Tecnologías con DNA
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Transcript of Tecnologías con DNA
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• Artificial selectionArtificial selection
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Recombinant DNA TechnologyRecombinant DNA Technology
• Isolates and amplifiesIsolates and amplifies• specific sequences of DNAspecific sequences of DNA• incorporates them into incorporates them into vectorvector DNA molecules DNA molecules
• Resulting Resulting recombinant DNArecombinant DNA • is propagated and amplified is propagated and amplified (cloned)(cloned)• in organisms such as in organisms such as E. coliE. coli
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Recombinant DNA VectorsRecombinant DNA Vectors
• Naturally occurring circular bacteria DNA Naturally occurring circular bacteria DNA molecules molecules (plasmids)(plasmids)
• Bacterial viruses Bacterial viruses (bacteriophages)(bacteriophages)
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Genetically Altered BacteriaGenetically Altered Bacteria
• Produce important human protein productsProduce important human protein products• insulininsulin• growth hormonegrowth hormone• tissue plasminogen activator (TPA)tissue plasminogen activator (TPA)• tissue growth factor-beta (TGF- tissue growth factor-beta (TGF- ββ))• clotting factor VIIIclotting factor VIII• Dornase Alpha (DNase)Dornase Alpha (DNase)
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Genentech- Humulin 1982
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Restriction EnzymesRestriction Enzymes
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Fig. 15-2, p. 325
Plasmid from a bacterium
DNA of interest from another organism
1 Plasmid and DNA from another organism are cut by the same restriction enzyme (in this example, Hin dIII). This produces molecules with complementary single-stranded ends.
Clonable DNA fragment
2 Mix two types of molecules so their sticky ends pair. DNA ligase then forms covalent bonds at junctions, linking fragments.
Recombinant DNA
3 Transfer recombinant DNA molecule to host cell, where it is copied and turned on to produce gene product.
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PlasmidsPlasmids
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Gen reportero
Gen de interéscDNA
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Vector Adenovirall
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Fig. 15-15, p. 340
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Fig. 15-16, p. 341
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Fig. 15-17b, p. 342
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Genetically Engineered PlantGenetically Engineered Plant
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Libraries (1)Libraries (1)
• Genomic DNA libraryGenomic DNA library• thousands of DNA fragmentsthousands of DNA fragments• all DNA of an organismall DNA of an organism
• Chromosome libraryChromosome library• all DNA fragments of a specific chromosome all DNA fragments of a specific chromosome
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Chromosome Chromosome LibraryLibrary
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cDNA LibrarycDNA Library
• Complementary DNA (cDNA)Complementary DNA (cDNA)• produced using produced using reverse transcriptasereverse transcriptase• makes DNA copies of eukaryotic mRNAmakes DNA copies of eukaryotic mRNA
• Copies are incorporated into recombinant Copies are incorporated into recombinant DNA DNA vectorsvectors
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cDNAcDNA
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Genetic ProbeGenetic Probe
• Radioactive DNA or RNA sequenceRadioactive DNA or RNA sequence• used to screen recombinant DNA molecules used to screen recombinant DNA molecules
in bacterial cellsin bacterial cells• to find specific colony with DNA of interestto find specific colony with DNA of interest
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Genetic ProbeGenetic Probe
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Animation: Use of a Radioactive Animation: Use of a Radioactive ProbeProbe
CLICKTO PLAY
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Polymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)
• Automated Automated in vitroin vitro technique technique• targets a particular DNA sequence by specific targets a particular DNA sequence by specific
primers primers • clones it using heat-resistant DNA clones it using heat-resistant DNA
polymerasepolymerase
• Used to analyze tiny DNA samplesUsed to analyze tiny DNA samples• from crime scenes, archaeological remainsfrom crime scenes, archaeological remains
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Fig. 15-7, p. 329
