Masking techniques: masking selected sequence variation by incorporating mismatches into melting analysis probes

Hybridization probe melting analysis can be complicated by the presence of sequence variation (non-pathogenic polymorphisms or other mutations) near the targeted mutation. We investigated the use of 'masking' probes to differentiate alleles with similar probe melting temperatures. Materials and Methods: Selected sequence variation was masked by incorporating deletions, unmatched (non-complementary) 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 proto-oncogene and HPA6 mutation detection using unlabeled hybridization probes, a saturating dsDNA dye, and high-resolution melting analysis.