Polymerase chain reaction—single strand conformation polymorphism or how to detect reliably and efficiently each sequence variation in many samples and many genes

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Abstract
A simple and fast method with high reliability is necessary for the identification of mutations, polymorphisms and sequence variants (MPSV) within many genes and many samples, e.g. to clarify the genetic background of individuals with multifactorial diseases. We evaluated polymerase chain reaction—single strand conformation polymorphism (PCR‐SSCP) analysis to identify MPSV in several genes, which are thought to be involved in the pathogenesis of multifactorial autoimmune diseases like multiple sclerosis. The method is based on the property, that the electrophoretic mobility of single‐stranded nucleic acids depends not only on its size but also on its sequence. The target sequence was amplified, digested into fragments ranging from 50–200 bp, heat‐denatured and analyzed on native gels. The analysis of 55 PCR systems, including a total of 145 fragments demonstrates, that the detection rate of MPSV depends primarily on the fragment lengths. Appropriate dilutions of samples enhances the proportion of ssDNA compared to dsDNA. Changing the gel conditions, glycerol concentrations and/or the addition of urea may increase fragment resolution in some cases. In general, the detection of MPSV is neither influenced by their location within the fragment nor by the type of substitution, i.e. transitions or transversions. The standard PCR‐SSCP system described here provides high reliability and detection rates and allows the efficient analysis of many samples and many genes.