A novel method of genotyping single nucleotide polymorphisms (SNP) using melt curve analysis on a capillary thermocycler

We report the development of a homogenous assay for the genotyping of single-nucleotide polymorphisms (SNPs), utilizing a fluorescent dsDNA-binding dye. Termed TM-shift genotyping, this method combines multiplex allele-specific PCR with sequence differentiation based on the melting temperatures of amplification products. Allele-specific primers differing in length were used with a common reverse primer in a single-tube assay. PCR amplification followed by melt curve analysis was performed with a fluorescent dsDNA-binding dye on a real-time capillary thermocycler. Genotyping was carried out in a single-tube homogeneous assay in 25 minutes. We compared the accuracy and efficiency of this T-M genotyping method with conventional restriction fragment genotyping of a novel single nucleotide polymorphism in the Jagged1 (JAG1) gene. The flexibility, economy and accuracy of this new method for genotyping polymorphisms could make it useful for a variety of research and diagnostic applications.