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Gel Gel ElectrophoresisElectrophoresis
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Fig. 15-8a, p. 330
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Fig. 15-8a, p. 330
DNACut with restriction enzyme
100 base pairs 200 base pairs 300 base pairs
Mixture placed in well Standards of
known size
+ –Origin
Direction of movement 200 base pairs
300 base pairs
100 base pairs
Gel
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Fig. 15-8b, p. 330
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Southern Southern BlotBlot
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RNA and ProteinsRNA and Proteins
• Northern BlotNorthern Blot • RNA moleculesRNA molecules separated by electrophoresis separated by electrophoresis• transferred to a membranetransferred to a membrane
• Western BlotWestern Blot• Proteins or polypeptidesProteins or polypeptides previously separated previously separated
by gel electrophoresisby gel electrophoresis
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DNA Microarrays (2)DNA Microarrays (2)
• Cancer and other diseases exhibit altered Cancer and other diseases exhibit altered patterns of gene expressionpatterns of gene expression
• DNA microarrays DNA microarrays identify disease-causing identify disease-causing genes (or the proteins they code for)genes (or the proteins they code for)
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DNA DNA MicroarrayMicroarray
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Fig. 15-13, p. 336
1 Prepare microarray. Each microdot contains multiple copies of a specific single-stranded cDNA.
Treated cell Untreated (control) cell2 Prepare cDNA from
two cell populations (treated and control).
Mature mRNA Mature mRNAReverse transcriptase
Reverse transcriptase
cDNA copy of mRNA
cDNA copy of mRNA
3 Tag each cDNA with different fluorescent dye.
cDNA mRNA (discard) cDNA mRNA (discard)
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Fig. 15-13, p. 336
4 Hybridize two cDNA populations to array.
Laser 1 Scan array to identify fluorescence where hybridization has occurred.
5Laser 2
Gene that was inactive in both treated and untreated cells
Emissions6 Computer analysis
produces color-coded readout.
Gene in treated cell that increased activity, compared to controlGene in treated cell that decreased activity, compared to controlGene that was active in both treated and untreated cells
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DNA SequencingDNA Sequencing
• Yields information about gene structureYields information about gene structure• and amino acid sequence of encoded proteinsand amino acid sequence of encoded proteins
• Geneticists compare DNA sequencesGeneticists compare DNA sequences• with other sequences stored in databaseswith other sequences stored in databases
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DideoxynucleotideDideoxynucleotide
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Chain Termination MethodChain Termination Method
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Fig. 15-11a, p. 334
Single-strand DNA fragment to be sequenced
+ddATP +ddCTP +ddGTP +ddTTP
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Fig. 15-11b, p. 334
Radioactive primer
Direction of synthesis+ddATP
Reaction products from mixture containing dideoxyATP
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Fig. 15-11c, p. 334
Larger fragments
Smaller fragments
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Fig. 15-11d, p. 334
CA TG
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Automated DNA SequenceAutomated DNA Sequence
CLICKTO PLAY
CLICKTO PLAY
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DNA FingerprintingDNA Fingerprinting
• Analysis of individual’s DNAAnalysis of individual’s DNA• based on based on short tandem repeats (STRs)short tandem repeats (STRs)
(molecular markers, highly (molecular markers, highly polymorphicpolymorphic))
• Applications inApplications in• law enforcementlaw enforcement• disputed parentagedisputed parentage• tracking tainted foodstracking tainted foods
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Animation: DNA FingerprintingAnimation: DNA Fingerprinting
CLICKTO PLAY
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Fig. 15-14, p. 339
1 2 3 4 5 6 7From blood at
crime scene
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Transgenic OrganismsTransgenic Organisms
• Foreign DNAForeign DNA• incorporated into genetic material incorporated into genetic material
• Transgenic livestockTransgenic livestock• produce foreign proteins in milkproduce foreign proteins in milk
• Transgenic plantsTransgenic plants• have great potential in agriculturehave great potential in agriculture