Gotoh K, Hata M, Miyajima M, Yokota H
Kazusa DNA Research Institute, Kisarazu, Chiba, Japan.
Biochem Biophys Res Commun. 2000 Feb 16;268(2):535-40. doi: 10.1006/bbrc.2000.2174.
We propose a procedure for detecting unknown, subtle DNA changes throughout the entire bacterial genome by a combination of MutS and RDA. Current techniques detect subtle mutations after PCR amplification of the target regions, so the mutation detection is done between amplified PCR fragments. In this paper, genome-wide subtle mutation scanning in bacteria was performed by combining the MutS and RDA techniques. Our strategy for cloning a small mutation region is composed of two steps: an enrichment of fragments containing subtle mutations using MutS, followed by an RDA subtraction procedure for further enrichment. We successfully identified small mutations such as a four-base insertion, a two-base insertion, and transition mutations in bacteria.
我们提出了一种通过MutS和代表性差异分析（RDA）相结合的方法来检测整个细菌基因组中未知的、细微的DNA变化。目前的技术是在对目标区域进行PCR扩增后检测细微突变，因此突变检测是在扩增的PCR片段之间进行的。在本文中，通过结合MutS和RDA技术对细菌进行全基因组范围的细微突变扫描。我们克隆小突变区域的策略包括两个步骤：使用MutS富集含有细微突变的片段，然后通过RDA消减程序进一步富集。我们成功地在细菌中鉴定出了如四碱基插入、两碱基插入和转换突变等小突变。
