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Abstract

Various methods are described that provide for high resolution melt (HRM) genotyping. The embodiments include providing a locus specific primer and two allele specific primers each having a 5′ end with a short tail, providing a nucleic acid having a single nucleotide polymorphism (SNP) base located within 1-20 base pairs of the 3′ end of nucleic acid, hybridizing the locus specific primer and the allele specific primers to the nucleic acid, amplifying the sample using pyrophosphorolysis activated polymerization (PAP) PCR enzyme, and determining the Tm of the amplicons using HRM. In other embodiments, reactions mixtures and kits for HRM genotyping are provided and disclosed. These kits comprise a locus specific primer, one or more allele specific primers each having a 5′ end with a short tail, a nucleic acid, and a pyrophosphorolysis activate polymerization (PAP) PCR enzyme.

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First Claim