodologiacutea RT-PCR a tiempo real
Cuantificacioacuten relativa necesario realizar una recta patroacuten con un ADN estaacutendar (iacutentegro) de concentracioacuten conocida
Ventajas- concentracioacuten e integridad mediante el caacutelculo de los valores de ciclo de umbral o Ct
- coste elevado metodologiacutea maacutes compleja interpretacioacuten de los resultados
CONCENTRACIOacuteN DE ARN
- Fluorimetriacutea
Inconvenientes
Los fluoroacuteforos pueden ser de dos tipos a) fluoroacuteforos con afinidad por el ADN b) sondas especificas para fragmentos del ADN
Fundamento determinacioacuten de la concentracioacuten en funcioacuten de la fluorescencia emitida por un fluoroacuteforo de unioacuten al ADN sintetizado
Hay que tener en cuenta que el tamantildeo de los fragmentos amplificados es limitado
- deteccioacuten de contaminacioacuten por ADN- funcionalidad
- no aporta informacioacuten sobre presencia de contaminantes en la muestra
PUREZA
Capacidad de absorbancia a una λ determinada de las posibles sustancias contaminantes presentes en una solucioacuten de ADN
RELACIOacuteN DE ABSORBANCIAS
A280 proteiacutenas fenol (A270)A230 sales caotroacutepicas fenol hidratos de carbono
wwwbancoadnorg
A260A280Contaminacioacuten c aromaacuteticoslt 16
18 ndash 20 PUREZA OacutePTIMAgt 21 Contaminacioacuten ARN
A260A230 20-22 PUREZA OacutePTIMA
ADN
A260A280 20 - 21
A260A230 ~20
ge 18 Pureza aceptablePUREZA OacutePTIMA
PUREZA OacutePTIMAARN
A260280 187
A260230 246
A260280 204
A260230 168
ADN alta pureza
PUREZA
ARN alta pureza
Precipitacioacuten con etanol y sales
A260280 190
A260230 237
ngmicrol 1400
A260280 168
A260230 125
ngmicrol 2551
Contaminacioacuten con fenol
Relacioacuten A260230
A230sales caotroacutepicas fenolhidratos de carbono
Puede resultar muy variable dependiendo del tampoacuten que utilicemos como blanco en la medida del espectrofotoacutemetro
muestras concentracioacuten A260280 A26023010-3 5618 191 -6610-4 5736 197 -6810-5 577 191 -72510-6 567 187 -63910-8 548 188 -595
muestras concentracioacuten A260280 A26023010-3 5578 19 22510-4 5598 201 23910-5 5541 194 22610-6 5483 201 2310-8 532 203 235
Buffer TE
Buffer kit bolas magneacuteticas
A260280 189A260230 226
ngmicrol 1316
A260280 186A260230 172
ngmicrol 111
dilucioacuten 110
Si diluimos una muestra a la mitad de concentracioacuten en un tampoacuten salino larelacioacuten 260280 se mantiene constante (la muestra tiene la misma pureza) sinembargo el ratio 260230 disminuye por el efecto del incremento proporcional desales respecto al ADN
INTEGRIDAD
ELECTROFORESIS EN GEL DE AGAROSA
28 S18 S
gDNA
agarosa 07 agarosa 1
integridad bandas ribosomales 28S y 18S discretas en proporcioacuten 21 21
integridad una uacutenica banda definidaen la parte superior del gel
pureza deteccioacuten de presencia de ADN en la parte superior del gel
pureza deteccioacuten de presencia de ARN en la parte inferior del gel
ge 50 ng
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
- Espectrofotometriacutea
1 unidad de absorbancia en 1cm de trayectoria oacuteptica para ADN de doble cadena = 50 microg ml y para ARN = 40 microg ml
Cada moleacutecula absorbe la energiacutea radiante a una longitud de ondaespeciacutefica a partir de la cual es posible extrapolar la concentracioacuten de unsoluto en una solucioacuten
Ley de Lambert-Beer una relacioacuten lineal entre absorbancia A (DO) y concentracioacuten
A = DO = αlc (1)
α coeficiente de extincioacuten molar (probabilidad de que el fotoacuten sea absorbido por el material a esa λl distancia que recorre la luz para atravesar el materialc concentracioacuten
CONCENTRACIOacuteN
-basa la cuantificacioacuten en la medida de absorbancia 260 nmde todos los nucleoacutetidos
- concentracioacuten y pureza
- Espectrofotometriacutea Nanodrop ND1000
Ventajas
- sencillo raacutepido y econoacutemico
Incovenientes
La capacidad de absorcioacuten a una λ especiacutefca de lasposibles sustancias contaminantes
ADN degradadoADN monocatenariooligonucleoacutetidosARN
-No aporta informacioacuten sobre la integridad
CONCENTRACIOacuteN
-No discrimina entre ADN y ARN
Fundamento determinacioacuten de la concentracioacuten en funcioacuten de la fluorescenciaemitida por un fluoroacuteforo de unioacuten especiacutefica al ADN de doble cadena
Cuantificacioacuten relativa necesario realizar una recta patroacuten con un ADN estaacutendar (iacutentegro) de concentracioacuten conocida
PicoGreenreg SYBR Green reg QuantiFluortrade
Ventajas- concentracioacuten e integridad
Unioacuten exclusiva a ADN de doble cadena
- coste elevado metodologiacutea maacutes compleja (gran precisioacuten)
-No aporta informacioacuten sobre la pureza del ADN
RELACIOacuteN PicoGreenreg Nanodrop ND1000R lt 1
CONCENTRACIOacuteN DE ADN
- Fluorimetriacutea
Inconvenientes
CONCENTRACIOacuteN DE ADN
muestra oacuteptima de ADN R 08gt
Picogreen PromediosPicogreen
MEDIDAS DE CONCENTRACION (ngul))MEDIDAS DE CONCENTRACION (ngul))
ltgt 1 2 3 ltgtA 9829 9678 9740 A 9749B 16827 17030 16198 B 16685C 40833 41898 41583 C 41438D 21674 21724 21649 D 21682E 9611 9224 9088 E 9308
Promedios NanodropMEDIDAS DE CONCENTRACIOacuteN (ngul))ltgtA 11800B 18730C 47950D 25650E 10480
Ratios piconano
A 083B 089C 086D 085E 089
Meacutetodo precipitacioacuten con salesldquosalting outrdquo
A4 B3 B6 B9 B10 C2 C3085 076 074 025 024 055 074
RELACIOacuteN PicoGreenreg Nanodrop
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
A4 B3 B6 B9 B10 C2 C3ng microl 588 658 68 50 455 455 42
A4 B3 B6 B9 B10 C2 C3ng microl 50 50 50 125 11 25 312
concentracioacuten PicoGreenreg
concentracioacuten Nanodrop ND1000
Metodologiacutea RT-PCR a tiempo real
Cuantificacioacuten relativa necesario realizar una recta patroacuten con un ADN estaacutendar (iacutentegro) de concentracioacuten conocida
Ventajas- concentracioacuten e integridad mediante el caacutelculo de los valores de ciclo de umbral o Ct
- coste elevado metodologiacutea maacutes compleja interpretacioacuten de los resultados
CONCENTRACIOacuteN DE ARN
- Fluorimetriacutea
Inconvenientes
