A single nucleotide polymorphism genotyping method using phosphate-affinity polyacrylamide gel electrophoresis |
| |
Authors: | Kinoshita Eiji Kinoshita-Kikuta Emiko Koike Tohru |
| |
Affiliation: | Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8553, Japan. kinoeiji@hiroshima-u.ac.jp |
| |
Abstract: | To date, various methods have been developed to facilitate the genotyping of a single nucleotide polymorphism (SNP) for aiding in the diagnosis and treatment of inherited diseases. The most commonly used method for SNP genotyping is an allele-specific hybridization procedure using an expensive fluorochrome-labeled oligonucleotide probe and a specialized fluorescence analyzer. Here, we introduce a simple and reliable genotyping method using a 1:1 mixture of 5'-phosphate-labeled and nonlabeled allele-specific polymerase chain reaction (PCR) primers. The method is based on the difference in mobility of the phosphorylated and nonphosphorylated PCR products (in the same number of basepairs) on phosphate-affinity polyacrylamide gel electrophoresis. The phosphate-affinity site is a polyacrylamide-bound dinuclear zinc(II) complex, which preferentially captures the 5'-phosphate-labeled allele-specific product compared with the corresponding nonlabeled product. The obtained DNA migration bands can be visualized by ethidium bromide staining. We demonstrate the genotyping of a SNP reported in a human cardiac sodium channel gene, SCN5A, using this novel procedure. |
| |
Keywords: | SNP genotyping Allele-specific PCR Phosphate-affinity PAGE Zinc |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|