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The iCycler iQ Detection System for TaqMan Assays, Rev B

Faye Boeckman, Keith Hamby, and Larissa Tan, Bio-Rad Laboratories,
2000 Alfred Nobel Drive, Hercules, CA 94547 USA


Introduction
Specific fluorescent oligonucleotide probes allow for real-time monitoring of the polymerase chain reaction (PCR*). One popular probe strategy is the TaqMan assay (PE Biosystems), which capitalizes on the 5' exonuclease activity of Taq polymerase to cleave a labeled hybridization probe during the extension phase of PCR (Holland et al. 1991). In a fluorescent TaqMan assay, the probe is labeled at the 5' end with a fluorescent reporter molecule such as fluorescein and at the 3' end with another fluorescent molecule, usually a tetramethyl- rhodamine derivative, which acts as a quencher for the reporter (Heid et al. 1996). When the two fluorophores are fixed at opposite ends of the 2030 nt probe and the reporter fluorophore is excited by an outside light source, the normal fluorescence of the reporter is absorbed by the nearby quencher, and no reporter fluorescence is detected. When Taq polymerase encounters the bound probe during extension from one of the primers, it digests the probe, freeing the reporter from the quencher, and the reporter fluorescence can be detected and measured.

The fluorescence of the reporter molecule increases as products accumulate with each successive round of amplification. In early cycles of amplification, the change in the reporters fluorescence is usually undetectable, but at some point during amplification, the accumulation of product results in a measurable change in the total fluorescence of the reaction mixture. This point, at which the fluorescence rises appreciably above the background, has been
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