Los fluoroacuteforos pueden ser de dos tipos a) fluoroacuteforos con afinidad por el ADN b) sondas especificas para fragmentos del ADN
Fundamento determinacioacuten de la concentracioacuten en funcioacuten de la fluorescencia emitida por un fluoroacuteforo de unioacuten al ADN sintetizado
Hay que tener en cuenta que el tamantildeo de los fragmentos amplificados es limitado
- deteccioacuten de contaminacioacuten por ADN- funcionalidad
- no aporta informacioacuten sobre presencia de contaminantes en la muestra
PUREZA
Capacidad de absorbancia a una λ determinada de las posibles sustancias contaminantes presentes en una solucioacuten de ADN
RELACIOacuteN DE ABSORBANCIAS
A280 proteiacutenas fenol (A270)A230 sales caotroacutepicas fenol hidratos de carbono
wwwbancoadnorg
A260A280Contaminacioacuten c aromaacuteticoslt 16
18 ndash 20 PUREZA OacutePTIMAgt 21 Contaminacioacuten ARN
A260A230 20-22 PUREZA OacutePTIMA
ADN
A260A280 20 - 21
A260A230 ~20
ge 18 Pureza aceptablePUREZA OacutePTIMA
PUREZA OacutePTIMAARN
A260280 187
A260230 246
A260280 204
A260230 168
ADN alta pureza
PUREZA
ARN alta pureza
Precipitacioacuten con etanol y sales
A260280 190
A260230 237
ngmicrol 1400
A260280 168
A260230 125
ngmicrol 2551
Contaminacioacuten con fenol
Relacioacuten A260230
A230sales caotroacutepicas fenolhidratos de carbono
Puede resultar muy variable dependiendo del tampoacuten que utilicemos como blanco en la medida del espectrofotoacutemetro
muestras concentracioacuten A260280 A26023010-3 5618 191 -6610-4 5736 197 -6810-5 577 191 -72510-6 567 187 -63910-8 548 188 -595
muestras concentracioacuten A260280 A26023010-3 5578 19 22510-4 5598 201 23910-5 5541 194 22610-6 5483 201 2310-8 532 203 235
Buffer TE
Buffer kit bolas magneacuteticas
A260280 189A260230 226
ngmicrol 1316
A260280 186A260230 172
ngmicrol 111
dilucioacuten 110
Si diluimos una muestra a la mitad de concentracioacuten en un tampoacuten salino larelacioacuten 260280 se mantiene constante (la muestra tiene la misma pureza) sinembargo el ratio 260230 disminuye por el efecto del incremento proporcional desales respecto al ADN
INTEGRIDAD
ELECTROFORESIS EN GEL DE AGAROSA
28 S18 S
gDNA
agarosa 07 agarosa 1
integridad bandas ribosomales 28S y 18S discretas en proporcioacuten 21 21
integridad una uacutenica banda definidaen la parte superior del gel
pureza deteccioacuten de presencia de ADN en la parte superior del gel
pureza deteccioacuten de presencia de ARN en la parte inferior del gel
ge 50 ng
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
-basa la cuantificacioacuten en la medida de absorbancia 260 nmde todos los nucleoacutetidos
- concentracioacuten y pureza
- Espectrofotometriacutea Nanodrop ND1000
Ventajas
- sencillo raacutepido y econoacutemico
Incovenientes
La capacidad de absorcioacuten a una λ especiacutefca de lasposibles sustancias contaminantes
ADN degradadoADN monocatenariooligonucleoacutetidosARN
-No aporta informacioacuten sobre la integridad
CONCENTRACIOacuteN
-No discrimina entre ADN y ARN
Fundamento determinacioacuten de la concentracioacuten en funcioacuten de la fluorescenciaemitida por un fluoroacuteforo de unioacuten especiacutefica al ADN de doble cadena
Cuantificacioacuten relativa necesario realizar una recta patroacuten con un ADN estaacutendar (iacutentegro) de concentracioacuten conocida
PicoGreenreg SYBR Green reg QuantiFluortrade
Ventajas- concentracioacuten e integridad
Unioacuten exclusiva a ADN de doble cadena
- coste elevado metodologiacutea maacutes compleja (gran precisioacuten)
-No aporta informacioacuten sobre la pureza del ADN
RELACIOacuteN PicoGreenreg Nanodrop ND1000R lt 1
CONCENTRACIOacuteN DE ADN
- Fluorimetriacutea
Inconvenientes
CONCENTRACIOacuteN DE ADN
muestra oacuteptima de ADN R 08gt
Picogreen PromediosPicogreen
MEDIDAS DE CONCENTRACION (ngul))MEDIDAS DE CONCENTRACION (ngul))
ltgt 1 2 3 ltgtA 9829 9678 9740 A 9749B 16827 17030 16198 B 16685C 40833 41898 41583 C 41438D 21674 21724 21649 D 21682E 9611 9224 9088 E 9308
Promedios NanodropMEDIDAS DE CONCENTRACIOacuteN (ngul))ltgtA 11800B 18730C 47950D 25650E 10480
Ratios piconano
A 083B 089C 086D 085E 089
Meacutetodo precipitacioacuten con salesldquosalting outrdquo
A4 B3 B6 B9 B10 C2 C3085 076 074 025 024 055 074
RELACIOacuteN PicoGreenreg Nanodrop
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
A4 B3 B6 B9 B10 C2 C3ng microl 588 658 68 50 455 455 42
A4 B3 B6 B9 B10 C2 C3ng microl 50 50 50 125 11 25 312
concentracioacuten PicoGreenreg
concentracioacuten Nanodrop ND1000
Metodologiacutea RT-PCR a tiempo real
Cuantificacioacuten relativa necesario realizar una recta patroacuten con un ADN estaacutendar (iacutentegro) de concentracioacuten conocida
Ventajas- concentracioacuten e integridad mediante el caacutelculo de los valores de ciclo de umbral o Ct
- coste elevado metodologiacutea maacutes compleja interpretacioacuten de los resultados
CONCENTRACIOacuteN DE ARN
- Fluorimetriacutea
Inconvenientes
Los fluoroacuteforos pueden ser de dos tipos a) fluoroacuteforos con afinidad por el ADN b) sondas especificas para fragmentos del ADN
Fundamento determinacioacuten de la concentracioacuten en funcioacuten de la fluorescencia emitida por un fluoroacuteforo de unioacuten al ADN sintetizado
Hay que tener en cuenta que el tamantildeo de los fragmentos amplificados es limitado
- deteccioacuten de contaminacioacuten por ADN- funcionalidad
- no aporta informacioacuten sobre presencia de contaminantes en la muestra
PUREZA
Capacidad de absorbancia a una λ determinada de las posibles sustancias contaminantes presentes en una solucioacuten de ADN
RELACIOacuteN DE ABSORBANCIAS
A280 proteiacutenas fenol (A270)A230 sales caotroacutepicas fenol hidratos de carbono
wwwbancoadnorg
A260A280Contaminacioacuten c aromaacuteticoslt 16
18 ndash 20 PUREZA OacutePTIMAgt 21 Contaminacioacuten ARN
A260A230 20-22 PUREZA OacutePTIMA
ADN
A260A280 20 - 21
A260A230 ~20
ge 18 Pureza aceptablePUREZA OacutePTIMA
PUREZA OacutePTIMAARN
A260280 187
A260230 246
A260280 204
A260230 168
ADN alta pureza
PUREZA
ARN alta pureza
Precipitacioacuten con etanol y sales
A260280 190
A260230 237
ngmicrol 1400
A260280 168
A260230 125
