SNP Genotyping by Gel-immobilized ssDNA and Biolumometric Assay Coupled with Allele-specific Primer Extension Reaction

The new gel-immobilization method for preparing a single stranded DNA (ssDNA) template has been described for single nucleotide polymorphisms (SNPs) genotyping by bioluminometric assay based on allele-specfic extension reaction. In this method, PCR products amplified by a primer modified with acrylamide group at the 5´-terminal is copolymerized with acrylamide monomer to form gel. After the ssDNA immobilized on gel being denatured, the impurities are removed by electrophoresis. SNPs genotyping is performed by allele-specific primer extension reaction. The SNP type was identified by comparing the signal intensities from the extension by two allele specific primers hybridized on gel-mobilized ssDNA. If the 3´ end of a primer is complementary to the template, polymerase extension occurs and pyrophosphate (PPi) released. The signal is produced by using PPi conversion and luciferase reactions. As many bases are extended in one extension reaction, the sensitivity of the method is very high. This new strategy of the ssDNA immobilization is robust, simple, and inexpensive. It can be used for various application of DNA analysis, such as SNP genotyping and pyrosequencing.