DETECTION OF POLYMORPHISMS OF HUMAN DNA BY GEL-ELECTROPHORESIS AS SINGLE-STRAND CONFORMATION POLYMORPHISMS
DETECTION OF POLYMORPHISMS OF HUMAN DNA BY GEL-ELECTROPHORESIS AS SINGLE-STRAND CONFORMATION POLYMORPHISMS
复制标题
DOI：
10.1073/pnas.86.8.2766
复制
发表时间：
1989-04-01
影响因子：
11.1
通讯作者：
SEKIYA, T
中科院分区：
文献类型：
--
作者：
ORITA, M;IWAHANA, H;SEKIYA, T
我们开发了中性聚丙烯酰胺凝胶电泳上单链 DNA 的迁移率变动分析，以检测 DNA 多态性。该方法首先用限制性内切酶消化基因组 DNA，在碱性溶液中变性，然后在中性聚丙烯酰胺凝胶上进行电泳。转移到尼龙膜后，由于单链 DNA 片段的核苷酸取代而引起的迁移率变化可以通过与切口翻译的 DNA 片段杂交来检测，或更清楚地用在 DNA 片段的每条链上合成的 RNA 拷贝作为探针进行杂交。由于核苷酸取代引起的迁移率变化可能是由于单链DNA的构象变化所致，因此我们将单链DNA的特征称为单链构象多态性（SSCP）。与限制性片段长度多态性 (RFLP) 一样，SSCP 被发现是真正孟德尔性状的等位基因变体，因此它们应该是有用的遗传标记。而且，SSCP分析比RFLP分析的优势在于可以检测DNA片段多个位置的DNA多态性和点突变。据估计，人类基因组中每隔几百个核苷酸就会出现 DNA 多态性，因此 SSCP 可以提供许多遗传标记。
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 of DNA fragments. Since DNA polymorphisms have been estimated to occur every few hundred nucleotides in the human genome, SSCP may provide many genetic markers.