ngmicrol 2551
Contaminacioacuten con fenol
Relacioacuten A260230
A230sales caotroacutepicas fenolhidratos de carbono
Puede resultar muy variable dependiendo del tampoacuten que utilicemos como blanco en la medida del espectrofotoacutemetro
muestras concentracioacuten A260280 A26023010-3 5618 191 -6610-4 5736 197 -6810-5 577 191 -72510-6 567 187 -63910-8 548 188 -595
muestras concentracioacuten A260280 A26023010-3 5578 19 22510-4 5598 201 23910-5 5541 194 22610-6 5483 201 2310-8 532 203 235
Buffer TE
Buffer kit bolas magneacuteticas
A260280 189A260230 226
ngmicrol 1316
A260280 186A260230 172
ngmicrol 111
dilucioacuten 110
Si diluimos una muestra a la mitad de concentracioacuten en un tampoacuten salino larelacioacuten 260280 se mantiene constante (la muestra tiene la misma pureza) sinembargo el ratio 260230 disminuye por el efecto del incremento proporcional desales respecto al ADN
INTEGRIDAD
ELECTROFORESIS EN GEL DE AGAROSA
28 S18 S
gDNA
agarosa 07 agarosa 1
integridad bandas ribosomales 28S y 18S discretas en proporcioacuten 21 21
integridad una uacutenica banda definidaen la parte superior del gel
pureza deteccioacuten de presencia de ADN en la parte superior del gel
pureza deteccioacuten de presencia de ARN en la parte inferior del gel
ge 50 ng
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
Fundamento determinacioacuten de la concentracioacuten en funcioacuten de la fluorescenciaemitida por un fluoroacuteforo de unioacuten especiacutefica al ADN de doble cadena
Cuantificacioacuten relativa necesario realizar una recta patroacuten con un ADN estaacutendar (iacutentegro) de concentracioacuten conocida
PicoGreenreg SYBR Green reg QuantiFluortrade
Ventajas- concentracioacuten e integridad
Unioacuten exclusiva a ADN de doble cadena
- coste elevado metodologiacutea maacutes compleja (gran precisioacuten)
-No aporta informacioacuten sobre la pureza del ADN
RELACIOacuteN PicoGreenreg Nanodrop ND1000R lt 1
CONCENTRACIOacuteN DE ADN
- Fluorimetriacutea
Inconvenientes
CONCENTRACIOacuteN DE ADN
muestra oacuteptima de ADN R 08gt
Picogreen PromediosPicogreen
MEDIDAS DE CONCENTRACION (ngul))MEDIDAS DE CONCENTRACION (ngul))
ltgt 1 2 3 ltgtA 9829 9678 9740 A 9749B 16827 17030 16198 B 16685C 40833 41898 41583 C 41438D 21674 21724 21649 D 21682E 9611 9224 9088 E 9308
Promedios NanodropMEDIDAS DE CONCENTRACIOacuteN (ngul))ltgtA 11800B 18730C 47950D 25650E 10480
Ratios piconano
A 083B 089C 086D 085E 089
Meacutetodo precipitacioacuten con salesldquosalting outrdquo
A4 B3 B6 B9 B10 C2 C3085 076 074 025 024 055 074
RELACIOacuteN PicoGreenreg Nanodrop
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
A4 B3 B6 B9 B10 C2 C3ng microl 588 658 68 50 455 455 42
A4 B3 B6 B9 B10 C2 C3ng microl 50 50 50 125 11 25 312
concentracioacuten PicoGreenreg
concentracioacuten Nanodrop ND1000
Metodologiacutea RT-PCR a tiempo real
Cuantificacioacuten relativa necesario realizar una recta patroacuten con un ADN estaacutendar (iacutentegro) de concentracioacuten conocida
Ventajas- concentracioacuten e integridad mediante el caacutelculo de los valores de ciclo de umbral o Ct
- coste elevado metodologiacutea maacutes compleja interpretacioacuten de los resultados
CONCENTRACIOacuteN DE ARN
- Fluorimetriacutea
Inconvenientes
Los fluoroacuteforos pueden ser de dos tipos a) fluoroacuteforos con afinidad por el ADN b) sondas especificas para fragmentos del ADN
Fundamento determinacioacuten de la concentracioacuten en funcioacuten de la fluorescencia emitida por un fluoroacuteforo de unioacuten al ADN sintetizado
Hay que tener en cuenta que el tamantildeo de los fragmentos amplificados es limitado
- deteccioacuten de contaminacioacuten por ADN- funcionalidad
- no aporta informacioacuten sobre presencia de contaminantes en la muestra
PUREZA
Capacidad de absorbancia a una λ determinada de las posibles sustancias contaminantes presentes en una solucioacuten de ADN
RELACIOacuteN DE ABSORBANCIAS
A280 proteiacutenas fenol (A270)A230 sales caotroacutepicas fenol hidratos de carbono
wwwbancoadnorg
A260A280Contaminacioacuten c aromaacuteticoslt 16
18 ndash 20 PUREZA OacutePTIMAgt 21 Contaminacioacuten ARN
A260A230 20-22 PUREZA OacutePTIMA
ADN
A260A280 20 - 21
A260A230 ~20
ge 18 Pureza aceptablePUREZA OacutePTIMA
PUREZA OacutePTIMAARN
A260280 187
A260230 246
A260280 204
A260230 168
ADN alta pureza
PUREZA
ARN alta pureza
Precipitacioacuten con etanol y sales
A260280 190
A260230 237
ngmicrol 1400
A260280 168
A260230 125
ngmicrol 2551
Contaminacioacuten con fenol
Relacioacuten A260230
A230sales caotroacutepicas fenolhidratos de carbono
Puede resultar muy variable dependiendo del tampoacuten que utilicemos como blanco en la medida del espectrofotoacutemetro
muestras concentracioacuten A260280 A26023010-3 5618 191 -6610-4 5736 197 -6810-5 577 191 -72510-6 567 187 -63910-8 548 188 -595
muestras concentracioacuten A260280 A26023010-3 5578 19 22510-4 5598 201 23910-5 5541 194 22610-6 5483 201 2310-8 532 203 235
Buffer TE
Buffer kit bolas magneacuteticas
A260280 189A260230 226
ngmicrol 1316
A260280 186A260230 172
ngmicrol 111
dilucioacuten 110
Si diluimos una muestra a la mitad de concentracioacuten en un tampoacuten salino larelacioacuten 260280 se mantiene constante (la muestra tiene la misma pureza) sinembargo el ratio 260230 disminuye por el efecto del incremento proporcional desales respecto al ADN
INTEGRIDAD
ELECTROFORESIS EN GEL DE AGAROSA
28 S18 S
gDNA
agarosa 07 agarosa 1
integridad bandas ribosomales 28S y 18S discretas en proporcioacuten 21 21
integridad una uacutenica banda definidaen la parte superior del gel
pureza deteccioacuten de presencia de ADN en la parte superior del gel
pureza deteccioacuten de presencia de ARN en la parte inferior del gel
ge 50 ng
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
CONCENTRACIOacuteN DE ADN
muestra oacuteptima de ADN R 08gt
Picogreen PromediosPicogreen
MEDIDAS DE CONCENTRACION (ngul))MEDIDAS DE CONCENTRACION (ngul))
ltgt 1 2 3 ltgtA 9829 9678 9740 A 9749B 16827 17030 16198 B 16685C 40833 41898 41583 C 41438D 21674 21724 21649 D 21682E 9611 9224 9088 E 9308
