SNP Genotyping by Gel-Immobilized RCA Product and Biolumometric Assay Coupled with Allele-Specific Primer Extension Reaction

A novel approach with the dual-specificity and dual-amplification for detecting the known single nucleotide variation was reported in this study. In the approach, a pair of padlock probes is specifically designed for single base variant, and the padlock probe can be circularized by the DNA ligase when its 3' end matches the variant. The circularized probe can be amplified by rolling circle amplification (RCA) with a 5'-terminal acrylamide-modified primer. Then the RCA products are immobilized on three dimension (3-D) polyacrylamide gel by copolymerizing with acrylamide monomer. Following this step, a pair of allele-specific primers, which contain different bases in 3' end, is respectively hybridized with the RCA products for conducting the extension reactions: a powerful light signal is produced if the allele-specific primer is complementary to the RCA products since a large number of bases extend in the extension reaction, but no light emitted when the allele-specific primer does not match the template. Thus, the information of the DNA sequence variants is translated into the "on-off" light signal amplification. The principle experimental results show this method is feasible with high specificity. The approach may be used for many critical researches and diagnostic applications, which focus on the high specificity and sensitivity to detect single nucleotide variations.