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oai:hiroshima.repo.nii.ac.jp:02006205 2025-02-21T06:56:44Z 1730444907710

A single nucleotide polymorphism genotyping method using phosphate-affinity polyacrylamide gel electrophoresis Kinoshita, Eiji Kinoshita Eiji Kinoshita-Kikuta, Emiko Kinoshita-Kikuta Emiko Koike, Tohru Koike Tohru open access Copyright (c) 2007 Elsevier Ltd. SNP genotyping Allele-specific PCR Phosphate-affinity PAGE Zinc 460 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 non-labeled allele-specific PCR primers. The method is based on the difference in mobility of the phosphorylated and nonphosphorylated PCR products (in the same number of base pairs) 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 non-labeled 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. Elsevier 2007-02-15 eng journal article AO https://hiroshima.repo.nii.ac.jp/records/2006205 10.1016/j.ab.2006.11.032 17196923 http://dx.doi.org/10.1016/j.ab.2006.11.032 0003-2697 AA00524867 Analytical Biochemistry Analytical Biochemistry 361 2 294 294 298 https://hiroshima.repo.nii.ac.jp/record/2006205/files/AnalBiochem_361_294.pdf 312.8 KB