Single-Strand Conformational Polymorphism Analysis of DNA Topoisomerases I and II
互联网
570
Single-strand conformational polymorphism (SSCP) analysis is a method for screening for mutations. This method is based on observations of Orita and coworkers that single strands of DNA assume unique conformations depending on their primary sequence. The uniqueness of each three-dimensional conformation, in turn, dictates a unique migration rate following electrophoresis through a nondenaturing polyacrylamide gel of the single-stranded fragments (1 ,2 ). Shown schematically in Fig. 1 , differences in migration rates reflect differences in primary sequences. Since complementary strands of DNA have different primary sequences, under optimal conditions, each fragment of double-stranded DNA analyzed by SSCP will appear as two distinct bands that migrate at different rates through a nondenaturing gel (Fig. 2 ). Usually, double-stranded radiolabeled PCR or RT-PCR products are generated, and then electrophoresed on polyacrylamide gels using a sequencing apparatus and detected by autoradiography. Nondenaturing conditions are essential to allow each DNA fragment to retain its three-dimensional conformation.
Fig. 1. Schematic representation of detection of mutations by SSCP analysis. Difference in secondary structure of mutant allele causes mobility shift.
Fig. 2. Autoradiogram of SSCP gel showing migration of each of several RT-PCR products from CEM cells: Lane 1, “Motif B” dinucleotide binding sequence of topoisomerase IIα; Lane 2, Motif B sequence of topoisomerase IIβ; Lane 3, region adjacent to tyrosine 805 of topoisomerase IIα; and, Lane 4, the Motif A sequence of topoisomerase IIα (3 ). Reprinted with permission of Cancer Res.
