Mutation Detection by Single Strand Conformation Polymorphism and Heteroduplex Analysis
نویسندگان
چکیده
Single strand conformation polymorphism (SSCP) and heteroduplex analysis (HDA) are two of the most popular electrophoresis-based mutation detection methods. Coupled to DNA amplification of the sequence to be analyzed, these techniques have become the methods of choice for a number of molecular diagnostic laboratories. This can be explained mainly by the numerous advantages, namely their technical simplicity and relatively high specificity for the detection of sequence variations, the low operation costs, and the potential for automation for high-throughput mutation analysis. If fluorescently labeled primers are employed during DNA amplification, SSCP analysis can be also performed in gelor capillary electrophoresis-based automated sequencers (F-SSCP, CE-SSCP), hence allowing for precise, reproducible and high-throughput analysis of the genomic variation. There are several factors that influence sensitivity, and therefore need to be taken into account in order to obtain reproducible results as well as to maximize the sensitivity of mutation detection. In the following pages, the theory and practice of both SSCP and HDA will be discussed. In particular, emphasis will be given to the principle, the parameters influencing the sensitivity and reproducibility of the results, the available detection schemes, and the limitations of both techniques. Finally, a number of applications for screening genomic loci in order to investigate the underlying molecular heterogeneity are also discussed.
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