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        Estimation of SNP Allele Frequencies by SSCP Analysis of Pooled DNA

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        The single strand conformation polymorphism (SSCP) method is a sensitive technique used to detect subtle sequence differences in PCR-amplified DNA fragments as separated peaks in electrophoretic analysis. In this chapter, we focus on SSCP analysis for quantifying polymorphic alleles rather than scanning for mutations. Short fragments carrying single nucleotide polymorphisms are amplified from individual and pooled DNA samples, then the products are labeled with fluorescent dyes and analyzed by automated capillary electrophoresis under nondenaturing conditions. Dedicated software, QSNPlite, interprets trace data of the electrophoresis to identify alleles of individuals and quantify these alleles in the pool. The software can also incorporate sequencing data to assign alleles at the nucleotide level. The procedures described here are being used in association studies that compare allele frequencies between cases and controls to identify genes responsible for common diseases.
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