SSCP Analysis by Capillary Electrophoresis with Laser-Induced Fluorescence Detector

Abstract

Among the various techniques developed for mutation detection, single-strand conformation polymorphism (SSCP) analysis has become the most popular method for the screening of unknown mutation in small stretches of DNA. Its widespread use is related to its simplicity and low cost. SSCP analysis is based on the principle that ssDNA with a single base substitution (or containing a small sequence deletion or insertion) often assumes an altered folded conformation to an equivalent wild-type sequence lacking the base substitution. The single base substituted and wild-type ssDNA molecules therefore may migrate with different mobility from each other in a nondenaturing electrophoresis gel (1). This is demonstrated in Fig. 1, in which the conventional SSCP procedure is shown. Here, the gene DNA fragment of interest is amplified by use of the polymerase chain reaction (PCR). The DNA fragments are [³²P]-radiolabeled (or using radiolabeled primers). Then following nondenaturing gel electrophoresis, the mobility (position) of the ssDNA is detected by autoradiography (2-4). In spite of the simplicity of the technique, conventional SSCP analysis remains both labor-intensive and time-consuming, involving the preparation of large-format polyacrylamide gels for multiple sample handling, the long run time of the electrophoresis gels, and the manipulation of radioactive compounds and radioactive wastes.