restriction enzymes. restriction endonucleases also called restriction enzymes 1962: “molecular...

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Restriction Enzymes Restriction Enzymes

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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

http://www.dnai.org/b/index.html