FRET (Förster Resonance Energy Transfer) works with two differently labeled probes. The first oligonucleotide is labeled at the 3'-end (usually with fluorescein) and the second oligonucleotide is labeled at the 5'-end with a FRET acceptor (e. g. Cyanine 5 or TAMRA). The first oligonucleotide hybridizes to the target in such a way that its 3'-end is separated from the 5'-end of the second oligonucleotide by no more than 1 base. When no complementary sequence is available, only the fluorescence of the donor is visible. If the target is present, the labeled probes will hybridize with the target and FRET can occur. The fluorescence of the acceptor is mediated by the quencher that emits fluorescence at a longer wavelength than that of the acceptor. The fluorescence intensity is proportional to the amount of PCR product formed during the early exponential phase of PCR (threshold value). The 3'-ends of both probes have to be protected against chain elongation during PCR.