Promedios NanodropMEDIDAS DE CONCENTRACIOacuteN (ngul))ltgtA 11800B 18730C 47950D 25650E 10480
Ratios piconano
A 083B 089C 086D 085E 089
Meacutetodo precipitacioacuten con salesldquosalting outrdquo
A4 B3 B6 B9 B10 C2 C3085 076 074 025 024 055 074
RELACIOacuteN PicoGreenreg Nanodrop
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
A4 B3 B6 B9 B10 C2 C3ng microl 588 658 68 50 455 455 42
A4 B3 B6 B9 B10 C2 C3ng microl 50 50 50 125 11 25 312
concentracioacuten PicoGreenreg
concentracioacuten Nanodrop ND1000
Metodologiacutea RT-PCR a tiempo real
Cuantificacioacuten relativa necesario realizar una recta patroacuten con un ADN estaacutendar (iacutentegro) de concentracioacuten conocida
Ventajas- concentracioacuten e integridad mediante el caacutelculo de los valores de ciclo de umbral o Ct
- coste elevado metodologiacutea maacutes compleja interpretacioacuten de los resultados
CONCENTRACIOacuteN DE ARN
- Fluorimetriacutea
Inconvenientes
Los fluoroacuteforos pueden ser de dos tipos a) fluoroacuteforos con afinidad por el ADN b) sondas especificas para fragmentos del ADN
Fundamento determinacioacuten de la concentracioacuten en funcioacuten de la fluorescencia emitida por un fluoroacuteforo de unioacuten al ADN sintetizado
Hay que tener en cuenta que el tamantildeo de los fragmentos amplificados es limitado
- deteccioacuten de contaminacioacuten por ADN- funcionalidad
- no aporta informacioacuten sobre presencia de contaminantes en la muestra
PUREZA
Capacidad de absorbancia a una λ determinada de las posibles sustancias contaminantes presentes en una solucioacuten de ADN
RELACIOacuteN DE ABSORBANCIAS
A280 proteiacutenas fenol (A270)A230 sales caotroacutepicas fenol hidratos de carbono
wwwbancoadnorg
A260A280Contaminacioacuten c aromaacuteticoslt 16
18 ndash 20 PUREZA OacutePTIMAgt 21 Contaminacioacuten ARN
A260A230 20-22 PUREZA OacutePTIMA
ADN
A260A280 20 - 21
A260A230 ~20
ge 18 Pureza aceptablePUREZA OacutePTIMA
PUREZA OacutePTIMAARN
A260280 187
A260230 246
A260280 204
A260230 168
ADN alta pureza
PUREZA
ARN alta pureza
Precipitacioacuten con etanol y sales
A260280 190
A260230 237
ngmicrol 1400
A260280 168
A260230 125
ngmicrol 2551
Contaminacioacuten con fenol
Relacioacuten A260230
A230sales caotroacutepicas fenolhidratos de carbono
Puede resultar muy variable dependiendo del tampoacuten que utilicemos como blanco en la medida del espectrofotoacutemetro
muestras concentracioacuten A260280 A26023010-3 5618 191 -6610-4 5736 197 -6810-5 577 191 -72510-6 567 187 -63910-8 548 188 -595
muestras concentracioacuten A260280 A26023010-3 5578 19 22510-4 5598 201 23910-5 5541 194 22610-6 5483 201 2310-8 532 203 235
Buffer TE
Buffer kit bolas magneacuteticas
A260280 189A260230 226
ngmicrol 1316
A260280 186A260230 172
ngmicrol 111
dilucioacuten 110
Si diluimos una muestra a la mitad de concentracioacuten en un tampoacuten salino larelacioacuten 260280 se mantiene constante (la muestra tiene la misma pureza) sinembargo el ratio 260230 disminuye por el efecto del incremento proporcional desales respecto al ADN
INTEGRIDAD
ELECTROFORESIS EN GEL DE AGAROSA
28 S18 S
gDNA
agarosa 07 agarosa 1
integridad bandas ribosomales 28S y 18S discretas en proporcioacuten 21 21
integridad una uacutenica banda definidaen la parte superior del gel
pureza deteccioacuten de presencia de ADN en la parte superior del gel
pureza deteccioacuten de presencia de ARN en la parte inferior del gel
ge 50 ng
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
A4 B3 B6 B9 B10 C2 C3085 076 074 025 024 055 074
RELACIOacuteN PicoGreenreg Nanodrop
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
A4 B3 B6 B9 B10 C2 C3ng microl 588 658 68 50 455 455 42
A4 B3 B6 B9 B10 C2 C3ng microl 50 50 50 125 11 25 312
concentracioacuten PicoGreenreg
concentracioacuten Nanodrop ND1000
Metodologiacutea RT-PCR a tiempo real
Cuantificacioacuten relativa necesario realizar una recta patroacuten con un ADN estaacutendar (iacutentegro) de concentracioacuten conocida
Ventajas- concentracioacuten e integridad mediante el caacutelculo de los valores de ciclo de umbral o Ct
- coste elevado metodologiacutea maacutes compleja interpretacioacuten de los resultados
CONCENTRACIOacuteN DE ARN
- Fluorimetriacutea
Inconvenientes
Los fluoroacuteforos pueden ser de dos tipos a) fluoroacuteforos con afinidad por el ADN b) sondas especificas para fragmentos del ADN
Fundamento determinacioacuten de la concentracioacuten en funcioacuten de la fluorescencia emitida por un fluoroacuteforo de unioacuten al ADN sintetizado
Hay que tener en cuenta que el tamantildeo de los fragmentos amplificados es limitado
- deteccioacuten de contaminacioacuten por ADN- funcionalidad
- no aporta informacioacuten sobre presencia de contaminantes en la muestra
PUREZA
Capacidad de absorbancia a una λ determinada de las posibles sustancias contaminantes presentes en una solucioacuten de ADN
RELACIOacuteN DE ABSORBANCIAS
A280 proteiacutenas fenol (A270)A230 sales caotroacutepicas fenol hidratos de carbono
wwwbancoadnorg
A260A280Contaminacioacuten c aromaacuteticoslt 16
18 ndash 20 PUREZA OacutePTIMAgt 21 Contaminacioacuten ARN
A260A230 20-22 PUREZA OacutePTIMA
ADN
A260A280 20 - 21
A260A230 ~20
ge 18 Pureza aceptablePUREZA OacutePTIMA
PUREZA OacutePTIMAARN
A260280 187
A260230 246
A260280 204
A260230 168
ADN alta pureza
PUREZA
ARN alta pureza
Precipitacioacuten con etanol y sales
A260280 190
A260230 237
ngmicrol 1400
A260280 168
A260230 125
ngmicrol 2551
Contaminacioacuten con fenol
Relacioacuten A260230
A230sales caotroacutepicas fenolhidratos de carbono
Puede resultar muy variable dependiendo del tampoacuten que utilicemos como blanco en la medida del espectrofotoacutemetro
muestras concentracioacuten A260280 A26023010-3 5618 191 -6610-4 5736 197 -6810-5 577 191 -72510-6 567 187 -63910-8 548 188 -595
muestras concentracioacuten A260280 A26023010-3 5578 19 22510-4 5598 201 23910-5 5541 194 22610-6 5483 201 2310-8 532 203 235
Buffer TE
Buffer kit bolas magneacuteticas
A260280 189A260230 226
ngmicrol 1316
A260280 186A260230 172
ngmicrol 111
dilucioacuten 110
