from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

In contrast to hydrolysis probes, the fluorescence from hybridization probes is reversible and depends only on probe hybridization. The first hybridization probes used in real-time PCR were dual hybridization probes consisting of two oligonucleotides, one labeled at the 3'-end the other at the 5'-end. Upon hybridization to their complementary sequences and fluorescent excitation, FRET increases. Signal generation with dual hybridization probes requires annealing of four oligonucleotides (two primers and two probes), suggesting even better specificity than hydrolysis probes. Later, single hybridization probe designs were developed, including FRET between an internally labeled primer and a single-labeled probe and deoxyguanosine quenching of a single-labeled probe (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). In contrast to hydrolysis probes that are consumed during amplification, the fluorescence of hybridization probes is reversible, enabling melting analysis. The first FDA-approved genetic tests in the US (F5 and F2 single base variants) used dual hybridization probes and melting analysis for genotyping.