Restriction Enzymes and Gel Electrophoresis

1. Restriction Enzymes and Gel Electrophoresis Genetics 335

2. 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

4. 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

5. 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

6. A B C D E F G

7. Videos • http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html • http://www.youtube.com/watch?v=lM8npqHw-mk

8. 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