Restriction EnzymesRestriction Enzymes
Restriction EndonucleasesRestriction 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
Restriction EndonucleasesRestriction Endonucleases
Named for bacterial genus, species, strain, and type
Example: EcoR1
Genus: EscherichiaSpecies: coliStrain: ROrder discovered: 1
Restriction EndonucleasesRestriction Endonucleases
Recognition sites have symmetry (palindromic)Recognition sites have symmetry (palindromic)
“Able was I, ere, I saw Elba”
Bam H1 site:5’-GGATCC-3’3’-CCTAGG-5’
Restriction EndonucleasesRestriction Endonucleases
Enzymes recognize specific 4-8 bp sequencesEnzymes recognize specific 4-8 bp sequences
Some enzymes cut in a staggered fashion - “sticky ends”Some enzymes cut in a staggered fashion - “sticky ends”
EcoRI 5’…GAATTC…3’ EcoRI 5’…GAATTC…3’
3’…CTTAAG…5’3’…CTTAAG…5’
Some enzymes cut in a direct fashion – “blunt ends”Some enzymes cut in a direct fashion – “blunt ends”
PvuII 5’…CAGCTG…3’PvuII 5’…CAGCTG…3’
3’…GTCGAC…5’3’…GTCGAC…5’
Restriction EndonucleasesRestriction Endonucleases
Why don’t bacteria destroy their own DNA with their restriction enzymes?
MethylationMethylation
Uses for Restriction EnzymesUses for Restriction Enzymes
RFLP analysis (Restriction Fragment Length Polymorphism)RFLP analysis (Restriction Fragment Length Polymorphism)
DNA sequencingDNA sequencing
DNA storage – librariesDNA storage – libraries
Transformation Transformation
Large scale analysis – gene chipsLarge scale analysis – gene chips
Human DNA cleaved with EcoRI Corn DNA cleaved with EcoRI
5’-C-G-G-T-A-C-T-A-G-OH3’-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4
PO4-A-A-T-T-C-A-G-C-T-A-C-G-3’ HO-G-T-C-G-A-T-G-C-5’
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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’
Complementary base pairing
+ DNA Ligase, + rATP
recombinant DNA molecule
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’
Restriction Enzymes for TransformationRestriction Enzymes for Transformation
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DNA is negatively charged from the phosphate backbone
Visualize DNA with ethidium bromide – fluoresces ONLY when bound to DNA
Restriction Enzymes for RFLPRestriction Enzymes for RFLP
Restriction EndonucleasesRestriction Endonucleases
Restriction enzyme animation
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