There are two popular probe configurations, TaqMan probe and molecular beacon.
TaqMan probe has reporter dye on a 5’ end of the probe and a quencher on a 3’ end. When a probe is intact, because of its proximity between the reporter dye and the quencher, it has undetectable fluorescent light. During the PCR cycle, at the extension step, the probe binds to a complementary target sequence. When Taq polymerase extends the annealed primers and reaches the probe, the enzyme’s 5’-3’ exonuclease activity will digest the bound probes into an individual nucleotide. The process releases the reporter dye from the quencher quenching effect, and the fluorescent light from reporter dye can be detected.
Molecular Beacon has a similar labeling configuration as TaqMan—a 5’ end has a reporter dye and a 3’ end has a quencher. The difference between a molecular beacon and a TaqMan probe is that a molecular beacon has a stem-loop structure, as shown below:
The loop sequence is a 18-30 base pair long and complementary to the target sequence. The stem is formed by both ends of the loop, which have 5-7 base pair complementary sequences. With such a structure, the quencher can prevent fluorescent dye from emitting light.
In contrast to the TaqMan probe, a molecular beacon releases fluorescent light during the annealing step of the PCR. During annealing step, the loop sequence of the probe will bind to target the sequence, and the probe is unfolded to a linear form. Such a change stretches the distance between the reporter dye and the quencher and the molecular beacon fluoresce.
[Insert molecular beacon animation: Beacon_chemistry.swf]
Real-time PCR has a wide range of applications and they include:
