Real-time PCR is based on the detection of a fluorescent signal produced proportionally during amplification of a PCR. The TaqMan© Probe is an oligonucleotide design with a high-energy dye termed a Reporter at the 5 end, and a low-energy molecular termed a Quencher at the 3 end. When this probe is intact and excited by a light source, the Reporter dyes emission is suppressed by the Quencher dye as a result of the close proximity of the dyes.

1. A TaqMan© Probe is added to the PCR reagent master mix. The probe is designed to anneal to a specific sequence of template between the forward and reverse primers.
2. During DNA amplification, Taq polymerase adds bases to a growing chain of DNA and removes DNA that is downstream, impeding its capability to synthesize the new strand.
3. As the TaqMan© Probe sits in the path of the enzymes, when the enzyme reaches the annealed probe the 5 exonuclease activity of the enzyme cleaves the probe.
4. The distance between the Reporter and the Quencher increases causing the fluorescent emissions of the Reporter increase and the Quencher decrease.
   The increase in Reporter signal is captured by the Sequence Detection instrument and display by the software.
5. An increase in the Reporter signal over time is recorded. The amount of Reporter signal increase is proportional to the amount of product being produced for a given sample.
6. The number of PCR cycles requires to achieve an arbitrary fluorescence threshold (Ct) provides a measure of the amount of target DNA. The lower the cycle number, the higher the amount of target DNA in sample.

Advantages of Real-time PCR

• Amplification can be monitored in real-time
• Post-amplification detection steps not needed, shorter time to result
• Measuring the kinetics of the reaction in situ in the machine
• Ability to quantify target DNA
• Detection is capable down to a 2-fold change