Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms.

Abstract

We developed mobility shift analysis of single-stranded DNAs on neutral polyacrylamide gel electrophoresis to detect DNA polymorphisms. This method follows digestion of genomic DNA with restriction endonucleases, denaturation in alkaline solution, and electrophoresis on a neutral polyacrylamide gel. After transfer to a nylon membrane, the mobility shift due to a nucleotide substitution of a single-stranded DNA fragment could be detected by hybridization with a nick-translated DNA fragment or more clearly with RNA copies synthesized on each strand of the DNA fragment as probes. As the mobility shift caused by nucleotide substitutions might be due to a conformational change of single-stranded DNAs, we designate the features of singlestranded DNAs as single-strand conformation polymorphisms (SSCPs). Like restriction fragment length polymorphisms (RFLPs), SSCPs were found to be allelic variants of true Mendelian traits, and therefore they should be useful genetic markers. Moreover, SSCP analysis has the advantage over RFLP analysis that it can detect DNA polymorphisms and point mutations at a variety of positions in DNA fragments. Since

Missense Mutation of the MET Gene Detected in Human Glioma

“…We analyzed exons 15,16,17,18, and 19, the most commonly affected regions of the MET gene, for mutations via SSCP and sequencing. One of 11 cases (Case 3) exhibited an aberrant band in SSCP (Fig.…”

“…PCR-SSCP analysis was performed by the modification of the method of Orita et al (18) with intronic sequences designed to amplify exons 15 to 19 from tumor and normal DNA. Each PCR sample contained 1.0 L of template DNA, 10 pmol of each primer, 20 nmol each of dGTP, dATP, dTTP, dCTP, 15 mM MgCl 2 , 0.1 unit of Taq DNA polymerase (Perkin-Elmer, Foster City, CA), 0.05 L of [ 32 P] dCTP (6000 Ci/mmol), and 1 L of 10ϫ buffer in a total volume of 10 L. PCR was performed for 35 cycles: denaturation at 94°C for 40 sec, annealing at 52 to 54°C for 40 sec, and extension at 72°C for 40 sec.…”

Isolation of a distinct class of gain-of-function SHP-2 mutants with oncogenic RAS-like transforming activity from solid tumors

“…Mutation screening was carried out by PCR-SSCP analysis (Orita et al, 1989). PCR was performed in a reaction volume of 20 ml containing 0.1 mg genomic DNA, 1 pmol of each primer, 4 nmol dNTPs, 0.1 unit Ex Taq polymerase (TaKaRa, Shiga, Japan).…”

Extent and distribution of linkage disequilibrium around the SLC11A1 locus

“…The genotypes of 16 markers were determined for the collected DNA samples using single-strand conformation polymorphism (SSCP) 72 analysis of 13 PCR fragments (excluding SLC1) in a conventional vertical electrophoresis tank as described by Yip. 73 The gel composition, buffer temperature and duration of electrophoresis specific for each fragment are summarized in Table 2.…”