chapter 20 weinstein

chapter 20 weinstein - Chapter 20 DNA Technology and...

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1 Chapter 20 DNA Technology and Genomics • Manipulation of Nucleic Acids • Isolation of Mammalian Genes • Gene Amplification by PCR • Use of Genes in Biotechnology and Therapeutics Plasmid - circular, extrachromosomal DNA has origin of DNA replication replicates autonomously carries a small number of genes beneficial to bacteria R (resistance) plasmids contain genes for antibiotic resistance multiple resistance genes may be present on a single plasmid - readily transferred major medical problem! Overuse of antibiotics selects for bacteria that are resistant The chemical structure of ampicillin, a commonly used antibiotic Basic strategies RESTRICTION ENZYMES (RE): cut DNA at specific sites called restriction sites hundreds of different enzymes cut different sites in DNA allow for very fine dissection of DNA sequences Bacterial defense mechanisms bacterial restriction enzymes digest unmodified (unmethylated) DNA bacterial DNA is modified, protected viral DNA is not modified, digested Host DNA Viral DNA Viral DNA m m m m m m Host DNA m m m m m m
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2 Restriction sites: 4-8 base-pair recognition sites always palindromic Can leave overhang (“sticky end”) Restriction sites: 4-8 base-pair recognition sites always palindromic 5 ʼ -G GATC C-3 ʼ 3 ʼ -C CTAG G-5 ʼ 5 ʼ -G GATC C-3 ʼ 3 ʼ -C CTAG G-5 ʼ 5 ʼ -G 3 ʼ -C CTAG GATCC-3 ʼ G-5 ʼ + Doesn’t have to leave overhang (“blunt end”) 5 ʼ -GATATC-3 ʼ 3 ʼ -CTATAG-5 ʼ 5 ʼ -GAT ATC-3 ʼ 3 ʼ -CTA TAG-5 ʼ 5 ʼ -GAT 3 ʼ -CTA ATC-3 ʼ TAG-5 ʼ + EcoRV EcoRI RI RI Treat with EcoRI - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + + + + + Inside, a gel is like a maze Shorter molecules of DNA move faster Larger molecules move slower Separation of DNA fragments by size using gel electrophoresis
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3 Ligating restriction fragments to create molecules of recombinant DNA Genomic library: collection of bacterial clones each clone contains a different recomb. plasmid
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chapter 20 weinstein - Chapter 20 DNA Technology and...

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