High-resolution melt analysis for SNP discovery, linkage mapping and analysis of DNA methylation

High Resolution Melt analysis (HRM) is a closed-tube method of genotyping that does not require use of fluorescent probes, fragment fractionation or amplicon sequence information. Recent advancements in florescent-detection instruments (such as the Corbett Rotor-Gene 6000) and the use of fully saturating intercalating dyes have made HRM analysis considerably more sensitive. The flexibility of the system allows it to be adapted for a wide range of uses including SNP genotyping, mutation detection, screening for loss of heterozygosity, DNA fingerprinting, characterization of haplotype blocks, species classification, somatically acquired mutations studies, linkage and physical mapping, and DNA methylation analysis. Here, we describe the first use of high-resolution melt analysis to generate STS markers based on Single Nucleotide Polymorphisms (SNPs) and microsatellite length polymorphisms for use in linkage mapping, using white lupin (Lupinus albus, x = 25) as a case study. The described strategy is rapid and low-cost, and circumvents the need for labeled primers or amplicon fractionation. We also illustrate the use of HRM analysis for the detection and/or quantification of the presence of, and relative abundance of, methylated nucleic acid bases within the double-stranded molecule without any prior chemical modification of the target DNA.