Si diluimos una muestra a la mitad de concentracioacuten en un tampoacuten salino larelacioacuten 260280 se mantiene constante (la muestra tiene la misma pureza) sinembargo el ratio 260230 disminuye por el efecto del incremento proporcional desales respecto al ADN
INTEGRIDAD
ELECTROFORESIS EN GEL DE AGAROSA
28 S18 S
gDNA
agarosa 07 agarosa 1
integridad bandas ribosomales 28S y 18S discretas en proporcioacuten 21 21
integridad una uacutenica banda definidaen la parte superior del gel
pureza deteccioacuten de presencia de ADN en la parte superior del gel
pureza deteccioacuten de presencia de ARN en la parte inferior del gel
ge 50 ng
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
Metodologiacutea RT-PCR a tiempo real
Cuantificacioacuten relativa necesario realizar una recta patroacuten con un ADN estaacutendar (iacutentegro) de concentracioacuten conocida
Ventajas- concentracioacuten e integridad mediante el caacutelculo de los valores de ciclo de umbral o Ct
- coste elevado metodologiacutea maacutes compleja interpretacioacuten de los resultados
CONCENTRACIOacuteN DE ARN
- Fluorimetriacutea
Inconvenientes
Los fluoroacuteforos pueden ser de dos tipos a) fluoroacuteforos con afinidad por el ADN b) sondas especificas para fragmentos del ADN
Fundamento determinacioacuten de la concentracioacuten en funcioacuten de la fluorescencia emitida por un fluoroacuteforo de unioacuten al ADN sintetizado
Hay que tener en cuenta que el tamantildeo de los fragmentos amplificados es limitado
- deteccioacuten de contaminacioacuten por ADN- funcionalidad
- no aporta informacioacuten sobre presencia de contaminantes en la muestra
PUREZA
Capacidad de absorbancia a una λ determinada de las posibles sustancias contaminantes presentes en una solucioacuten de ADN
RELACIOacuteN DE ABSORBANCIAS
A280 proteiacutenas fenol (A270)A230 sales caotroacutepicas fenol hidratos de carbono
wwwbancoadnorg
A260A280Contaminacioacuten c aromaacuteticoslt 16
18 ndash 20 PUREZA OacutePTIMAgt 21 Contaminacioacuten ARN
A260A230 20-22 PUREZA OacutePTIMA
ADN
A260A280 20 - 21
A260A230 ~20
ge 18 Pureza aceptablePUREZA OacutePTIMA
PUREZA OacutePTIMAARN
A260280 187
A260230 246
A260280 204
A260230 168
ADN alta pureza
PUREZA
ARN alta pureza
Precipitacioacuten con etanol y sales
A260280 190
A260230 237
ngmicrol 1400
A260280 168
A260230 125
ngmicrol 2551
Contaminacioacuten con fenol
Relacioacuten A260230
A230sales caotroacutepicas fenolhidratos de carbono
Puede resultar muy variable dependiendo del tampoacuten que utilicemos como blanco en la medida del espectrofotoacutemetro
muestras concentracioacuten A260280 A26023010-3 5618 191 -6610-4 5736 197 -6810-5 577 191 -72510-6 567 187 -63910-8 548 188 -595
muestras concentracioacuten A260280 A26023010-3 5578 19 22510-4 5598 201 23910-5 5541 194 22610-6 5483 201 2310-8 532 203 235
Buffer TE
Buffer kit bolas magneacuteticas
A260280 189A260230 226
ngmicrol 1316
A260280 186A260230 172
ngmicrol 111
dilucioacuten 110
Si diluimos una muestra a la mitad de concentracioacuten en un tampoacuten salino larelacioacuten 260280 se mantiene constante (la muestra tiene la misma pureza) sinembargo el ratio 260230 disminuye por el efecto del incremento proporcional desales respecto al ADN
INTEGRIDAD
ELECTROFORESIS EN GEL DE AGAROSA
28 S18 S
gDNA
agarosa 07 agarosa 1
integridad bandas ribosomales 28S y 18S discretas en proporcioacuten 21 21
integridad una uacutenica banda definidaen la parte superior del gel
pureza deteccioacuten de presencia de ADN en la parte superior del gel
pureza deteccioacuten de presencia de ARN en la parte inferior del gel
ge 50 ng
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
PUREZA
Capacidad de absorbancia a una λ determinada de las posibles sustancias contaminantes presentes en una solucioacuten de ADN
RELACIOacuteN DE ABSORBANCIAS
A280 proteiacutenas fenol (A270)A230 sales caotroacutepicas fenol hidratos de carbono
wwwbancoadnorg
A260A280Contaminacioacuten c aromaacuteticoslt 16
18 ndash 20 PUREZA OacutePTIMAgt 21 Contaminacioacuten ARN
A260A230 20-22 PUREZA OacutePTIMA
ADN
A260A280 20 - 21
A260A230 ~20
ge 18 Pureza aceptablePUREZA OacutePTIMA
PUREZA OacutePTIMAARN
A260280 187
A260230 246
A260280 204
A260230 168
ADN alta pureza
PUREZA
ARN alta pureza
Precipitacioacuten con etanol y sales
A260280 190
A260230 237
ngmicrol 1400
A260280 168
A260230 125
ngmicrol 2551
Contaminacioacuten con fenol
Relacioacuten A260230
A230sales caotroacutepicas fenolhidratos de carbono
Puede resultar muy variable dependiendo del tampoacuten que utilicemos como blanco en la medida del espectrofotoacutemetro
muestras concentracioacuten A260280 A26023010-3 5618 191 -6610-4 5736 197 -6810-5 577 191 -72510-6 567 187 -63910-8 548 188 -595
muestras concentracioacuten A260280 A26023010-3 5578 19 22510-4 5598 201 23910-5 5541 194 22610-6 5483 201 2310-8 532 203 235
Buffer TE
Buffer kit bolas magneacuteticas
A260280 189A260230 226
ngmicrol 1316
A260280 186A260230 172
ngmicrol 111
dilucioacuten 110
Si diluimos una muestra a la mitad de concentracioacuten en un tampoacuten salino larelacioacuten 260280 se mantiene constante (la muestra tiene la misma pureza) sinembargo el ratio 260230 disminuye por el efecto del incremento proporcional desales respecto al ADN
INTEGRIDAD
ELECTROFORESIS EN GEL DE AGAROSA
28 S18 S
gDNA
agarosa 07 agarosa 1
integridad bandas ribosomales 28S y 18S discretas en proporcioacuten 21 21
integridad una uacutenica banda definidaen la parte superior del gel
pureza deteccioacuten de presencia de ADN en la parte superior del gel
pureza deteccioacuten de presencia de ARN en la parte inferior del gel
ge 50 ng
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
A260280 187
A260230 246
A260280 204
A260230 168
ADN alta pureza
PUREZA
ARN alta pureza
Precipitacioacuten con etanol y sales
A260280 190
A260230 237
ngmicrol 1400
A260280 168
A260230 125
ngmicrol 2551
Contaminacioacuten con fenol
Relacioacuten A260230
A230sales caotroacutepicas fenolhidratos de carbono
Puede resultar muy variable dependiendo del tampoacuten que utilicemos como blanco en la medida del espectrofotoacutemetro
