Fig. 2.

Mediator probe PCR cycle with mediator probe sequence interactions (dotted ovals). The mediator probe (MP) and universal reporter (UR) with corresponding sequence modifications are presented in the box. The different steps of MP PCR are given below (1–5). In the presence of its complementary target DNA sequence, MP anneals to the sequence (1) and is subsequently cleaved during primer extension (2 + 3). The mediator is set free to hybridize to the UR mediator hybridization site (4). Through the subsequent extension of the mediator at UR, the fluorophore and quencher are separated and a fluorescence signal is generated (5). If no target DNA or an abundant amount (e.g., initial PCR cycles) of target DNA is present, the uncleaved mediator probe hybridizes to UR but is not extended. (Modified from [14]).