Chapter 4 - Mutation Detection by Single Strand Conformation Polymorphism and Heteroduplex Analysis

Abstract

This chapter deals with the single strand conformation polymorphism (SSCP) and heteroduplex analysis (HDA) for the detection of nucleotide sequence changes in genomic loci. These methods can detect single nucleotide substitutions, insertions, and deletions of a short nucleotide sequence accurately and in a relatively short time. Therefore, they are used for DNA analysis of human cancers and other genetic disorders. When coupled with DNA amplification of the sequence to be analyzed, these techniques become the methods of choice for a number of molecular diagnostic laboratories, which can be explained by the numerous advantages: technical simplicity, high specificity for low operation costs, and potential of automation for high-throughput mutation analysis. If fluorescently labeled primers are employed during DNA amplification, SSCP analysis can be performed in gel or capillary electrophoresis-based automated sequencers (F-SSCP, CE-SSCP), hence allowing for precise, reproducible, and high-throughput analyses of the genomic variation. Several factors that influence sensitivity need to be taken into account in order to obtain reproducible results as well as to maximize the sensitivity of mutation detection. SSCP and HDA continue to be popular electrophoresis-based mutation detection methods and substantially contribute to the growing knowledge of human genetic variation as well as that of other organisms.