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

Abstract

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 Δ H 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 Δ H vH. Using the LightCycler480, the influence of dye (ResoLight) and scanning speed was assessed. The model calculations show that only Δ H 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 °C, while lowering Δ H vH. Using a 4-bp deletion in a 200-bp amplicon as a model, the miscalling rate improved substantially, when using Δ H 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, Δ H 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.