Phosphate-Affinity Polyacrylamide Gel Electrophoresis for SNP Genotyping

We introduce a genotyping method which relies on the use of a 1:1 mixture of 5′-phosphate-labeled and nonlabeled allele-specific primers for polymerase chain reaction (PCR). The method is based on the difference in mobility of the phosphorylated and nonphosphorylated PCR products (possessing the same number of base pairs) during phosphate-affinity polyacrylamide gel electrophoresis (PAGE). The phosphate-affinity site in the gel is represented by an immobilized phosphate-binding tag molecule [i.e., a polyacrylamide-bound dizinc(II) complex], which selectively captures the 5′-phosphate-labeled allele-specific product compared with the corresponding nonlabeled one. The DNA migration bands obtained can be visualized by ethidium bromide staining. We demonstrate the genotyping of a single-nucleotide polymorphism reported in a human cardiac sodium channel gene, SCN5A , using the phosphate-affinity PAGE.