Step 1: During the annealing step, hybridization of molecular beacon probes to target sequences separate the fluorescent dye (D) and quencher (Q), resulting in fluorescence that is detected by the real-time PCR instrument. Probes that do not hybridize to target DNA sequences will reform the hairpin structure and will not fluoresce.
Step 2: The polymerase extends from the primers and begins DNA synthesis.
Step 3: When the polymerase reaches the molecular beacon, the probe is displaced without being degraded. Therefore, molecular beacons can participate in multiplex rounds of annealing.
Step 4: The polymerase continues extension of the primers to complete synthesis of the DNA strand.