Figure 1.

Principle of real-time PCR genotyping using displacing probes. A pair of displacing probes is included in the reaction. In the presence of wild-type templates, only Fam-labeled wild-type probes hybridize to the amplicons and generate green fluorescence (fluorescent cross-annealing), whereas the Texas Red-labeled mutant probes retain their duplex and cannot produce red fluorescence (non-fluorescent self-annealing). In the presence of heterozygous templates, both wild-type and mutant probes hybridize to amplicons and generate both green and red fluorescence. In the presence of mutant templates, only the Texas Red-labeled mutant probes hybridize to the amplicons, generating red fluorescence, whereas Fam-labeled wild-type probes remain dark.