DNA Concentration Can Specify DNA Melting Point in a High-Resolution Melting Analysis Master Mix

Abstract

To the Editor: High-resolution melting analysis (HRMA)1 can be used to discriminate sequence variants of PCR-amplified DNA fragments (1). We attempted to develop an HRMA-based procedure to discriminate multilocus sequence typing (MLST) (2) alleles from a divergent lineage of Staphylococcus aureus (3) using a Corbett Rotorgene 6000. In the case of the 573-bp glycerol uptake facilitator (glpF) fragment, there were 4 known alleles in our culture collection. Allele discrimination performed with the nonsaturating dye SYBR Green was not reliable. We therefore tested the saturating dye LCGreenPlus (Biofire Diagnostics) (4). Initial experiments revealed very poor allele discrimination due to lack of correlation between the melting temperature ( T m) and allele identity/allele percentage of guanine plus cytosine (%G+C) content. Here we report an investigation of the basis for this poor performance. Our initial results with LCGreenPlus suggested that the T m was primarily a function of the PCR yield rather than the allele sequence. This was tested by subjecting a glpF allele 321 sample to PCR in LCGreenPlus master mix, making a dilution series of the reaction products in the same master mix, and then subjecting the dilutions to HRMA. As predicted, the T m values in both melting domains were inversely correlated with DNA concentration (Fig. 1 …