Genotyping of Single-Nucleotide Polymorphisms by High-Resolution Melting of Small Amplicons

Abstract

Background: High-resolution melting of PCR amplicons with the DNA dye LCGreen TM I was recently introduced as a homogeneous, closed-tube method of genotyping that does not require probes or real-time PCR. We adapted this system to genotype single-nucleotide polymorphisms (SNPs) after rapid-cycle PCR (12 min) of small amplicons (<50 bp). Methods: Engineered plasmids were used to study all possible SNP base changes. In addition, clinical protocols for factor V (Leiden) 1691G>A, prothrombin 20210G>A, methylenetetrahydrofolate reductase (MTHFR) 1298A>C, hemochromatosis (HFE) 187C>G, and ␤-globin (hemoglobin S) 17A>T were developed. LCGreen I was included in the reaction mixture before PCR, and high-resolution melting was obtained within 2 min after amplification. Results: In all cases, heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves. Approximately 84% of human SNPs involve a base exchange between A::T and G::C base pairs, and the homozygotes are easily genotyped by melting temperatures (T m s) that differ by 0.8 -1.4°C. However, in ϳ16% of SNPs, the bases only switch strands and preserve the base pair, producing very small T m differences between homozygotes (<0.4°C). Although most of these cases can be genotyped by T m , one-fourth (4% of total SNPs) show nearest-neighbor symmetry, and, as predicted, the homozygotes cannot be resolved from each other. In these cases, adding 15% of a known homozygous genotype to unknown samples allows melting curve separation of all three genotypes.

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“…HRM a post-PCR technique proved to be very simple, fast and cost-effective technique for screening samples with unknown mutations or SNPs on a large scale with rapid turn-around time requiring just PCR, DNA dye and melting instrument. It can reduce unnecessary load of sequencing of samples [22]. It is a closed technique so very less chances of contamination and also it is non-destructive so the same sample can be used to analyse further.…”

“…Smaller amplicons have better sensitivity as they give better differentiation. The error rate depends upon length of amplicon and type of base change but not on the position of base change [26,27]. DNA contamination was thought to be the reason for showing false positive results in study.…”

Molecular Characterization of Jatropha curcas Resources and Identification of Population-Specific Markers

“…The single nucleotide polymorphism (SNP) marker is recognized as a powerful marker system, and its polymorphism can be analyzed rapidly with high-resolution melting (HRM) analysis [17]. Taking advantage of the thermodynamic variations of the amplicons, genetic variants with differences in base composition can be detected by their characteristic melting curves [21].…”

Co-amplification at Lower Denaturation-temperature PCR Combined with Unlabled-probe High-resolution Melting to Detect KRAS Codon 12 and 13 Mutations in Plasma-circulating DNA of Pancreatic Adenocarcinoma Cases

“…Next, the unlabeled probe designed complementary to the wild-type sequence could detect the potential existence rather than the types of KRAS mutations. In addition, the most common types of KRAS mutations in PA were found to be 35G>A (46%) and 35G>T (32%) with a decreased Tm (Liew et al, 2004), while some type of G>C mutation could be missed in spite of the use of the fast COLD-PCR step.…”