Because both homozygous and heterozygous variants can be distinguished from wild type, specific loci can be genotyped by amplicon melting. Protocols are identical to mutation scanning except that small amplicons are recommended to increase the melting curve differences between SNP genotypes (Liew et al., 2004). Heterozygotes are always easy to identify because low melting heteroduplexes change the shape of the melting curve. The homozygotes of most SNPs are readily distinguished from wild type with Tm differences of about 1°C. However, about 12% of human SNPs are challenging with differences of about 0.2°C and 4% are impossible because of thermodynamic symmetry. The SNPs that are difficult can be predicted by the type of SNP (Liew et al., 2004). The impossible SNPs can be genotyped after mixing with wild type sample either before or after PCR. When mixed after PCR, equal amounts of wild type and unknown amplicons are mixed, denatured, annealed, and melted again. If the unknown is wild type, no change in the melting curve occurs. If the unknown is homozygous mutant, the new melting curve appears heterozygous. Slightly more elegant is mixing before PCR. In this case, the ideal amount of wild-type DNA is one part added to six parts of unknown DNA. The melting curves of all three genotypes are separated based on the proportion of heteroduplexes formed. When mixing before PCR, only one melting analysis is needed and genotyp-ing remains closed-tube.
Because the Tm differences between homozygous mutant and wild type may be smaller than the temperature variation across 96- or 384-well blocks, only the HR-1™ is recommended for amplicon genotyping (Herrmann et al., 2006). The ability of HR-1™ genotyping to distinguish different heterozygotes was recently demonstrated (Graham et al., 2005). Twenty-one out of 21 heteroduplex pairs tested were distinguishable by high-resolution melting of small amplicons.
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