Application of High-Resolution Melting to Large-Scale, High-Throughput SNP Genotyping: A Comparison with the TaqMan® Method

Abstract

IntroductIon Single-Nucleotide Polymorphisms (snps) are the most common dna variants in the human genome, accounting for about 1/1000 bp changes. more or less, 65% of the substitutions are transitions, equally represented by a/g and c/t mutations, whereas 35% are transversions, and all the a/c, a/t, c/g, and g/t variants have the same frequency.1,2 today, snps are widely investigated in several fields-that is, as prognostic markers in pharmacogenetics, in studies on susceptibility to cancer and other complex diseases, as genetic markers (tagsnps) in genome-wide scans, and in many diagnostic protocols. associated with their high frequencies, the increasing popularity of snps is due to the availability of large-scale, high-throughput genotyping methods that have allowed the description of more than 7 million common variants worldwide. 2 to date, many real-time pcr-based approaches have been used for snp-typing in large-scale studies. one of the most commonly applied is the taqman ® method, also known as the 5′ exonuclease assay, which makes use of 2 fluorescently labeled probes for allelic discrimination. the presence of allele-specific probes guarantees a high sensitivity, offering appreciable advantages in terms of time and labor savings. other classical pcr-based genotyping techniques, such as the allele-specific oligonucleotide pcr (aso-pcr) assay or the restriction fragment length polymorphism pcr (rFlp-pcr) assay, still remain the most affordable for low-budget studies. high-resolution melting (hrm) analysis was first introduced in 1997 by ririe et al. 3 as the natural extension of a realtime monitoring process of pcr reactions. in brief, melting profiles of pcr fragments describe the amount of doublestranded dna that dissociates into single-stranded dna during incubation at increasing temperatures, monitored by measuring the fluorescence emitted by an intercalating dye. in particular, melting temperature (t m ) is defined as the temperature at which half of the dna amount is denatured. the quantity of information available from melting analyses has exponentially increased with the introduction of highly saturating intercalating dyes such as lcgreen (idaho technology, inc., salt lake city, ut) or evagreen (Biotium, inc., hayward, ca), which can be added at high concentration without inhibiting pcr reactions.during the past few years, hrm has been successfully applied to mutation detection, presequence scanning, methylation studies, and snp-typing. Because of the wide use of single-nucleotide polymorphisms (snps) as markers of genetic variation, several high-throughput genotyping methods have been developed and applied during the past decades. high-resolution melting (hrm) is a very attractive, advanced, fast, and cost-effective snp genotyping technology based on the analysis of the melting profile of pcr products, using intercalating fluorescent dyes to monitor the transition from unmelted to melted dna. the authors used hrm for genotyping 215 human dna samples for snps in the aBcB1, nQo1, and slc19a1 genes ...

Rapid Genotyping of Shigella sonnei by Use of Multiplex High-Resolution Melting

“…A typing scheme based on high-resolution melting (HRM) of six chromosomal single nucleotide polymorphisms (SNPs) has been described to identify all lineages/ sublineages (4). HRM detects SNPs in small DNA fragments based on the melting patterns (5,6). HRM involves minimal labor, with a rapid turnaround time and a decreased risk of PCR carryover contamination (7).…”

A high-resolution melt (HRM) assay to characterize CYP51 haplotypes of the wheat pathogen Mycosphaerella graminicola

“…The sensitivity of our assay was 100% while the overall accuracy was 96.9%. Especially in large-scale studies, sensitivity and accuracy around 95% can be considered adequate, with variation in this range having negligible effects on the results of the study (Martino et al, 2010). New mutations can be detected using this method, when aberrant melting curves are found.…”

Mutation Detection by Analysis of DNA Heteroduplexes in TILLING Populations of Diploid Species

“…a SNP, is easily detected because of the formation of the heteroduplexes for which an altered melting profile is observed. Because heteroduplexes are less stable than homoduplexes, a loss of fluorescence is observed at lower temperatures than for homoduplexes (Ishikawa et al 2010;Hondow et al 2011;Martino et al 2010). Differences between homozygous samples can be observed as a shift in melting temperature, whereas changes in the shape of the melting curves (which are composites of the homo-and heteroduplex) demonstrate different heterozygous samples (Gundry et al 2003).…”