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PCR-SSCP Analysis of Polymorphism 327
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PCR-SSCP Analysis of Polymorphism
A Simple and Sensitive Method for Detecting Differences
Between Short Segments of DNA
Mei Han and Mary Ann Robinson
1. Introduction
Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP)
(1) is a simple method that allows one to rapidly determine whether there are sequence
differences between relatively short stretches of DNA. Coupled with sequence analysis,
SSCP is an extremely useful method for both identifying and characterizing genetic
polymorphisms and mutations. The theory of SSCP is that the primary sequence and
the length of a single stranded DNA fragment determine its conformation when it is
resolved in a nondenaturing polyacrylamide gel. Even single-base differences can cause
different secondary conformations and thus result in different migration rates of the
DNA strands. Radioactive nucleotides are incorporated into the DNA strands by PCR,
making it possible to detect the DNA by autoradiography. SSCP has been widely used
to identify mutations in host genes such as p53 (2–5) and in viruses, such as simian
immuno-deficiency virus (SIV), during the course of infection (6). SSCP has been used
to identify and characterize polymorphisms in a variety of genes (7–9) and was effective
in characterizing alleles of linked genes present in individual sperm (10).
Orita and colleagues (11) developed the SSCP method in 1989 and since then, it has
been applied to screen for sequence differences in either genomic or complementary
DNA (cDNA) samples. Figure 1 schematically shows the steps of the procedure. First,
the region of interest (the gene or cDNA) can be PCR amplified by using primers
corresponding to the desired sequence. Because migration differences are better
resolved using shorter DNA fragments, success is more likely with primers selected
to amplify fragments in 100- to 300-bp range. The PCR mixture contains 32 P-dCTP
to radioactively label the amplification product, which is important for visualizing the
migration differences in later steps of the procedure. The second step is to heat diluted
amplified samples, which will denature the double-stranded DNA into single-stranded
DNA. The samples are mixed with loading buffer containing formamide to hinder
reannealing of the DNA and dye to visually follow the migration of samples through
the gel. The third step is to resolve the single-stranded DNA samples by nondenaturing
polyacrylamide gel electrophoresis. The length of time necessary for running the
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
327