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							      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
                                         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                     DNA transferred
fragments contain same or similar             to filter paper
DNA sequences.                                = 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




                                          (DNA binding filter paper)
 Mapping Chromosomal
 Rearrangement
 Breakpoints
Genomic Southern blots

                         Reciprocal Translocation




          Fig. 9-13