Heterozygote PCR Product Melting Curve Prediction

Abstract

Melting curve prediction of PCR products is limited to perfectly complementary strands. Multiple domains are calculated by recursive nearest neighbor thermodynamics. However, the melting curve of an amplicon containing a heterozygous single-nucleotide variant (SNV) after PCR is the composite of four duplexes: two matched homoduplexes and two mismatched heteroduplexes. To better predict the shape of composite heterozygote 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 (depending on SNV class) and averaged 88 ± 16.4%. Based on uMelt (www.dna.utah.edu/umelt/umelt.html) with an expanded nearest neighbor thermodynamic set that includes mismatched base pairs, uMelt HETS calculates helicity as a function of temperature for homoduplex and heteroduplex products, as well as the composite curve expected from heterozygotes. It is an interactive Web tool for efficient genotyping design, heterozygote melting curve prediction, and quality control of melting curve experiments. The application was developed in Actionscript and can be found online at http://www.dna.utah.edu/hets/.

Identification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by righ-resolution melting analysis

“…It may also identify and discriminate various infectious agents by melting curves [16–17] because amplicons of different sequences may be distinguished on the basis of the curve shape even when they share the same melting temperature (Tm). In this technique, the use of saturation dyes enables detection of Tm differences as small as 0.01°C during the acquisition of fluorescence [18].…”

Assessment of high resolution melting analysis as a potential SNP genotyping technique in forensic casework

“…Two SNPs (rs1800407 and rs16891982) proved difficult to genotype consistently. The melting profile produced for SNP rs1800407 has more melting domains (peaks) than expected from in silico prediction tools such as uMelt HETS (Supporting Information Fig. 2).…”

“…There are a number of useful in silico tools available for the prediction of high‐resolution melting curves (e.g. uMelt and uMelt HETS ). However, we note that while uMelt HETS predicted small temperature differences for the alternate genotypes of the symmetrical SNP rs16891982, it was unable to predict the complex melting transitions that we observed for SNP rs1800407.…”

“…uMelt and uMelt HETS ). However, we note that while uMelt HETS predicted small temperature differences for the alternate genotypes of the symmetrical SNP rs16891982, it was unable to predict the complex melting transitions that we observed for SNP rs1800407. Therefore, SNPs with these characteristics could be avoided during SNP selection when designing HRM SNP assays (e.g.…”

COLD-HRM: A Combination of Methods to Infer the Nature of Somatic Mutations

“…It is possible to calculate the Tc using software which can predict the melting temperature of hetero duplexes therefore removing the need to individually test each target. 30 Another caveat is that this method depends on the presence of a minor allele. In most cases, a mutant allele will be the minor allele due to the presence of stroma within a tumour.…”