Nuclease Probes

"5'-Nuclease probe" (or TaqMan® probe) assays utilize the inherent endo-nucleolytic activity of Taq DNA polymerase to generate a fluorescence signal (4). 5'-Nuclease probes are single-stranded hybridization probes labeled with a donor-acceptor fluorophore pair that interact via FRET (see Fig. 1B). The acceptor molecule can also be a nonfluorescent quencher molecule. Probes that are free in solution form "random coils," in which the fluorophore is often close to the quencher molecule, enabling energy transfer from the donor fluorophore to the acceptor molecule, resulting in a low fluorescence signal from the donor fluorophore. The probe is designed to hybridize to its target DNA strand at the same time as the polymerase chain reaction primer. When Taq DNA polymerase extends the primer, it encounters the probe, and as a result of its 5'-nuclease activity, it cleaves the probe. Cleavage of the probe results in the separation of the donor fluorophore and acceptor molecule, and leads to an increase in the intensity of the fluorescence signal from the donor fluorophore, because FRET can no longer take place. With each cycle of

Fig. 1. Schematic overview of energy transfer and fluorescence signal generation in fluorescent hybridization probes.

amplification, a new round of hybridization occurs and additional fluorophores are cleaved from their probes, resulting in higher fluorescence signals, indicating the accumulation of target DNA molecules.

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