Influencing factors of dsDNA dye (high-resolution) melting curves and improved genotype call based on thermodynamic considerations.

High-resolution melting of dsDNA using suitable dyes is a simple and cost-effective alternative for mutation scanning. Analytical variation can result from salt and template concentration (C(T)). To overcome this problem the van't Hoff transition enthalpy DeltaH(vH) from dsDNA melting curves was estimated and used for robust genotype calling or mutation scanning. Model calculations show the effect of salt, C(T), and temperature resolution on (1) T(m), (2) difference plots, (3) melting peaks, and (4) calculated DeltaH(vH). Using the LightCycler480, the influence of dye (ResoLight) and scanning speed was assessed. The model calculations show that only DeltaH(vH) is not influenced by salt and C(T). Higher amplicon enthalpy ameliorates the ability to discriminate mutations. Temperature resolution is important for peak- but not for curve-based genotyping. ResoLight increases T(m) by 3.4 degrees C, while lowering DeltaH(vH). Using a 4-bp deletion in a 200-bp amplicon as a model, the miscalling rate improved substantially, when using DeltaH(vH) instead of difference plots. Melting curves of duplex DNA are influenced by dye and salt and less so by duplex concentrations. As predicted from theory, DeltaH(vH) is a robust measure for mutation detection in two-state melting. The influence of dyes on enthalpy is of general impact for PCR assays.