Restriction Enzymes and Gel Electrophoresis

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Restriction Enzymes and Gel Electrophoresis

• Genetics 335

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Step 1 Restriction enzymes/endonucleases

• Enzymes that cut DNA into fragments of different
  sizes
• Cleave DNA at specific phosphodiester bonds
• Named after bacterial strains
• E.g., BamHI, EcoRI, HAEIII, HindII, MstII
• Can cleave straight (flush) or staggered
• Blunt or sticky ends

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Why cut up DNA?

• If different alleles can be cut up and identified
  (using GE), we can identify unknown persons
  (e.g., criminals, biological relatives), and
  document unknown genotypes (e.g., Huntingtons
  Disease)
• http//www.youtube.com/watch?vKVnFBCzg8Ms

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Gel Electrophoresis

• You have a vial of undescribed DNA fragmentsnow
  what?
• Samples pipetted into wells on one end of a gel
  (e.g., agarose)
• Electricity is added to the gel
• DNA fragments move through the gel at different
  rates, away from the negative and toward the
  positive end
• Smaller fragments move easier and further from
  well, larger fragments more cumbersome and move
  shorter distances from well
• The resulting fingerprint of fragments can be
  matched to a known sample or investigated for
  specific alleles

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A B C D E F G
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Videos

• http//www.sumanasinc.com/webcontent/animations/co
  ntent/gelelectrophoresis.html
• http//www.youtube.com/watch?vlM8npqHw-mk

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Review

• Restriction enzymes cut up DNA into pieces unique
  to your DNA
• DNA pieces are a function of your genetics and
  the restriction enzyme used
• DNA fragment soup placed in Gel well and
  distributes based on fragment/sequence length
• Resulting gel is uniquelike a fingerprint