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Appendix A: Molecular Genetics in Brief
band high on the gel. This band corresponds to the long fragment between
the two flanking restriction sites. Homozygotes for the presence of the site
show two separate bands lower down on the gel. These correspond to the
two smaller fragments defined by the flanking sites and separated by the
interior restriction site. Heterozygotes show all three bands. If the probe
falls to one side of the polymorphic restriction site, then at most two bands
appear, but it is still possible to distinguish all three genotypes.
The single base pair differences revealed by RFLPs must fall within a re-
striction site for some restriction enzyme. These sites are naturally rare, and
it is convenient to exploit single base pair differences wherever they occur.
This can be accomplished by sequencing a small segment of DNA around
a polymorphic site and designing short probes (or oligonucleotides) that
match the sequence of the segment except at the site. At the site, each
probe matches one of the dominant bases appearing in the population. Be-
cause annealing of a short probe requires a perfect match, Southern blots
with different probes detect different alleles. This technique is particularly
useful in screening for common mutations in disease loci.
The biallelic markers generated by single base pair differences exhibit
limited polymorphism. Short tandem repeat markers are often much
more polymorphic. For instance, the dinucleotide CA is repeated a random
number of times in many regions of the human genome. Repeat numbers
in a repetitive sequence ···CACACACACA··· often vary from person to
person. If a probe closely flanks a repeat region, then Southern blotting
with the probe will reveal allelic differences in the number of repeat units
as length differences in the fragments highlighted by the probe.
Radiation hybrids and somatic cell hybrids are physical mapping
techniques covered in detail in Chapters 11 and 13, respectively. Flu-
orescence in situ hybridization (FISH) and pulsed-field gel elec-
trophoresis are two other competing physical techniques with good reso-
lution. In FISH, probes are directly annealed to chromosomes during the in-
terphase period of cell division. Because chromosomes are less contracted
during interphase, map resolution to within 100,000 bases is possible. In a
recent variation of FISH, probes are annealed to DNA filaments stretched
on a glass slide. In pulsed-field gel electrophoresis, the electric field applied
to a gel is occasionally reversed. This permits large DNA fragments to un-
tangle and slowly migrate down the gel without breaking. If two different
probes anneal to the same large fragment, then presumably they coexist on
the fragment. Using a sufficient number of fragments, closely spaced loci
defined by well-defined probes can be ordered.
One advantage of physical mapping is that it does not require polymor-
phic loci. It is usually harder to find polymorphisms than it is to construct a
probe from unique sequence DNA. For example, geneticists can easily iden-
tify expressed sequence tags by sequencing complementary DNA
(cDNA). Because cDNA is synthesized from messenger RNA, an expressed
sequence tag is guaranteed to be in the coding region of some gene. (Re-