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BIOL 200 Notes After Midterm.pdf

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Biology (Sci)
BIOL 200
Thomas Bureau

Molecular Genetics Methodologies II Some historical perspective into Ecoli and bacteriophages Dr Paul Berg and others were the first to successfully introduce exogenous DNA fragments into bacteria to produce a recombinant gene productUprising of Recombinant DNA technology Bacterial restriction enzymes cut DNA at specific sites Polylinkers are engineered with many restriction enzyme sites to facilitate insertion of DNA EcoRI cuts DNA at a specific palindromic sequence GAATC Note Vector is a specialized plasmid which carries out specific functions upon removal of nonfunctional regionsHowever if the Polylinker undergoes digestion by 2 different enzymes direction cloning results thus it only inserts in one orientation DNA libraries permanent collection of genes 1 Genomic library contain copies of DNA present in the genomicchromosomal context intergenic regions introns exons repetitive sequence 2 cDNA library represent mRNA tissue specificity abundanceAfter RT uses oligoT primer polydT to initiate thessDNA synthesis that is complementary to the mRNA template RNA is then removed and a polydG adapterGGG is annealed to the 3 end A polydC primerCCC is used to initiate synthesis ndof the 2 DNA strandEcoli DNA polymerase I progresses to extend any remaining hybrid regions DNA synthesis stops at the 3 end Note Alkaline may serve as RNase H to break hydrogen bonds and degrade RNA Then PolydG and PolydC are attachedAlmost no one uses cDNA libraries nowbecause Studying Genes of interest RTPCR Since all mRNAs present at a given cell will have a polyA tail then poly T primerscan be used to prime a cDNA using RTPCR The resulting cDNA molecules in a reaction solution after several cycles should be a reasonable prediction of various starting levels of each species The population can be screened using degenerate primers Bacterial Expression Vectors over expression of recombinant protein ex Insulin ThelacZ promoterunder control of IPTG provides an effective means of inducing gene expressionFor example it can drive exogenous T7 RNA polymerase which expresses recombinant gene under the control of T7 promoterEndogenous native to the organism Exogenous introduced from another organism
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