Distinguishing Different DNA Heterozygotes by High-Resolution Melting

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Abstract
High-resolution melting was recently introduced as a technique to genotype single-nucleotide polymorphisms (SNPs) within small amplicons (1). This closed-tube method (including rapid-cycle PCR) can be completed in <15 min and does not require real-time PCR instruments (2), allele-specific PCR (3), or fluorescently labeled oligonucleotides (4)(5)(6). The process is made possible by heteroduplex-detecting DNA dyes that can be used at saturating concentrations without inhibiting PCR (7). Wild-type and homozygous mutant samples are distinguished by melting temperature ( T m) shifts. Heterozygous samples are best distinguished from homozygotes, not by T m, but by altered curve shape. Heterozygous samples produce heteroduplexes that melt at lower temperatures than homoduplexes. Melting curves of amplified heterozygotes include 2 homoduplexes and 2 heteroduplexes, giving a skewed composite melting curve easily distinguished from the curves for homozygotes by curve shape (1). However, it was not clear whether different heterozygotes within the same amplicon could be distinguished from each other based on curve shape differences. Four different classes of SNPs have been defined based on the homo- and heteroduplexes that are produced after amplification (1). Because the heteroduplex mismatches of SNPs in different classes are different, their melting curves should be distinguishable if the resolution of the instrumentation is sufficient. It is also possible that different heterozygotes in the same class may be distinguishable. Although the mismatches are the same, nearest-neighbor stability parameters depend on the bases adjacent to the mismatches; therefore, the predicted stabilities are often different. We selected DNA samples retrospectively from those submitted to ARUP Laboratories for standard clinical genotyping of HFE , factor V Leiden, and factor II polymorphisms. DNA was extracted from blood samples by use of the MagNa Pure instrument (Roche) and genotyped by use of adjacent hybridization probe (HybProbe™) methods (4)(5) on a LightCycler®. …