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—the fluorescence is measured by the real-time instrument.
These steps are repeated for each PCR cycle. When using intercalating dyes, such as SYBR Green I (Thermo Fisher Scientific), primer-dimers and non-specific products will also contribute to fluorescence. In contrast, the 5′ nuclease assay is specific and fluorescence will only be produced for the DNA sequence to which the probe and primers hybridize.