muestras concentracioacuten A260280 A26023010-3 5618 191 -6610-4 5736 197 -6810-5 577 191 -72510-6 567 187 -63910-8 548 188 -595
muestras concentracioacuten A260280 A26023010-3 5578 19 22510-4 5598 201 23910-5 5541 194 22610-6 5483 201 2310-8 532 203 235
Buffer TE
Buffer kit bolas magneacuteticas
A260280 189A260230 226
ngmicrol 1316
A260280 186A260230 172
ngmicrol 111
dilucioacuten 110
Si diluimos una muestra a la mitad de concentracioacuten en un tampoacuten salino larelacioacuten 260280 se mantiene constante (la muestra tiene la misma pureza) sinembargo el ratio 260230 disminuye por el efecto del incremento proporcional desales respecto al ADN
INTEGRIDAD
ELECTROFORESIS EN GEL DE AGAROSA
28 S18 S
gDNA
agarosa 07 agarosa 1
integridad bandas ribosomales 28S y 18S discretas en proporcioacuten 21 21
integridad una uacutenica banda definidaen la parte superior del gel
pureza deteccioacuten de presencia de ADN en la parte superior del gel
pureza deteccioacuten de presencia de ARN en la parte inferior del gel
ge 50 ng
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
Precipitacioacuten con etanol y sales
A260280 190
A260230 237
ngmicrol 1400
A260280 168
A260230 125
ngmicrol 2551
Contaminacioacuten con fenol
Relacioacuten A260230
A230sales caotroacutepicas fenolhidratos de carbono
Puede resultar muy variable dependiendo del tampoacuten que utilicemos como blanco en la medida del espectrofotoacutemetro
muestras concentracioacuten A260280 A26023010-3 5618 191 -6610-4 5736 197 -6810-5 577 191 -72510-6 567 187 -63910-8 548 188 -595
muestras concentracioacuten A260280 A26023010-3 5578 19 22510-4 5598 201 23910-5 5541 194 22610-6 5483 201 2310-8 532 203 235
Buffer TE
Buffer kit bolas magneacuteticas
A260280 189A260230 226
ngmicrol 1316
A260280 186A260230 172
ngmicrol 111
dilucioacuten 110
Si diluimos una muestra a la mitad de concentracioacuten en un tampoacuten salino larelacioacuten 260280 se mantiene constante (la muestra tiene la misma pureza) sinembargo el ratio 260230 disminuye por el efecto del incremento proporcional desales respecto al ADN
INTEGRIDAD
ELECTROFORESIS EN GEL DE AGAROSA
28 S18 S
gDNA
agarosa 07 agarosa 1
integridad bandas ribosomales 28S y 18S discretas en proporcioacuten 21 21
integridad una uacutenica banda definidaen la parte superior del gel
pureza deteccioacuten de presencia de ADN en la parte superior del gel
pureza deteccioacuten de presencia de ARN en la parte inferior del gel
ge 50 ng
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
Relacioacuten A260230
A230sales caotroacutepicas fenolhidratos de carbono
Puede resultar muy variable dependiendo del tampoacuten que utilicemos como blanco en la medida del espectrofotoacutemetro
muestras concentracioacuten A260280 A26023010-3 5618 191 -6610-4 5736 197 -6810-5 577 191 -72510-6 567 187 -63910-8 548 188 -595
muestras concentracioacuten A260280 A26023010-3 5578 19 22510-4 5598 201 23910-5 5541 194 22610-6 5483 201 2310-8 532 203 235
Buffer TE
Buffer kit bolas magneacuteticas
A260280 189A260230 226
ngmicrol 1316
A260280 186A260230 172
ngmicrol 111
dilucioacuten 110
Si diluimos una muestra a la mitad de concentracioacuten en un tampoacuten salino larelacioacuten 260280 se mantiene constante (la muestra tiene la misma pureza) sinembargo el ratio 260230 disminuye por el efecto del incremento proporcional desales respecto al ADN
INTEGRIDAD
ELECTROFORESIS EN GEL DE AGAROSA
28 S18 S
gDNA
agarosa 07 agarosa 1
integridad bandas ribosomales 28S y 18S discretas en proporcioacuten 21 21
integridad una uacutenica banda definidaen la parte superior del gel
pureza deteccioacuten de presencia de ADN en la parte superior del gel
pureza deteccioacuten de presencia de ARN en la parte inferior del gel
ge 50 ng
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
A260280 189A260230 226
ngmicrol 1316
A260280 186A260230 172
ngmicrol 111
dilucioacuten 110
Si diluimos una muestra a la mitad de concentracioacuten en un tampoacuten salino larelacioacuten 260280 se mantiene constante (la muestra tiene la misma pureza) sinembargo el ratio 260230 disminuye por el efecto del incremento proporcional desales respecto al ADN
INTEGRIDAD
ELECTROFORESIS EN GEL DE AGAROSA
28 S18 S
gDNA
agarosa 07 agarosa 1
integridad bandas ribosomales 28S y 18S discretas en proporcioacuten 21 21
integridad una uacutenica banda definidaen la parte superior del gel
pureza deteccioacuten de presencia de ADN en la parte superior del gel
pureza deteccioacuten de presencia de ARN en la parte inferior del gel
ge 50 ng
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
INTEGRIDAD
ELECTROFORESIS EN GEL DE AGAROSA
28 S18 S
gDNA
agarosa 07 agarosa 1
integridad bandas ribosomales 28S y 18S discretas en proporcioacuten 21 21
integridad una uacutenica banda definidaen la parte superior del gel
pureza deteccioacuten de presencia de ADN en la parte superior del gel
pureza deteccioacuten de presencia de ARN en la parte inferior del gel
ge 50 ng
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
CONCENTRACIOacuteN E INTEGRIDAD
Agilent 2200 TapeStation system
-Permite evaluar la concentracioacuten y la integridad de una muestra
-Requiere muy poca cantidad de muestra
-Uso de geles multi-liacutenea y microfluiacutedos que permiten un ensayo semiautomaacutetico
-Simplifica el manejo de muestras
Quantitative range25-500 pg microl10-100 ng microl
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
Integridad del ADN DINDIN DNA integrity number
Valor del 1 al 10 que define la integridad de una muestra de ADN
10 valor de una muestra iacutentegra
1 valor de una muestra con alto nivel de degradacioacuten
20445 DIN 8 9 amplifica todas las bandas por Long PCR muacuteltiple (1705 kb)
Amplif 5000 pb DIN 62 amplifica hasta 5 kb
13-21 FFPE DIN 15 amplifica deacutebilmente 600 y 300 pb Amplifica con intensidad 200 pb
4B-04 Qiagen DIN 15 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
5B-43 fC DIN 13 amplifica deacutebilmente 300 pb Amplifica con intensidad 200 pb
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
Integridad del ARN RINe
RINe RNA integrity number
