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

TLDR

High‑resolution melting of PCR amplicons with the DNA dye LCGreen I is a homogeneous, closed‑tube genotyping method that does not require probes or real‑time PCR, and while most human SNPs (≈84 %) produce distinct melting temperatures, about 16 % have minimal T(m) differences that make homozygote discrimination difficult. The authors adapted the LCGreen I system to genotype SNPs by performing rapid‑cycle PCR (≤12 min) of small (≤50 bp) amplicons, adding the dye before amplification, and then obtaining high‑resolution melting curves within 2 min, allowing heterozygotes to be distinguished by heteroduplex‑induced curve shape changes; engineered plasmids and clinical protocols for several disease‑associated SNPs were also developed. The method successfully genotyped most SNPs, but for about 4 % of cases with nearest‑neighbor symmetry the homozygotes could not be resolved; adding 15 % of a known homozygous sample enabled separation of all three genotypes, a strategy applied to the HFE 187C>G protocol but unnecessary for the other four, and overall the assay proved simple, rapid (<2 min post‑PCR), inexpensive, and probe‑free.

Abstract

High-resolution melting of PCR amplicons with the DNA dye LCGreen 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).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 beta-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.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 degrees C. However, in approximately 16% of SNPs, the bases only switch strands and preserve the base pair, producing very small T(m) differences between homozygotes (<0.4 degrees 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. This approach was used for the HFE 187C>G protocol, but, as predicted from the sequence changes, was not needed for the other four clinical protocols.SNP genotyping by high-resolution melting analysis is simple, rapid, and inexpensive, requiring only PCR, a DNA dye, and melting instrumentation. The method is closed-tube, performed without probes or real-time PCR, and can be completed in less than 2 min after completion of PCR.