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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’
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Methylation sites
Most Restriction Enzymes Recognize Palindromes
GATATC||||||||||||||||||| CTATAG
5’
5’3’
3’
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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
