Biology Reference
In-Depth Information
DSCP markers could provide markers for species determination, kinship and
paternity analysis, as well as other aspects of population genetics that require
variation from a rapidly evolving region of DNA. For example, Atkinson
and Adams (1997) analyzed the mitochondrial control region of the termite
Nasutitermes corniger by DSCP using PCR products and discovered highly vari-
able markers for population studies. Higher levels of polymorphism were found
by DSCP than by RFLP analysis. The DSCP data suggested that some termite colo-
nies contained unrelated queens, each of which produced workers.
13.5.5 Heteroduplex Analysis (HDA)
Heteroduplex analysis combines some of the advantages of allele-specific PCR
and RFLP methods with the advantage of direct sequence analysis to detect new
alleles ( Tang and Unnasch 1997 ). This method originally was used to identify
virus isolates or detect immune genotypes in humans. HDA detects changes in
mobility on an electrophoresis gel of heteroduplex products formed between
the strands of a probe DNA and a test DNA molecule. The number and type of
mismatched bases within a given heteroduplex product determines the confor-
mation and mobility of the DNA duplex during electrophoresis. HDA is suffi-
ciently sensitive to detect single-base changes in fragments up to 500 bp.
HDA involves obtaining PCR products from the probe DNA and test DNA. The
DNAs are mixed, and then denatured by heating; heteroduplex and homodu-
plex products are formed during the cooling of the sample. This results in four
products: the probe and sample DNA because the probe and sample strands
reanneal to themselves (homoduplexes); and two heteroduplex products,
each comprising one strand from the probe and one from the test DNA. These
homo- and heteroduplex DNAs are separated by polyacrylamide gel electro-
phoresis and the separated products are detected by ethidium bromide stain-
ing. The relative amount of retardation of the heteroduplex products compared
to the homoduplexes reflects the number and type of mismatched nucleo-
tides between the probe and test DNA. This allows new alleles to be detected,
which can be further analyzed by sequencing to determine their relationship
to previously identified sequences. HDA is sufficiently rapid that multiple indi-
viduals can be screened, allowing one to determine the allele frequency in the
population.
Tang and Unnasch (1997) suggested HDA be applied to medically important
vectors of disease. They argued that HDA is simple, rapid, inexpensive, and capa-
ble of detecting small differences among DNA sequences. Disadvantages to HDA
include the difficulty of measuring differences in the mobility of the sequences
on the gels; such differences are only a rough estimate of the relative genetic
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