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

TLDR

SSCPs are allelic variants of Mendelian traits that arise from conformational changes in single‑stranded DNA. The study aimed to develop a mobility‑shift analysis of single‑stranded DNA on neutral polyacrylamide gel electrophoresis to detect DNA polymorphisms. The method digests genomic DNA with restriction enzymes, denatures it, runs it on a neutral polyacrylamide gel, transfers it to nylon, and detects mobility shifts by hybridization with labeled probes. SSCPs can detect point mutations at many positions and are therefore useful genetic markers, offering advantages over RFLP analysis.

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 single-stranded 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 DNA polymorphisms have been estimated to occur every few hundred nucleotides in the human genome, SSCPs may provide many genetic markers.