Impact of Mutation Type and Amplicon Characteristics on Genetic Diversity Measures Generated Using a High-Resolution Melting Diversity Assay

We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric FIRM score. Herein, we determined the impact of mutation types and amplicon characteristics on FIRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion < 1-bp change < 3-bp insertion < 9-bp insertion). The impact of mutations on FIRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impart at 50%. The G/C content altered melting temperature values of amplicons but had no impact on FIRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology. (J Mol Diagn 2013, 15:130-137; http://dx.doi.org/10.1016/j.jmoldx.2012.08.008)