Heterozygote PCR Product Melting Curve Prediction

Melting curve prediction of PCR products is limited to perfectly complementary strands. Multiple do- mains are calculated by recursive nearest neighbor ther- modynamics. However, the melting curve of an ampli- con containing a heterozygous single-nucleotide variant (SNV) after PCR is the composite of four duplexes: two matched homoduplexes and two mismatched heterodu- plexes. To better predict the shape of composite het- erozygote melting curves, 52 experimental curves were compared with brute force in silico predictions varying two parameters simultaneously: the relative contribution of heteroduplex products and an ionic scaling factor for mismatched tetrads. Heteroduplex products contributed 25.7 ± 6.7% to the composite melting curve, varying from 23%-28% for different SNV classes. The effect of ions on mismatch tetrads scaled to 76%-96% of normal (depend- ing on SNV class) and averaged 88 ± 16.4%. Based on uMelt (www.dna.utah.edu/umelt/umelt.html) with an ex- panded nearest neighbor thermodynamic set that includes mismatched base pairs, uMelt HETS calculates helicity as a function of temperature for homoduplex and heterodu- plex products, as well as the composite curve expected from heterozygotes. It is an interactive Web tool for effi- cient genotyping design, heterozygote melting curve pre- diction, and quality control of melting curve experiments. The application was developed in Actionscript and can be