John M. S. Bartlett.pdf - Bio-Nica.info

PCR-SSCP Analysis of Polymorphism 329
Fig. 2. Sample SSCP pattern. The human CD1a gene has two alleles. The DNA sample in
band A derived from an individual homozygous for allele 1, in lane B from a heterozygous
individual, and in lane C from an individual homozygous for allele 2. In this example, one of the
DNA strands assumes two different conformations as it migrates through the gel and results in two
bands. Notice the intensity of the top band compared with the lower bands showing that the top
band corresponds to one strand in one conformation and the lower bands to two conformations
for one strand. The pattern of the heterozygote is a composite of the homozygous patterns. The
single-stranded DNA patterns are shown in this figure. Double-stranded DNA corresponding to
nondenatured PCR product would be found migrating more rapidly on the gel.
corresponds to the upper strand of DNA and the other the lower strand of the amplified
fragment. It is also possible for a segment of single-stranded DNA to assume two
different conformations as it migrates through the gel, and this will result in two bands
for that strand of DNA on a SSCP autoradiogram. Examples of such SSCP patterns are
shown in Fig. 2. There are two alleles of the human CD1a gene (9). DNA samples in
lane A derives from an individual homozygous for allele 1, in lane C, the DNA is from
an individual homozygous for allele 2, and in lane B the DNA is from an individual
heterozygous for alleles 1 and 2. The bands corresponding to alleles 1 and 2 migrate
at different rates, and each has one strand that assumes two different conformations.
The pattern for a heterozygous individual is the composite of the two individual
allele’s patterns.
It was possible to make determinations about the assignment of CD1a alleles because
the analysis was coupled with sequence analysis. To obtain material for sequencing,
the same reaction mixture as used for the SSCP procedure is amplified using an
amplification buffer that does not contain radioactivity for cloning. Many systems are
commercially available for cloning and sequence analysis. In addition, SSCP bands
can be excised from the gel and re-PCR amplified using the same set of primers, which
provides a very convenient way to directly sequence the altered gene segment.
A successful approach to screen for genetic polymorphisms has been to examine
DNA samples from 10 unrelated individuals of diverse ethnic backgrounds. Genetic
polymorphism is likely to be found in such a sampling; however, this does depend upon
allele frequencies and distributions. Although SSCP is quite efficient in identifying