| TaqMan* fluorescent PCR is a recently developed technique enabling the measurement of an accumulating PCR product in real-time by utilizing a dual-labelled TaqMan fluorogenic probe. The fluorogenic 5'-nuclease PCR assay utilizes the endogenous 5'-3' nuclease activity of Taq polymerase to digest the TaqMan probe, which hybridizes to the target sequence during PCR. When the probe is intact the reporter fluorophore emission is suppressed by the quencher fluorophore. During PCR, the probe anneals to the DNA template and the 5'-3'-nuclease activity of Taq polymerase releases the reporter from the vicinity of the quencher dye resulting in increased reporter fluorescence. The remaining probe dissociates from the target sequence allowing the polymerase reaction to continue. The intensity of fluorescence is directly related to the amount of input target DNA. The fluorescent signal is captured using the ABI Prism 7000 or 7700 Sequence Detection System (PE Applied Biosystems) allowing rapid screening in a 96-well format. The instrument determines the initial copy number of the target template by analyzing the cycle-to-cycle changes in fluorescence signal as a result of the amplification of template during PCR. The fewer cycles it takes to reach a detectable level of fluorescence the greater the initial copy number. Both primer and probe must hybridize to the target for amplification and cleavage to occur. The fluorescence signal is generated only if the target sequence for the probe is amplified during PCR. Because of these requirements, non-specific amplification is not detected. *TaqMan is a registered trademark of Roche Molecular System, Inc. |
