Biology Reference
In-Depth Information
many more binding sites than 20-base primers. Several to many primers
can be used. These primers work throughout the genome and attach at
points where their structure matches that of a DNA segment. Primer
pairs that attach with an orientation toward each other define the seg-
ments that are to be amplified.Amplification is carried out by PCR, using
a procedure similar to that in AFLP. Binding sites flanking DNA segments
up to about 2,000 or so base pairs in length can be amplified.The result-
ing amplified fragments are from unknown locations and are considered
to be of random origin within the genome. Within these amplified seg-
ments, differences in DNA arrangements may exist. After amplification,
the DNA segments are separated by electrophoresis and stained. When a
series of many primers is used, up to 100-200 electrophoretic bands typ-
ically can be seen, some or many of which will show polymorphisms. For
example, in a study of variability of water spinach in Florida, Van and
Madeira (1998) used 18 primers and found 188 different bands, 31% of
which were polymorphic. Analyses such as this allow both the degree of
polymorphism and the similarity of samples from different populations to
be assessed.
Microsatellite Analysis
Another indicator of genetic variability is variation in DNA microsatel-
lites, which are present in the genomes of many organisms. Microsatellites
are sections of DNA in which a sequence of one to about six nucleotides
is repeated along DNA strands. For example, the bases ATT (adenine-
thymine-thymine) might be repeated along a strand of DNA in a
sequence of 12 successive units (ATTATTATT ...ATT). These
sequences do not code for protein. They tend to be localized in cen-
tromeric or noncoding regions of the chromosomes. Although their ori-
gin and function are still not fully understood, evidence is accumulating
that microsatellites have organizational or regulatory functions within
the genome (Li et al. 2002). The number of microsatellite units at a par-
ticular location varies, however, so they can be analyzed in a manner
analogous to alleles of a DNA gene. Microsatellites mutate by changes in
the number of repeated units. The mutation rate of these units is much
higher than the rate of point mutation at nuclear gene loci, leading to
extensive variation in the number of repeats.These mutations are believed
to occur either by slippage of complementary DNA strands during repli-
cation or by unequal recombination between complementary DNA
strands (Li et al. 2002).
