Single nucleotide discrimination requires a match between the primary probe (1691A or 1691G) and the target sequence at the base to be discriminated, as well as invasion of at least one base by the Invader probe. (A), formation of overlapping invasive complex between 1691A probe and 1691A template, releasing the 5′ flap, which in turn serves as an invader probe in the secondary reaction, producing fluorescence. (B), the hybridization of 1691G probe to 1691A template does not generate the invasive structure recognized by the Cleavase VIII enzyme; therefore, cleavage of the primary probe does not occur. Mut, mutant; WT, wild type; F, fluorescein; Q, quencher