PrimeTime™ Dual-labeled DNA Probes

Real-time systems for PCR were improved by probe-based PCR product detection. The 5' nuclease assay provides a real-time method for detecting only specific amplification products. During amplification, annealing of the probe to its target sequence generates a substrate that is cleaved by the 5' nuclease activity of Taq polymerase when the enzyme extends from an upstream primer into the region of the probe. This dependence on polymerization ensures that cleavage of the probe occurs only if the target sequence is being amplified.

The probe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. While the probe is intact, the proximity of the quencher greatly reduces the fluorescence emitted by the reporter dye by Fluorescence Resonance Energy Transfer (FRET).

Fluorescence Resonance Energy Transfer (FRET) is a process where energy from an excited fluorophore is transferred to a neighboring acceptor molecule without the release of light by the fluorophore (quenching). In FRET, the efficiency of this energy transfer process is dependent upon the physical distance between the reporter fluorophore and the quencher and the degree of overlap between reporter emission and quencher absorption spectra. When reporter and quencher are close, quenching is efficient and the oligo is dark. When reporter and quencher are distant, quenching is reduced or eliminated and the oligo is bright. The range over which quenching occurs is unique for each reporter/quencher pair but is commonly between 30 and 70 Å (the distance where quenching is 50% efficient is defined as the Förster radius for that R/Q pair). The quencher (energy acceptor) molecule can be another fluorescent dye such as TAMRATM or a non-fluorescent dark quencher.

Dark Quenchers Provide Superior Performance

• Probes incorporating dark quenchers have lower background fluorescence, providing greater sensitivity.

• Since dark quenchers absorb broadly and do not emit light, multiple reporter dyes can be used with a given quencher, expanding the options available for multiplex assays.

• Dark quenchers simplify detection, making them compatible with a broad range of image analysis instruments.

Absorbance Range of Dark Quenchers

Advantages of fluorescent probes in real-time PCR

The advantage of fluorogenic probes over DNA binding dyes is that specific hybridization between probe and target is required to generate fluorescent signal. Thus, with fluorogenic probes, non-specific amplification due to mis-priming or primer-dimer artifact does not generate signal. Another advantage of fluorogenic probes is that they can be labeled with different, distinguishable reporter dyes. By using probes labeled with different reporters, amplification of two distinct sequences can be detected in a single PCR reaction.

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