Hydrolysis probes are fluorescently labeled DNA oligonucleotides that are designed to bind downstream of one of the primers during PCR

Hydrolysis probes are fluorescently labeled DNA oligonucleotides that are designed to bind downstream of one of the primers during PCR. The 5′-end of probe is labeled with a fluorescent reporter molecule while the 3′- end is labeled with a quencher molecule (66). Their mechanism is based on the 5′- to 3′- exonuclease activity of the Taq polymerase that degrades the bound probe during amplification. The cleavage of the probe separates the reporter molecule from the rest of the probe allowing the reporter molecule to fluoresce. When the reporter and quencher are in close proximity to one another fluorescence is suppressed (70). The hydrolysis probe is designed to increase specificity; it binds to a specific region of the DNA target (64). These probes reduce the background fluorescence and have larger dynamic range because of the efficiency of reporter quenching (66)

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