Polymerase Chain Reaction PCR Use to exponentially amplify discrete regions of DNA. Need to know the DNA sequence of two short regions, not too far apart, but don’t need to know the sequence of the entire region to be amplified. Make two oligonucleotide primers (ea. ~20 bases long), complementary to opposite strands at the ends of the region to be amplified.

- Gel Electrophoresis + Separates molecules (DNA, RNA, or proteins) on the basis of size. Gels: Agarose or Acrylamide DNA gels: - Load DNA samples into ‘wells’ at one end of gel. - Apply electric current through gel. - DNA pushed toward + electrode. - Smaller molecules travel faster through gel, resulting in separation of DNA fragments on basis of size - + Ethidium Bromide binds to DNA & fluoresceses when exposed to UV

Restriction Mapping Digest DNA with restriction enzymes: 17 kb Restriction Mapping Digest DNA with restriction enzymes: Single enzyme digests & Double enzyme digests (two enzymes together) Determine sizes of restriction fragments Deduce locations of restriction enzyme recognition sites

Soak gel in NaOH Run DNA on gel Southern Blots Determine whether two DNA fragments contain same or similar DNA sequences. DNA transferred to filter paper = Southern blot Hybridize Southern blot with radioactive probe (same as in library screening) Wash filter & expose X-ray film Only restriction fragments on Southern blot that contain sequences complementary to probe will show up as bands on autoradiogram

Transfer DNA from gel to piece of DNA binding filter paper

Mapping Chromosomal Rearrangement Breakpoints Genomic Southern blots Reciprocal Translocation Fig. 9-13