Lec 14 Recombinant DNA - Recombinant DNA, PCR and DNA...

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Unformatted text preview: Recombinant DNA, PCR and DNA sequencing • Cloning vectors • Restriction endonucleases • Gel electrophoresis • Recombinant DNA libraries • Southern blots • Polymerase chain reaction • Site-directed mutagenesis • Sanger and Next Gen sequencing • Themes In real estate, it’s location, location, location. In molecular genetics, it’s amplification, amplification… Early 1970s: routine to purify proteins according to solubility, size and charge But how to go about purifying a gene? Problem 1: genes are covalently linked to other genes Problem 2: DNA is uniform in solubility, shape and charge Problem 3: Only up to 4 copies/cell (G 2 of a diploid) The advantages of viral genomes and bacterial plasmids Unlinked to host genome Very small (just a handful of genes within a few thousand bases) Amplified to many copies per cell Light bulb moment: insert a cellular DNA fragment into a plasmid or viral genome. But how to cut and paste? What to use for scissors and glue? Creating and propagating recombinant DNA molecules restriction endonuclease plasmid vector recombinant plasmid transform competent E. coli with ampicillin selection for recombinant plasmid propagation and amplification mouse genomic DNA DNA ligase a m p r o r i a m p r o r i mouse DNA insert plasmid host chromosome E. coli Cloning Vector Insert size Bacterial plasmid < 20,000 bp Lambda < 20,000 bp Cosmid < 40,000 bp Bacterial artificial chromosome < 200,000 bp Yeast artificial chromosome < 1 million bp Stanley Cohen Herbert Boyer Paul Berg Restriction Endonucleases host restriction endonuclease CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 phage DNA degraded; methylation protects host chromosome Restriction endonuclease protect bacteria from viruses by recognizing and degrading nonmethylated foreign DNA. Werner Arber-G-C-T-T-A-A A-A-T-T-C- G--G-C-T-T-A-A A-A-T-T-C- G--G-C-T-T-A-A A-A-T-T-C- G--G-C-T-T-A-A A-A-T-T-C- G--G-C-T-T-A-A A-A-T-T-C- G- CH 3 CH 3 EcoRI cleavage blocked by methylation Used by natural host to protect its own DNA from cleavage by cognate restriction endonuclease 3’ OH 3’ OH 5’ P 5’ P sticky ends EcoRI EcoRI anneal DNA ligase EcoRI methylase nick nick Restriction Endonucleases Sticky vs. blunt ends; cut site frequency as a function of lengthSticky vs....
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This note was uploaded on 07/17/2011 for the course BIO 151 taught by Professor Grinblat during the Summer '08 term at University of Wisconsin Colleges Online.

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Lec 14 Recombinant DNA - Recombinant DNA, PCR and DNA...

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