restriction endonucleases david peterson. structure of dna and rna

RestrictionEndonucleases

David Peterson

Structure of DNA and RNA

DNA RNA Protein

Information Flow in Cells

DNA is a Double Helix

Phage

48,512 bp dsDNA virus that grows on E. coli.

Life cycle ~ 30 min at 37°C

typical burst ~ 100-300 particles

Bacteriophage infection: 1 → 300 → 10,000 → 3,000,000 → 109!

30’ 30’ 30’ 30’

Bacterium

Phage DNA

Typical phage infection cycle: 1. Phage DNA penetrates bacterial envelope2. Bacterial DNA is disrupted & phage DNA is replicated3. Phage structural proteins are synthesized4. Heads, tails, fibers made and assembled into particles

5. Cell lysis, release of ~ 100 – 300 phage particles

30 min

1000’s of phage particles attacking an E. coli cell

Bacteriophages Make Plaques

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Restriction Endonucleases

• Type 1 Cuts at random position relative to recognition sequence (sometimes far away)

• Type II Cuts at recognition sequence

• Type III Cuts at fixed distance from recognition sequence

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SV40 DNA Cut with Hin cII

Palindromes

•Madam, I’m Adam.

•Gary knits a stinky rag.

•Harass sensuousness, Sarah.

•Go hang a salami, I’m a lasagna hog.

Most Restriction Enzymes Recognize Palindromes

GATATC||||||||||||||||||| CTATAG

5’

5’3’

3’

EcoRV Bound to DNA

www.pdb.org1AZ0

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Methylation sites

Most Restriction Enzymes Recognize Palindromes

GATATC||||||||||||||||||| CTATAG

5’

5’3’

3’

EcoRV Bound to DNA

www.pdb.org1AZ0

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SV40 DNA Cut with Hin cII

Restriction Enzymes Aid in DNA Cloning

Dan Nathans and Hamilton Smith

Werner Arber

Nobel Prize in Physiology or Medicine 1978