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SSCP Analysis of Point Mutations by Multi-Color Capillary Electrophoresis

Published

Author(s)

K Hayashi, H M. Wenz, M Inazuka, T Tahira, Donald H. Atha

Abstract

Virtually all methods for the detection of mutations (polymorphism or variant) rely on polymerase chain reaction (PCR). Direct sequence determination of a PCR product is the gold standard for identifying mutaions. However, the vast majority of the signal in the sequencing data is derived from nonvariant sequence, and can be a source of noise. Thus, a womewhat high false positive rate is inevitalbe when rare mutations are searched for in a large genomic region or in a region of many genomes. Techniques to detect variants as positive signals have the advantage of intrinsically low false positive rate, and are suitable methods to preselect fragments that carry mutations among an excess of nonmutated fragments. Such techniques are especially useful, for example in surveying for possible mutations in genes suspected to be responsible for genetic diseases, or finding single-nucleotide polymorphisms (SNPs) in blindly amplified genomic segments.Single-strand conformation polymorphism (SSCP) analysis of PCR products is the most popular technique to positively detect mutations/ polymorphisms in genomic or cDNA sequence. In this analysis method, a PCR product is denatured to become single-stranded, and separated by gel electrophoresis using nondenaturing conditions. The mobility of a mutant fragment through a gel matrix is generally shifted from that of a reference fragment, because it has a folded structure that is different from that of the reference fragment. PCR-SSCP analysis is widely used in clinical or basic medical science, because it is less skill-demanding than other mutation detection methods, yet can detect mutations at a high sensitivity. Though relatively easy and simple to set up, the method still requires certain skill such as gel preparation and sample loading. Also, the judgment of a mobility shift often requires experience, and tends to be subjective. Capillary-electrophoresis SSCP (CE-SSCP) has been developed to overcome these disadvantages, and also to adapt the technique to the emerging demand of high throughput analysis in the age of SNP collection and typing. The first part of this chapter includes optimization of CE-SSCP analysis of PCR products using fluorescently labeled primers, and efforts of identifying several different mutations from their characteristic mobility shifts. These protocols are developed through collaborative work between PE Applied Biosystems and the National Institute of Standards and Technology. The second part covers post-PCR fluorescent labeling of amplification products and their use in a high-precision calibration of mobility that allows a simplified statistical discrimination between a reference fragment and variant fragments at a high sensitivity. The latter system is developed and is extensively used by Kyushu University group.
Citation
SSCP Analysis of Point Mutations by Multi-Color Capillary Electrophoresis
Volume
163
Publisher Info
Methods in Molecular Biology Book Chapter: Capillary Electrophoresis of Nucleic Acids, in Methods in Molecular Biology,

Keywords

capillary electrophoresis, DNA point mutations, single-strand conformational polymorphis, SSCP

Citation

Hayashi, K. , Wenz, H. , Inazuka, M. , Tahira, T. and Atha, D. (1999), SSCP Analysis of Point Mutations by Multi-Color Capillary Electrophoresis, Methods in Molecular Biology Book Chapter: Capillary Electrophoresis of Nucleic Acids, in Methods in Molecular Biology, (Accessed April 13, 2024)
Created October 31, 1999, Updated October 12, 2021