L53-07 DNA tech HANDOUT - L. 53 DNA Technology Studying...

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1 L. 53 DNA Technology Studying how gene functions often requires: ± single gene away from other genes in the genome - solution: restriction enzymes can cut DNA at speci f c sequence ± methods to produce large quantity of the gene so that its function can be observed easily - solution: vector such as plasmids in bacteria can produce many copies of the same gene Restriction Enzyme - Restriction enzymes are speci f c enzymes in bacteria that cut foreign DNA for defense - After being “restricted” the foreign DNA can be digested by exonucleases, enzymes that digest DNA from ends - In contrast, restriction enzymes are also called endonucleases endonucleases exonucleases - Restriction enzymes recognize speci f c sequences (restriction sites) of DNA and cut both strands in speci f c ways - The sequences recognized are 4 to 8 base pairs - The sequences are palindromes - The average frequency of restriction sites is 1/4 4 (1/256) to 1/4 8 (1/65536) - Restriction reaction creates reproducible DNA segments called restriction fragments Restriction Enzyme Mechanism Restriction Enzyme Cutting - Some RE cut create blunt ends - Some RE cut create staggered ends Blunt cutter Stagger cutters Fig. 20.3 - The staggered ends are often called sticky end for their tendency to re-associate with each other Eco RI
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2 Molecules cut by the same restriction enzyme can form base pairing Gap in sugar-phosphate backbone is sealed by adding ligase Restriction Enzyme Structure - Most restriction enzymes are homodimers GGATTC CTTAGG Bam H1 GAATTC CTTAAG Eco RI methylase GAATTC CTTAAG CH 3 CH 3 No cleavage Eco RI (restriction enzyme)
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This note was uploaded on 05/18/2008 for the course BIO G 104 taught by Professor Chen,k.c during the Spring '06 term at Cornell University (Engineering School).

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L53-07 DNA tech HANDOUT - L. 53 DNA Technology Studying...

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