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		The Real-Time TaqMan PCR and Applications in Veterinary Medicine



		· Introduction · From PacMan to TaqMan - a computer game revisited · The advantages of real-time TaqMan PCR over conventional quantitative PCR · Applications in Veterinary Medicine · Allelic discrimination · Discussion · The veterinarian and his relationship with the next-generation PCR technology · Acknowledgements · References
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From PacMan to TaqMan - a computer game revisited
The TaqMan process is based on the PacMan principle - a computer game introduced more than twenty years ago. Who remembers it? PacMan, a fictitious character, was moved with the aid of a joystick through a labyrinth containing thousands of tiny blue ghosts which at the same time had to be caught to add points to the player's score. The TaqMan probe follows the same principle. Continuing the analogy, PacMan is represented by the enzyme Taq DNA polymerase. The internal TaqMan probe has two fluorescent tags and is analogous to the ‘target' of the PacMan, thus theTaqMan probe is 'eaten up' by Taq DNA polymerase, causing release of the fluorescence which is coupled to the probe (Figure 1).

Fig.1 (Flash plug-in required)
An Animation of the TaqMan 5'-3' nuclease assay. PCR primers 1 and 2 and a TaqMan probe, labelled with a reporter dye, FAM, (R) and a quencher dye, TAMRA, (Q), bind to the DNA template. The 3' phosphate group (P) prevents extension of the TaqMan probe. The presence of the enzyme, Taq polymerase, enables extension of the primer which displaces the TaqMan probe. The displaced probe is cleaved by Taq DNA polymerase resulting in an increase in relative fluorescence of the reporter. Polymerisation is now complete.

Briefly, the method is based on the 5'-3' exonuclease activity of the Taq DNA polymerase, which results in cleavage of fluorescent dye-labelled probes during PCR; the intensity of fluorescence is then measured by a Sequence Detection System (SDS). The TaqMan probe is located between the two PCR primers and has a melting temperature 10°C higher than that of the primers: binding of the TaqMan probe prior to the primers is crucial because without it PCR products would be formed without generation of fluorescence intensity and thus without being detected. The TaqMan probe has two fluorescent tags attached to it. One is a reporter dye, such as 6-carboxyfluorescein (FAM), which has its emission spectra quenched due to the spatial proximity of a second fluorescent dye, 6-carboxy-tetramethyl-rhodamine (TAMRA). Degradation of the TaqMan probe, by the Taq DNA polymerase, frees the reporter dye from the quenching activity of TAMRA and thus the fluorescent activity increases with an increase in cleavage of the probe, which is proportional to the amount of PCR product formed (Figure 2). The ABI Prism 7700 is a laser-coupled spectrophotometer which monitors the position of the 96-well microtitre plate, 8 times per minute. Most of the data are stored in a true real-time determination and at the end of 40 cycles all the data for quantitative analysis are stored in a SDS file. A positive TaqMan PCR result may be visualised by at least two means (Figure 2 and 3). The amplification plot reflects the generation of the reporter dye during amplification and is directly related to the formation of PCR products (Figure 2).

Fig.2 Positive results from a TaqMan PCR are visualised by at least two means: the amplification plot reflects the generation of the reporter dye during amplification and is related directly to the formation of PCR products. The intersection between the amplification plot and the threshold is defined as the cycle threshold, or CT, value. The CT value is related directly to the amount of PCR product and, therefore, related to the original amount of target present in the PCR reaction.