Single-nucleotide polymorphism discrimination using high-resolution melting analysis for the genotyping of Bacillus anthracis

Abstract

High-resolution melting (HRM) is a post-PCR technique that determines with high precision the melt profile of PCR products using a new generation of double-stranded DNA-binding dyes and accurate fluorescence data acquisition over small temperature increments. The method can be used to interrogate small sets of single-nucleotide polymorphisms (SNPs). Here, we describe a simple and cost-effective HRM-based method for the screening of 14 phylogenetically informative SNPs within the genome of Bacillus anthracis that subtype the species into 13 major sublineages or subgroups. Fourteen monoplex and seven duplex SNP-discrimination assays have been designed. We detail the parameters most important for the successful application of HRM for B. anthracis genotyping.