Page 356 - Applied Probability

Appendix A: Molecular Genetics in Brief
346
than small fragments and consequently travel more slowly, just as in a
flowing stream, large stones travel more slowly than small ones. Once the
DNA fragments are separated by size, a Southern blot can be made. This
involves denaturing the fragments by the addition of alkali and transferring
the separated strands to a nitrocellulose or nylon membrane. After the
strands are fixed to the membrane by baking or chemical crossbinding,
radioactive probes are introduced to the membrane and anneal with specific
fragments. When the membrane is applied to an X-ray film, a sequence
of bands develops on the film highlighting those DNA fragments bound
to probes. Alternatively, if sufficient DNA is sampled, then fluorescent or
chemiluminescent probes can be substituted for radioactive probes.
Geneticists often work with minuscule amounts of DNA. For instance, in
genotyping human sperm cells, geneticists encounter single-copy DNA. The
polymerase chain reaction (PCR) permits enormous copy-number am-
plification of a short DNA sequence. The chromosome region surrounding
the target sequence is first denatured by heating it in a solution containing
the four DNA bases, two specially chosen primers, and a polymerase.
As the solution cools, the two primers anneal to the two strand-specific
3 regions flanking the target sequence. The polymerase then extends each

primer through the target sequence, creating a new strand that partially
complements one of the original strands. This constitutes the first cycle
of PCR and doubles the number of target sequences. Each subsequent cy-
cle of denaturation, primer annealing, and polymerase extension similarly
doubles the number of target sequences. Figure A.2 depicts the first cycle
of the process. Here the primers are shorter than they would be in practice.
Cloning is a kind of in vivo DNA amplification. DNA fragments iso-
lated by restriction enzymes are ligated into circular DNA molecules called
vectors and inserted into bacteria or yeast cells. Once inside the host cells,
vectors resemble viruses in their ability to harness the machinery of the cell
to replicate independently of the host chromosomes. Vast libraries of ran-
dom DNA clones can be maintained in this manner. These clone libraries
furnish the raw material for DNA sequencing.

A.4 Mapping Strategies

Linkage mapping is described in detail in earlier chapters. It is worth em-
phasizing here the nature of most modern markers. Restriction fragment
polymorphisms (RFLPs) exploit individual differences in the presence or
absence of restriction sites. Suppose a probe is constructed to straddle a
polymorphic restriction site for a particular restriction enzyme. If nonpoly-
morphic restriction sites flank the probe region on its left and right, then
Southern blots of appropriately digested DNA from random individuals fall
into three patterns. Homozygotes for the absence of the site show a single