Quadruplex genotyping of F5, F2, and MTHFR variants in a single closed tube by high-resolution amplicon melting.

BACKGROUND Multiplexed amplicon melting is a closed-tube method for genotyping that does not require probes, real-time analysis, asymmetric PCR, or allele-specific PCR; however, correct differentiation of homozygous mutant and wild-type samples by melting temperature (T(m)) analysis requires high-resolution melting analysis and controlled reaction conditions. METHODS We designed 4 amplicons bracketing the F5 [coagulation factor V (proaccelerin, labile factor)] 1691G>A, MTHFR (NADPH) 1298A>C, MTHFR 677C>T, and F2 [coagulation factor II (thrombin)] 20210G>A gene variants to melt at different temperatures by varying amplicon length and adding GC- or AT-rich 5' tails to selected primers. We used rapid-cycle PCRs with cycles of 19-23 s in the presence of a saturating DNA dye and temperature-correction controls and then conducted a high-resolution melting analysis. Heterozygotes were identified at each locus by curve shape, and homozygous genotypes were assigned by T(m). We blinded samples previously genotyped by other methods before analysis with the multiplex melting assay (n = 110). RESULTS All samples were correctly genotyped with the exception of 7 MTHFR 1298 samples with atypical melting profiles that could not be assigned. Sequencing revealed that these 5 heterozygotes and 2 homozygotes contained the unexpected sequence variant MTHFR 1317T>C. The use of temperature-correction controls decreased the T(m) SD within homozygotes by a mean of 38%. CONCLUSION Rapid-cycle PCR with high-resolution melting analysis allows simple and accurate multiplex genotyping to at least a factor of 4.