Figure 5.

Demonstration of mismatched “masking” probes to conceal polymorphisms in probe melting analysis. Two single base variant loci are considered. The locus of interest is a “mutation site”, while the other is a benign variant at the “polymorphism site”. The four different possible genotypes (green/red, solid/dotted lines) are shown hybridized to two different probes (thick grey lines). The probes are either, A) perfectly-matched to wild type (wild-type probe) or B) mismatched at the polymorphism site (masking probe). The filled circles indicate the masking position in the probe that can be a deletion, a mismatched base, or a universal base. The X represents the polymorphic variant base. Mismatched bases are enclosed by rectangles. With a wild-type probe as shown in (A), it is difficult to distinguish the mutation from the polymorphism on a derivative plot (bottom left), because both result in one mismatch. However, the masking probe shown in (B) is always mismatched at the polymorphism site so that the presence (2 mismatches) or absence (1 mismatch) of the mutation is always clear on derivative plots (bottom right).