The TaqMan® Probe is designed with a high-energy dye termed a Reporter at the 5 end, and a low-energy molecule termed a Quencher at the 3 end. When this probe is intact and excited by a light source, the Reporter dye’s emission is suppressed by the Quencher dye as a result of the close proximity of the dyes, Figure 15.
When the probe is cleaved by the 5 nuclease activity of the enzyme, the distance between the Reporter and the Quencher increases causing the transfer of energy to stop. The fluorescent emissions of the reporter increase and the quencher decrease.
Figure 15: Increased florescence activity due to the cleaved probe
The increase in reporter signal is captured by the Sequence Detection instrument and displayed by the software. Figure 16 shows an increase in the reporter signal over time. The amount of reporter signal increase is proportional to the amount of product being produced for a given sample.
Figure 16: Increase in Reporter Signal
The combination of FRET and the 5‵nuclease activity of AmpliTaq Gold® DNA Polymerase enables the 5‵nuclease assay and the SDS instrumentation to collect data in real time. When the fluorescent signal Reporter increases to a detectable level it can be captured and displayed as an Amplification Plot, Figure 17.
Figure 17: Amplification Curve（生物秀——努力给您最好！ www.bbioo.com）
Rn: measure of reporter signal
