Recombinant DNA

Recombinant DNA - Recombinant DNA Restriction Enzymes 1...

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Recombinant DNA
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Restriction Enzymes 1) First discovered from a study of the interactions between phage and bacteria. 2) Bacteria can produce an enzyme, called a restriction enzyme , that cleaves DNA at a specific sequence, called a restriction site . - Distinct from general nucleases, which cleave DNA randomly, either from the ends (exonucleases) or at internal sites (endonucleases). 3) The host cell modifies its own restriction sites for the restriction enzymes it makes via methylation that is catalyzed by a site- specific methylase.
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Restriction/Methylation Enzyme
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Res - Mod - λ Res + Mod + Re-infect with the few λ Lots of growth Lots of growth Res - Mod - Lots of growth λ Res + Mod + Almost no growth λ Restriction/Modification in E. coli Note: If a cell is restriction+, it MUST be modification+ or it will cleave its own DNA These phage are NOT methylated These phage are methylated
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Restriction Enzymes 1) First discovered from a study of the interactions between phage and bacteria 2) Bacteria can produce an enzyme, called a restriction enzyme , that cleaves DNA at a specific sequence, called a restriction site . - Distinct from general nucleases, which cleave DNA randomly, either from the ends (exonucleases) or at internal sites (endonucleases). 3) The host cell modifies its own restriction sites for the restriction enzymes it makes via methylation that is catalyzed by a site- specific methylase. 4) Restriction sites are generally palindromic. 5) Generally named by the bacterium from which they are derived.
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Eco RI Restriction Enzyme First restriction enzyme from E scherichia co li, so Eco R1 Single stranded “nick”
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Single Stranded Nicks in DNA Hydrolysis of this ester bond H 2 O P O O OH O - OH
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Restriction Enzyme Recognition Sites Restriction sites are general palindromic: “Able was I, ere, I saw Elba” Bam H1 site: 5’-GGATCC-3’ 3’-CCTAGG-5’
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Restriction Enzyme Recognition Sites Bgl II 5’ A-G-A-T-C-T T-C-T-A-G-A 5’ Sau 3A 5’ G-A-T-C C-T-A-G 5’ Bam HI 5’ G-G-A-T-C-C C-C-T-A-G-G 5’ All these sticky ends are compatible Isoschizomers : In certain cases, two or more different enzymes may recognize identical sites. (e.g. Mbo I also cleaves at GATC, and so is an isochizomer of Sau 3A.)
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Frequency of cutting of recognition enzymes Sau 3A (GATC) cuts (¼)(¼)(¼)(¼) = once every 256 base pairs (assuming G/C = A/T, which is often does not) Bam H1 (GGATCC) cuts (¼)(¼)(¼)(¼)(¼)(¼) = once every ~4Kb Hind II (GTPyPuAC) cuts (¼)(¼)(½)(½)(¼)(¼) = once every ~1Kb
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5’ overhang ( Eco RI) 5’-GAATTC-3’ 5’-G AATTC-3’ 3’-CTTAAG-5’ 3’-CTTAA G-5’ “Sticky” ends 5’ overhang ( Sma I) 5’-CCCGGG-3’ 5’-CCC GGG-3’ 3’-GGGCCC-5’ 3’-GGG CCC-5’ “Blunt” ends 3’ overhang ( Pst I) 5’-CTGCAG-3’ 5’-CTGCA G-3’ 3’-GACGTC-5’ 3’-G ACGTC-5’ + + + -OH HO- PO 4 - HO- HO- PO 4 - -OH -OH -PO 4 -PO 4 -PO 4 PO 4 -
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Human DNA cleaved with Eco RI Corn DNA cleaved with Eco RI 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 -PO 4 PO 4 - A-A-T-T-C- A-G-C-T-A-C-G -3’ HO- G- T-C-G-A-T-G-C -5’ Ligation of compatible sticky ends + 5’-
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