Principle Of The Taqman Assay

Like several eubacterial DNA polymerases, Thermus aquaticus (Taq) polymerase is a bifunctional enzyme consisting of a polymerization domain and a nuclease domain, and it is the latter that is crucial for the TaqMan assay.[1] In essence, the TaqMan assay is identical to a standard PCR reaction powered by Taq polymerase, except for the addition of the TaqMan probe, a nonextendable fluorescently labeled oligonucleotide hy bridizing on the target DNA between the forward and reverse primers[2-3] (Fig. 1A). The TaqMan probe is modified with a fluorescent reporter dye (R) at its 5' end and a fluorescence quencher dye (Q) at its 3' end. Because of the proximity of the two dyes in the intact TaqMan probe, fluorescence resonance energy transfer (FRET) takes place from the reporter dye (FRET donor) to quencher dye (FRET acceptor) (De Angelis, FRET chapter, EDGP, and Ref. [4]). This proximity results in low fluorescence emission from the reporter dye because most of its excited state energy is transferred to the quencher dye. During extension from the forward primer, Taq polymerase encounters the TaqMan probe hybridized to the sequence being amplified. Taq then displaces the first few bases of the TaqMan probe off the template (Fig. 1B), and subsequently cleaves the TaqMan probe with its 5' nuclease activity. While this was initially thought to be a 5'-3' exonuclease, subsequent work showed that it is an endonuclease that cleaves at the juncture between the unhybridized and hybridized portions of the probe.[1,5] This cleavage results in an increased separation of the reporter dye on the 5' end of the TaqMan probe from the acceptor (or quencher) on the 3' end, yielding increased donor fluorescence emission (Fig. 1C). Therefore, when the TaqMan probe hybridizes perfectly to a DNA sample in the PCR reaction, an increase in fluorescence proportional to accumulation of the cleaved TaqMan probe follows every amplification cycle, and this signal is monitored in real time. On the other hand, in the absence of a perfect match, the probe remains intact and there is no increase of fluorescence above background.

The first use of the 5' nuclease activity of Taq specifically designed to cleave a nonextendable, radio-actively labeled oligonucleotide probe positioned between forward and reverse primers in a PCR reaction, was described in 1991.[6] The introduction of duallabeled fluorescent probes[7] was a quantum leap that made the technology easier to use and safer; however, the analysis of fluorescence from accumulated cleaved probe in the sample still required some post-PCR reaction processing. The advent of the ABI PRISMâ„¢ 7700 system allowed continuous monitoring of fluorescence in real time.

111111111111 ffi*

Fig. 1 Schematic depicting three stages of the TaqMan assay. (A) Hybridization: The forward primer (indicated by the arrow) and the TaqMan probe, labeled at the 5' end with the reporter dye (R) and at the 3' end with the quencher dye (Q) are shown annealing to the template DNA. The x on the TaqMan indicates that the probe is nonextendable. Proximity of R and Q yields low fluorescence from R through FRET. (B) Extension: Taq polymerase extends the forward primer and encounters the TaqMan probe. C) Cleavage: Taq polymerase cleaves the TaqMan probe, thus releasing the reporter from the quencher and causing an increase in the fluorescence of the reporter, which is monitored by the RT-PCR instrumentation.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook

Post a comment