BICH107RestrictionEnz.ppt

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					  Restriction
Endonucleases
   David Peterson
Structure of DNA and RNA
 Information Flow in Cells




DNA       RNA          Protein
DNA is a Double Helix
Phage l


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’


   Phage DNA


   Bacterium




                                      30 min

Typical phage infection cycle:
1. Phage DNA penetrates bacterial envelope
2. Bacterial DNA is disrupted & phage DNA is replicated
3. Phage structural proteins are synthesized
4. Heads, tails, fibers made and assembled into particles
5. Cell lysis, release of ~ 100 – 300 phage particles
1000’s of phage l particles
attacking an E. coli cell
Bacteriophages Make Plaques
       QuickTime™ an d a
      None decompressor
are need ed to see this picture.
          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
SV40 DNA Cut with Hin cII


             QuickTime™ and a
         TIFF (LZW) decompressor
      are needed to see this picture.
            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



                                           3’
5’          GATATC
     |||||||||||||||||||
3’
            CTATAG                         5’
EcoRV Bound to DNA




                     www.pdb.org
                       1AZ0
       QuickTime™ an d a
      None decompressor
are need ed to see this picture.
Methylation sites
      Most Restriction Enzymes Recognize
                  Palindromes



                                           3’
5’          GATATC
     |||||||||||||||||||
3’
            CTATAG                         5’
EcoRV Bound to DNA




                     www.pdb.org
                       1AZ0
SV40 DNA Cut with Hin cII


             QuickTime™ and a
         TIFF (LZW) decompressor
      are needed to see this picture.
Restriction Enzymes Aid in DNA Cloning
                                        Werner Arber
Dan Nathans and Hamilton Smith




    Nobel Prize in Physiology or Medicine 1978

				
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posted:5/2/2013
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