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Restriction Enzymes

Restriction Enzymes. Restriction Endonucleases. Also called restriction enzymes 1962: “molecular scissors” discovered in in bacteria E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA

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Restriction Enzymes

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  1. Restriction Enzymes

  2. Restriction Endonucleases Also called restriction enzymes 1962: “molecular scissors” discovered in in bacteria E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA 3,000 enzymes have been identified, around 200 have unique properties, many are purified and available commercially

  3. Restriction Endonucleases Named for bacterial genus, species, strain, and type Example: EcoR1 Genus: Escherichia Species: coli Strain: ROrder discovered: 1

  4. “Able was I, ere, I saw Elba” 5’-GGATCC-3’ 3’-CCTAGG-5’ Bam H1 site: Restriction Endonucleases Recognition sites have symmetry (palindromic)

  5. Restriction Endonucleases Enzymes recognize specific 4-8 bp sequences Some enzymes cut in a staggered fashion - “sticky ends” EcoRI 5’…GAATTC…3’ 3’…CTTAAG…5’ Some enzymes cut in a direct fashion – “blunt ends” PvuII 5’…CAGCTG…3’ 3’…GTCGAC…5’

  6. Restriction Endonucleases Why don’t bacteria destroy their own DNA with their restriction enzymes?

  7. Methylation

  8. Uses for Restriction Enzymes RFLP analysis (Restriction Fragment Length Polymorphism) DNA sequencing DNA storage – libraries Transformation Large scale analysis – gene chips

  9. Complementary base pairing 5’-A-C-G-G-T-A-C-T-A-GA-A-T-T-C-A-G-C-T-A-C-G-3’ 3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5’ + DNA Ligase, + rATP 5’-C-G-G-T-A-C-T-A-G-OH 3’-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4 5’-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3’ 3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5’ recombinant DNA molecule Restriction Enzymes for Transformation Human DNA cleaved with EcoRICorn DNA cleaved with EcoRI + PO4-A-A-T-T-C-A-G-C-T-A-C-G-3’ HO-G-T-C-G-A-T-G-C-5’

  10. _ DNA is negatively charged from the phosphate backbone + Visualize DNA with ethidium bromide – fluoresces ONLY when bound to DNA Restriction Enzymes for RFLP

  11. Restriction Endonucleases Restriction enzyme animation http://www.dnai.org/b/index.html

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