(hybridization probes, HyProbe, adjacent probes, anchor & reporter probes, donor-acceptor probes or kissing probes) A pair of fluorescent probes placed in close proximity as in LightCycler (hybridizing) probes. Maximum fluorescence occurs with a one base separation between probes. The emission spectrum of one fluorophore overlaps significantly with the excitation spectrum of the other fluorophore. During FRET, the donor fluorophore is excited by a light source from the real-time PCR instrument, and transfers its energy to an acceptor fluorophore when positioned in the direct vicinity of the former. The acceptor fluorophore emits light of a longer wavelength, which is detected in specific channels by the instrument. The light source cannot excite the acceptor dye. The donor probe is labeled with fluorophore at the 3′ end and the acceptor probe at 5′ end (its 3′-end blocked to prevent extension). See an overview by Premier Biosoft, a presentation by BioChem, and a report on genotyping on LightCycler with FRET probes.