Kinoshita-Kikuta Emiko, Kinoshita Eiji, Koike Tohru
Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
Methods Mol Biol. 2009;578:169-82. doi: 10.1007/978-1-60327-411-1_10.
We introduce a method for the detection of single-nucleotide polymorphisms (SNPs) by polyacrylamide gel electrophoresis (PAGE) with an additive Zn2+-cyclen complex (cyclen is 1,4,7,10-tetraazacyclododecane), called "Zn2+-cyclen-PAGE." The method is based on the difference in mobility of mutant DNA (of the same length) in PAGE, which is due to Zn2+-cyclen binding to thymine bases accompanying a total charge decrease and a local conformation change of target DNA. In combination with a polymerase chain reaction based heteroduplexing technique, the method is more accurate than many other conventional PAGE-based methods, as shown by clear gel-shifting bands because of the separation of heteroduplex and homoduplex DNAs. We demonstrate SNP mapping and heterozygosity screening in a human cardiac sodium channel gene (i.e., SCN5A) that relates to inherited arrhythmia syndromes using Zn2+-cyclen-PAGE.
我们介绍了一种通过聚丙烯酰胺凝胶电泳（PAGE）检测单核苷酸多态性（SNP）的方法，该方法使用一种添加剂Zn2 + - 环烯配合物（环烯为1,4,7,10 - 四氮杂环十二烷），称为“Zn2 + - 环烯 - PAGE”。该方法基于PAGE中（相同长度的）突变体DNA迁移率的差异，这是由于Zn2 + - 环烯与胸腺嘧啶碱基结合，伴随目标DNA的总电荷减少和局部构象变化。与基于聚合酶链反应的异源双链技术相结合，该方法比许多其他传统的基于PAGE的方法更准确，如由于异源双链和同源双链DNA的分离而出现清晰的凝胶迁移带所示。我们使用Zn2 + - 环烯 - PAGE在与遗传性心律失常综合征相关的人类心脏钠通道基因（即SCN5A）中进行SNP定位和杂合性筛选。
