A single nucleotide polymorphism genotyping method using phosphate-affinity polyacrylamide gel electrophoresis

Kinoshita Eiji

Kinoshita-Kikuta Emiko

Koike Tohru

Analytical Biochemistry

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.

SNP genotyping

Allele-specific PCR

Phosphate-affinity PAGE

Zinc

Biology [ 460 ]

eng

Elsevier

Copyright (c) 2007 Elsevier Ltd.

[ISSN] 0003-2697

[DOI] 10.1016/j.ab.2006.11.032

[NCID] AA00524867

[PMID] 17196923

[DOI] http://dx.doi.org/10.1016/j.ab.2006.11.032 isVersionOf