title: A single nucleotide polymorphism genotyping method using phosphate-affinity polyacrylamide gel electrophoresis.
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.
authors.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
authors.forename: Eiji
authors.initials: E
authors.lastname: Kinoshita
authors.forename: Emiko
authors.initials: E
authors.lastname: Kinoshita-Kikuta
authors.forename: Tohru
authors.initials: T
authors.lastname: Koike
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004996:Ethidium / Q000737:chemistry; D005838:Genotype; D006801:Humans; D009124:Muscle Proteins / Q000235:genetics; D062554:NAV1.5 Voltage-Gated Sodium Channel; D010710:Phosphates / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D015222:Sodium Channels / Q000235:genetics
chemical_list: D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D010710:Phosphates; C568320:SCN5A protein, human; D015222:Sodium Channels; D004996:Ethidium