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

PCR-SSCP Analysis of Polymorphism 333
11. Usually the SSCP patterns can be read clearly after 16- to 20-h exposure. If the migration
bands are still very weak after exposed for 20 h, exposure time should be prolonged to
more than 48 h, sometimes even for as long as 2 wk.
12. SSCP gels are analyzed by observing the pattern of bands resulting on the autoradiograms.
The number of bands present is dependent upon the sequence amplified. When one
sequence is amplified, the most simple pattern that can be observed consists of two
bands, one corresponding to the upper strand of DNA, and the other to the lower strand
of the amplified fragment. If a segment of single-stranded DNA assumes more than one
conformation as it migrates through the gel, the result will be multiple bands (equal
to the number of conformers). Knowledge of the sequence being amplified provides a
control and a point of reference. Amplification of more than one sequence results in a
composite pattern of bands corresponding to those of the constituent sequences. A band
that corresponds to double-stranded DNA may also be visible lower in the gel as doublestranded
DNA migrates more rapidly. Inclusion of a sample of nondenatured DNA (not
heated) serves as a marker for double-stranded DNA.
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