Methods for DNA Transfer BT 201 Biotechnology Techniques I

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Text of Methods for DNA Transfer BT 201 Biotechnology Techniques I

  • Methods for DNA TransferBT 201 Biotechnology Techniques I

  • Transferring GenesVectors are used to move genes around Plasmids, Bacteriophage, Cosmids, YACs, BACs, Viruses are usedE. coli often used to express genes that have been transferredTransformation is a common method for gene transfer

  • TransformationRecipient cells take up foreign DNA from surrounding mediaOften accomplished using plasmid vectorsArtificially induced in laboratoriesAllows introduction of unrelated genes into bacterial expression systemsProducts of interest can be produced, extracted, and purified for use

  • Bacterial PlasmidsCircular pieces of DNA that can be replicated outside the bacterial chromosomeOccur in varying sizesCapable of carrying varying sizes and types of genesMay produce several hundred copies in a single cell

  • Vector CreationRestriction enzymes are used to cut DNA to be inserted into small fragmentsPlasmids are cut open with REs so DNA fragments may be insertedPlasmids, DNA fragments, and DNA ligase are mixed to put it all back together: cloningReady for transformation now

  • Restriction enzymerecognition sequenceRestriction enzymecuts the DNA intofragmentsAddition of a DNAfragment fromanother sourceDNAG

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    Sticky endTwo (or more)fragments sticktogether bybase-pairingDNA ligasepastes the strandRecombinant DNA moleculeG

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  • Vector Creation

  • TransformationTransformation was discovered in 1928 by Frederick Griffith using S. pneumoniae in mice: Transforming PrincipleIn 1944 a genetic basis was for process was discovered by Avery, McLeod, and McCartyThey named the process Transformation

  • Transformation of E. coliCells capable of taking up foreign DNA are competentSome cells are naturally competent, some cells have to be made competent E. coli not naturally competentArtificial competence induced using cold and cationic solutions (cold CaCl2)Plasmid introduced into iced solutionCells heat shocked to force plasmid uptakeSolution allowed to return to ambient temperature, pores close

  • Isolate DNAfrom two sourcesCut both DNAswith the samerestriction enzymeMix the DNAs;they join bybase-pairingAdd DNA ligaseto bond the DNA covalentlyRecombinant DNAplasmidE. coliPlasmidHuman cellSticky endsGene VDNAGene VPut plasmid into bacteriumby transformationRecombinantbacteriumClone the bacteriumBacterial clone carrying manycopies of the human gene

  • Detecting TransformationMany plasmids carry antibiotic resistance genes: R factorsUsed to select transformantspBestLuc plasmid contains ampr and luc genesAmpicillin resistance, Luciferase productionPotential transformants plated on ampicillin-containing media Colonies exposed to luciferin solution and observed for luminescence

  • Manipulating Genes for BiotechnologyTo make productsTo improve cropsTo improve livestockTo improve quality of lifeTo treat disease

  • Biotechnology ApplicationsRecombinant pharmaceutical productsTransgenic animals as pharmaceutical factories and organ sourcesTransgenic crops

  • BacterialchromosomePlasmidBacteriumPlasmidisolatedRecombinant DNA(plasmid)RecombinantbacteriumCopies of geneClone of cellsGene for pestresistanceinserted intoplantsGene used to alter bacteriafor cleaning up toxic wasteProtein used to dissolve bloodclots in heart attack therapyCopies of proteinCell multiplies withgene of interestProtein used tomake snowform at highertemperaturePlasmid put intobacterial cellDNAGene ofinterestGene insertedinto plasmidDNAisolatedCell containing geneof interest

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