１本鎖ＤＮＡ高次構造多型（ｓｉｎｇｌｅ‐ｓｔｒａｎｄ ｃｏｎｆｏｒｍａｔｉｏｎ ｐｏｌｙｍｏｒｐｈｉｓｍ， ＳＳＣＰ）法による遺伝子変異の解析

Abstract

We developed mobility shift analysis of single-stranded DNAs on neutral polyacrylamide gel electrophoresis to detect base changes in DNA. Originally this method follows digestion of genomic DNA with restriction endonucleases, denaturation in alkaline solution, and electrophoresis in 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. 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). Invention of the polymerase chain reaction (PCR) made it possible to amplify specific regions of genomic sequences efficiently. The method can simultaneously label amplified DNA fragments by using labeled substrates. After denaturation of PCR products, electrophoresis of obtained single-stranded DNA fragments in nondenaturing polyacrylamide gels and detection of mobility shifts by radioautography can reveal the presence of base substitutions. Most single base changes in up to 300-base fragments could be detected as mobility shifts. Thus the combined use of SSCP and PCR could provide a rapid and sensitive method for the detection of base changes in given sequences of genomic DNA. Many mutations resulted in activation of oncogenes and inactivation of tumor suppressor genes were detected by this PCR-SSCP technique.