Restriction enzymes and gel electrophoresis
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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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Restriction Enzymes and Gel Electrophoresis

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Restriction enzymes and gel electrophoresis

Restriction Enzymes and Gel Electrophoresis

Genetics 335


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


Restriction enzymes and gel electrophoresis

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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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