Genotyping by high-resolution melting analysis.

High-resolution melting (HRM) analysis is a simple, closed tube, post-PCR method used to identify genetic variation. The method is highly sensitive and can discriminate DNA sequence variants based on length (such as insertions or deletions), composition (such as single nucleotide polymorphisms, i.e., SNP) or strand complementarity (such as heterozygous or homozygous material). The technique involves PCR amplification of a target sequence in the presence of a fluorescent double-stranded DNA (dsDNA) binding dye, melting of the fluorescent amplicons, and subsequent interpretation of melt curve profiles. Here, we describe general considerations for assay design, PCR amplification, and HRM analysis.