Masking selected sequence variation by incorporating mismatches into melting analysis probes

Hybridization probe melting analysis can be complicated by the presence of sequence variations (benign polymorphisms or other mutations) near the targeted mutation. We investigated the use of 'masking' probes to differentiate alleles with similar probe melting temperatures. Selected sequence variation was masked by incorporating deletions, unmatched nucleotides, or universal bases into hybridization probes. Such masking probes create a probe:target mismatch with all possible alleles at the selected polymorphic location. Any allele with additional variation at another site is identified by a lower probe melting temperature than alleles that vary only at the masked position. This technique was applied to RET and HPA6 mutation detection using unlabeled hybridization probes, a saturating dsDNA dye and high-resolution melting analysis.