Kinoshita-Kikuta Emiko, Kinoshita Eiji, Koike Tohru
Department of Functional Molecular Science, Division of Medicinal Chemistry, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.
Nucleic Acids Res. 2002 Nov 15;30(22):e126. doi: 10.1093/nar/gnf126.
The analysis of single nucleotide polymorphisms (SNPs) is increasingly utilized in the study of various genetic determinants. Here, we introduce a simple, rapid, low-cost and accurate procedure for the detection of SNPs by polyacrylamide gel electrophoresis (PAGE) with a novel additive, the Zn2+- cyclen complex (cyclen = 1,4,7,10-tetraazacyclododecane). The method is based on the difference in mobility of mutant DNA (in 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. Various nucleotide substitutions (e.g. AT to GC) in DNA fragments (up to 150 bp) can be visualized with ethidium bromide staining. Furthermore, heteroduplex and homoduplex DNAs are clearly separated as different bands in the gel. We demonstrate the analysis of single- and multiple-nucleotide substitutions in a voltage-dependent sodium channel gene by using this novel procedure (Zn2+-cyclen-PAGE).
单核苷酸多态性（SNP）分析在各种遗传决定因素的研究中越来越受到广泛应用。在此，我们介绍一种简单、快速、低成本且准确的程序，通过聚丙烯酰胺凝胶电泳（PAGE）并添加一种新型添加剂Zn2 + - 轮环配体络合物（轮环配体 = 1,4,7,10 - 四氮杂环十二烷）来检测SNP。该方法基于PAGE中突变DNA（长度相同）迁移率的差异，这是由于Zn2 + - 轮环配体与胸腺嘧啶碱基结合，伴随目标DNA总电荷减少和局部构象变化所致。DNA片段（最长150 bp）中的各种核苷酸替换（如AT替换为GC）可用溴化乙锭染色进行可视化观察。此外，异源双链和同源双链DNA在凝胶中作为不同条带被清晰分离。我们通过使用这种新程序（Zn2 + - 轮环配体 - PAGE）展示了对电压依赖性钠通道基因中单核苷酸和多核苷酸替换的分析。
