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Restriction Enzymes and Gel Electrophoresis. Genetics 335. 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

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Presentation Transcript
step 1 restriction enzymes endonucleases
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
why cut up dna
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., Huntington’s Disease)
  • http://www.youtube.com/watch?v=KVnFBCzg8Ms
gel electrophoresis
Gel Electrophoresis
  • You have a vial of undescribed DNA fragments—now 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
videos
Videos
  • http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html
  • http://www.youtube.com/watch?v=lM8npqHw-mk
review
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 unique—like a fingerprint
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