In 1991, Holland and colleagues at the Cetus Corporation used the 5' to 3' exonuclease activity of Taq polymerase to detect amplification products post-PCR. An oligonucleotide probe complementary to the PCR product was used with a non-extendable 3'-end and a radioactively labeled 5'-end. During amplification the polymerase degraded the probe, releasing the radioactive label as smaller fragments of the probe. However, a post-PCR radiograph was required in order to visualize the degraded probe. By replacing the radioactive label with two fluorescent labels in a FRET relationship, successful allele discrimination and later real-time monitoring were achieved. These dual-labeled fluorescent probes were hydrolyzed by the 5' to 3' exonuclease activity of Taq during PCR, separating the fluorescent labels with a loss of FRET to generate fluorescence. Specificity was enhanced over dsDNA dyes because complementation to three independent oligonucleotides (two primers and one probe) was necessary for probe hydrolysis and signal generation. Hydrolysis probes (also known by the trademark TaqMan, among others) are the most commonly used probes today (Wittwer and Farrar, 211 in PCR Troubleshooting and Optimization). Their popularity was advanced by simplified design and a strong commercial effort to provide synthesis services. Signal generation is produced by probe hydrolysis and is irreversible and cumulative.
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