Ｎｏｎ‐ＲＩ法を用いた遺伝子解析 キャピラリー電気泳動およびＤＨＰＬＣによる遺伝子解析法

Abstract

PCR-based analysis of genetic alterations has been applied to the diagnosis of various diseases, however, for this technique to become widely accepted as a tool for clinical DNA diagnosis, the development of automated analytical system is essential. Capillary electrophoresis (CE) is a promising alternative to gel electrophoresis for high-speed and reproducible separation of DNA fragments, and its use is feasible for automated DNA analysis. Therefore, CE is applied for DNA sequencing and the detection of genetic changes including microsatellite instability and mutations. Recently, capillary array electrophoresis for the simultaneous analyses of many samples is applied for the automated high-throughput sequencing. Heteroduplex analysis has been reported to be a promising technique for detecting mutated sequences because of its higher sensitivity than that of SSCP analysis. In this analysis, heteroduplex DNA containing mutated sequences is separated by denaturing gradient gel electrophoresis (DGGE). However, in DGGE, PCR products joined to a GC clamp are necessary and the gel preparation is also complicated. Therefore, it is difficult to automate. Denaturing high-performance liquid chromatography (DHPLC) has been reported to be a novel automated high-throughput technique for separating heteroduplex and homoduplex DNA fragments amplified with primers without a GC clamp under controlled temperature conditions. Hetroduplex analysis involving DHPLC enable more accurate, more rapid and easier heterozygous mutation detection than SSCP analysis. Hence, it is suggested that CE is preferable for DNA sizing and sequencing analyses, and DHPLC is effective for screening of genetic mutations and polymorphisms.