Genotyping by high-resolution amplicon melting

Genotyping by high-resolution amplicon melting is a simple method using only PCR primers and double-stranded DNA binding dyes. Heterozygous genotypes are easily assigned based on derivative melting curve shape and width. Homozygous genotypes are assigned based on Tm differences that are usually 1 °C but can be less. One potential drawback to genotyping by amplicon melting is Tm variance that can be caused by solution chemistry differences and thermal variability of PCR instruments. To address the Tm variance, internal temperature standards were created using complimentary 3'-blocked oligonucleotides which were included in each reaction. Genotyping by amplicon melting lends itself to potential multiplexing, due to only primers being used for detection. A thrombophilia panel, including the F5, F2, and the MTHFR 677 and 1298 mutations, was used as a multiplex model because of the potential additive effects of mutations at multiple loci. The first set of experiments were done using the LightCycler 1.5 and HR-1 technologies, which performed well in a blinded study of 109 samples. This assay was further improved by conversion to a mutli-sample high-resolution platform combining the LightCycler and HR-1 technologies. This new process decreased hands-on manipulation as well as overall analysis time while maintaining the same resolution.