Step 1: The primers and probe hybridize in a sequence-dependent manner to the complementary DNA strand. Because the probe is intact, the fluorophore and quencher are in close proximity, and the quencher absorbs fluorescence emitted by the fluorophore.
Step 2: The polymerase extends from the primers and begins DNA synthesis.
Step 3: The polymerase reaches the probe, and the exonuclease activity of the polymerase cleaves the hybridized probe. As a result of cleavage, the fluorophore is separated from the quencher and fluoresces.
Step 4: Nucleotide extension is completed, and the emitted fluorescence is detected by the real-time instrument.
These steps are repeated for each PCR cycle and allow detection of specific products, because fluorescence is only detected for the DNA sequence to which the probe and primers hybridize.