10 valor de una muestra iacutentegra1 valor de una muestra con alto nivel de degradacioacuten
RINe= RIN 2100 Bioanlyzer (Agilent)
RIN lt6 ARN degradado
RIN gt 7 ArraysRIN gt 4-7 qRT-PCR
RINe es una medida repetitiva pero puede diferir
del valor de RIN (en la misma muestra)
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
FUNCIONALIDAD DEL ADN
DIGESTIOacuteN del ADN con ENZIMAS DE RESTRICCIOacuteN
1
λ-HindIII
23 kb
2
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
FUNCIONALIDAD E INTEGRIDAD DEL ADN
kb
94
23
231
6543
20
λ-Hind III
1 2 3 4 5 6 7 H20
175
30
2024
kb
5070
PUNTUACIOacuteN
intensidadfuerte deacutebil
10 95
65745
253
5
8 75
1 2 3 4 5 6 7
LONG PCR MUacuteLTIPLE
Calidad del ADN
10-95 muestra oacutetpima
ge 65 muestra aceptable
lsaquo 45 rechazar muestra
45 muestra calidad comprometida
45 muestra calidad comprometida
enviar al investigador informando de su calidad
5 parejas de oligonucleoacutetidos (primers) simultaacuteneamenteamplificacioacuten en 6 cromosomas diferentesamplicones entre 175 kb y 20 kb
Long PCR MuacuteltipleADN molde 10 ng microl
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
Funcionalidad del ADN
- PCR muacuteltiple (100 ng ADN molde) 4 amplicones de 100 200 300 400 pb
ADN
- ADN muy fragmentado
- modificaciones quiacutemicas del ADN por formalina
pb
11 6 22 2018 11 8 7 19 17 16
(van Beers et al 2006)
antildeos
7 muestras amplificaron fragmentos ge 200 pb
TEJIDOS FFPE
CGH array(comparative genomic hybridisation)
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
TEJIDOS FFPE6 muestras extraiacutedas de tejidos parafinados con el meacutetodo kit QiAampreg mini(Qiagen) diferentes edades de bloque
1000
300200100
500700900
pb
200 pb
M 11 10 7 6 2A 2B
Con este meacutetodo la capacidadde amplificacioacuten de 4 de las 6muestras estaacute limitada a 200pb
La edad del bloque no estariacutea relacionada con la capacidad de amplificacioacuten de la muestra
300200100
300 pb500700900
1000
pb M 11 10 7 6 2 A 2B
400600800
1000
pb11 10 7 6 2 A 2BM
600 pb
100 ng molde
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
FUNCIONALIDAD DE ARN
REACCIOacuteN DE RT-PCR
- funcionalidad siacutentesis de cDNA (ADN complementario) a partir de ARNmediante una retro-transcriptasa y amplificacioacuten de secuencias especiacuteficas deADN- pureza si hay ADN en la muestra la amplificacioacuten de una regioacuteninterexoacutenica daraacute lugar adicionalmente a un producto mayor del esperado
5rsquo 3rsquo gDNAintroacuten exoacuten
5rsquo 3rsquo RNA cDNAamplicoacuten
primer
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
M marcador tamantildeo molecular
Resultado PCR utilizando cDNA como molde
1 gDNA (control de la PCR)
2 cDNA
3 cDNA con contaminacioacuten deADN genoacutemico
1500 pb
400 pb
M 1 2 3
05
10
30
kb
FUNCIONALIDAD DE ARN
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
wwwbancoadnorg
Laboratory Integrated Management System
- Etiquetas y lectores decoacutedigos de barras
- Microtubos 2D-coded
- Automatizacioacuten (Robots)
TRAZABILIDAD
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
IDENTIFICACIOacuteN DE SEXO PCR de Sexo
SEXO amplificacioacuten gen ZF (Fredsten y Villesten 2004)
B1
λ-Hind III 1 2 3 4 5
hombre mujer
1560 pb1137 pb
PCR sexo
cromosoma X insercioacuten de un elemento Alu de 423 pb
cromosoma Y no existe elemento Alu
TRAZABILIDAD
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
PERFIL GENEacuteTICO por MICROSATELITES o STRacutes
STRacutes ldquo Short Tandem Repeatsrdquo
-secuencias cortas de 2-7 pb que se repiten a lo largo de todo el genoma
-el nuacutemero de repeticiones es uacutenico en cada individuo
-la amplificacioacuten de estas regiones constituye el perfil o huella geneacutetica del individuo
TRAZABILIDAD
CHLC (Cooperative Human Linkage Center) markers
Sheffield JC et al (1995) Gastier JM et al (1995)
5 pares de oligonucleoacutetidos PCR MUacuteLTIPLE
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
TRAZABILIDAD
50 Hi-Gel Matrix (Biotools) y 07 agarosa estaacutendar
310281 271
234
194
118
φx174 HaeIII1 2 3 4
VALIDACIOacuteN DEL PROTOCOLO
AND molde 100 ng
4 individuos en dos reacciones de PCR diferentes una con Tm a 55ordmC y otra con Tm a 58ordmC
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
2
A (11130)
A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
ADN en solucioacuten
iquestqueacute muestraes A
Ceacutelulas en cultivo
A 1 2
1
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A = 2
TRAZABILIDAD
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
MUCHAS GRACIAS
wwwbancoadnorg
bancoadnusales
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
EPICENTRE Forum 5 (3) |Multiplex PCR Amplification of STRs from DNA Isolated with the MasterAmptrade Buccal Swab DNA Extraction Kit| Haiying Grunenwald Epicentre Technologies Introduction Short Tandem Repeats (STRs) consist of short (3-7 bp) repetitive sequence elements that are distributed throughout the human genome The highly polymorphic nature of these markers makes them powerful tools for human identification STRs are often used by laboratories performing parentage determination forensic analysis genetic linkage map construction or any other application requiring genetic profiling1 Typically for genetic analysis at least two STRs are simultaneously amplified in a single-tube PCR reaction (multiplex PCR) Success in multiplex PCR depends on both the quality of the genomic DNA used in the analysis and the PCR amplification conditions Standard techniques used to obtain genomic DNA for STR analysis often involve blood draws and lengthy isolations We have shown that the MasterAmptrade Buccal Swab DNA Extraction Kit can be used to isolate human genomic DNA from buccal (cheek) cells in less than one hour without blood draws2 Here we demonstrate multiplex PCR amplification of five sets of STRs from four individuals using MasterAmp PCR reagents and human genomic DNA isolated with the MasterAmp Buccal Swab Kit Methods Isolation of human genomic DNA using the MasterAmp Buccal Swab DNA Extraction Kit Human genomic DNA was isolated from buccal cells of four different individuals using the MasterAmp Buccal Swab DNA Extraction Kit The procedure is outlined in Table 1 The extracted DNA was resolved on a 1 agarose gel and visualized by ethidium bromide staining Table 1 MasterAmp Buccal Swab DNA Extraction Protocol Outline
