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

After the introduction of hydrolysis and hybridization probes, several other probe designs were adapted to, or created for, real-time PCR. Some of these probes increase in fluorescence when their conformation changes with hybridization and may function by hybridization and/or hydrolysis mechanisms depending on the reaction conditions. Hairpin probes, or "molecular beacons" can be monitored in real-time. These probes have a central sequence complementary to the DNA target and flanking ends complementary to each other. This configuration creates a hairpin at low temperatures. At higher temperatures in the presence of target, the probe hybridizes preferentially to the target. One end of the probe is labeled with a fluorophore and the other with a quencher so that when hybridized to the target, fluorescence increases. Hairpin probes use quenchers that release transferred energy as heat rather than light (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Hairpins can also be attached to the 5'-end of a PCR primer to generate self-probing amplicons during PCR. A fluorophore/quencher pair in the hairpin stem is linked to the primer with a blocking agent that prevents PCR read-through. The loop of the hairpin is complementary to the extension product of the primer so that once extension occurs, intramolecular hybridization separates the fluorophore/quencher pair and a larger hairpin is formed. This intra-molecular hybridization of self-probing amplicons is faster than the intermolecular hybridization of other probes. Hairpin primers can also be made without a blocking agent, allowing PCR read-through and incorporation of the stem-loop into the product. During PCR, the quencher/reporter pair is separated and fluorescence increases.