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

摘要

Background: 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).Methods: Engineered plasmids were used to study all possible SNP base changes. In addition, clinical protocols for factor V (Leiden) 1691GA, prothrombin 20210GA, methylenetetrahydrofolate reductase (MTHFR) 1298AC, hemochromatosis (HFE) 187CG, and β-globin (hemoglobin S) 17AT 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 ms) that differ by 0.8–1.4 °C. However, in ～16% 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. This approach was used for the HFE 187CG protocol, but, as predicted from the sequence changes, was not needed for the other four clinical protocols.Conclusions: 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.