1 Thoroughly rinse subjects mouth twice with water 2 Press the brush firmly against the inside of the cheek and rotate the brush 20 times Move the brush to the
other cheek and repeat 3 Place the brush into the tube containing DNA Extraction Solution and rotate 4 Mix by vortexing and incubate the tube at 60degC for 30 minutes 5 Mix by vortexing and incubate the tube at 98degC for 10 minutes Repeat 6 Pellet debris by centrifugation at 4degC for 5 minutes 7 Carefully transfer the supernatant (containing the genomic DNA) to a new tube and store at -20degC
Multiplex PCR amplification of CHLC markers Cooperative Human Linkage Center (CHLC) markers are a group of STRs consisting of tri- and tetranucleotide repeats34 The CHLC marker loci are highly polymorphic Genomic DNA from different individuals are differentiated by the number of copies of these repeats contained within the amplified regions PCR products from CHLC loci range between 100-300 bp Since our study involved one female and three males we selected 5 sets of CHLC primers that amplify regions of both the X chromosome (four sets of primers) and the Y chromosome (one set of primers) These primers were also selected to minimize the possibility of generating PCR products of similar size The isolated human genomic DNA was PCR amplified using the following 5 sets of CHLC primers DXS7132 GATA31E08 DYS390 GATA175D03 and DXS6789 Primer sequences location and expected PCR product length for each primer set are listed in Table 2 To determine the optimal multiplex PCR conditions preliminary reactions were carried out with genomic DNA from one individual using the MasterAmp PCR Optimization Kit MasterAmp PCR PreMix A was the optimal PreMix for this multiplex PCR reaction (data not shown) Each 50 microl multiplex PCR reaction contained 25 pmoles of each primer 1X MasterAmp PCR PreMix A (50 mM Tris-HCl pH 83 50 mM KCl 15 mM MgCl2 200 microM each dNTP) 15 microl of buccal DNA (84-250 ng) and 125 units of MasterAmp Taq DNA Polymerase The samples were incubated at 94degC prior to the addition of MasterAmp Taq Polymerase Then the samples were incubated at 94degC for 2 minutes followed by 30 cycles of 1 minute at 94degC 1 minute at 55degC and 1 minute at 72degC followed by 4 minutes at 72degC Fifteen microliters of each reaction were resolved by electrophoresis on a 3 agarose gel and visualized by ethidium bromide staining
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
Results Figure 1 shows the human genomic DNA isolated using the MasterAmp Buccal Swab DNA Extraction Kit The extracted DNA has a high molecular weight
Figure 1 Human genomic DNA extracted from buccal cells Approximately 5 (85 microl) of the extracted human genomic DNA was separated on a 1 agarose gel DNA was visualized by ethidium bromide staining Lane M DNA ladder Lanes 1-4 human genomic DNA from 4 different individuals
Four of the five sets of CHLC primers DXS7132 GATA31E08 GATA175D03 and DXS6789 amplify regions of the X chromosome whereas DYS390 amplifies a region of the Y chromosome For the female sample we expected to see no amplification from primer set DYS390 (Y chromosome) and therefore no PCR product in the 205-221 bp range The other four primer sets were expected to result in either one or two PCR products depending on heterozygosity For the male samples a single PCR product from each of the five primer sets was expected Figure 2 shows the multiplex PCR results The amplification products from the female sample are shown in Lane 2 and amplification results from the three male samples are shown in Lanes 4 6 and 8 As expected for the female sample (Lane 2) there is no amplification product between 205-221 bp from DYS390 There is one PCR product in the 118-150 bp size range from DXS6789 two PCR products from GATA175D03 due to heterozygosity (in the size range 170-186 bp) one product at approximately 250 bp from GATA31E08 (226-254 bp) and two PCR products in the size range 283-299 bp from DXS7132 again due to heterozygosity For the male samples in Lanes 6 and 8 there is clearly a single PCR product from each of the five primer sets Lane 4 shows the appearance of a doublet at ~230 bp from DYS390 and GATA31E08 (DYS390 produces products between 205-221 bp and GATA31E08 produces products between 226-254 bp) and therefore this sample also shows amplification from each primer set Multiplex PCR analysis of these CHLC markers resulted in four distinct patterns for these four individuals (Figure 2) Further statistical analysis would be needed to determine the significance of these comparisons
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829
Figure 2 Multiplex PCR amplification of CHLC markers from buccal DNA Multiplex PCR of five sets of CHLC markers was carried out using human genomic DNA extracted from buccal cells as described in the text Fifteen microliters of each reaction were resolved on a 3 agarose gel and visualized by ethidium bromide staining Lanes 1 3 5 7 100 bp DNA ladder Lane 2 multiplex PCR from a female sample Lanes 4 6 and 8 multiplex PCR from the three males samples Multiplex PCR products in Lanes 2 4 6 and 8 correspond to the human genomic DNA samples shown in Figure 1 in Lanes 1 2 3 and 4 respectively
Summary Human genomic DNA isolated from buccal cells using the MasterAmp Buccal Swab DNA Extraction Kit was an excellent template for the multiplex PCR amplification of STRs This DNA extraction system is both fast and non-invasive In addition use of the MasterAmp PCR Optimization Kit made it easy to determine the optimal conditions for the multiplex PCR analysis References
1 Edwards A et al (1991) Am J Hum Genet 49 746 2 Watson J (1997) Epicentre Forum 4 (1) 6 3 Sheffield JC et al (1995) Hum Mol Genet 4 1837 4 Gastier JM et al (1995) Hum Mol Genet 4 1829