Quantitative real-time polymerase chain reaction (PCR)

The mechanism of quantitative real-time PCR, which is based upon a probe that contains a fluorogenic reporter dye at its 5' end and a quencher at the 3' end (shown as a black line with a white box and a Q at its 5' and 3' end, respectively). With this technique, the tagged probe, which is designed to hybridize to one strand of the target DNA sequence (blue line), is added to a normal PCR. The quencher blocks fluorescence emission as long as it is in close proximity to the reporter. As the primer extends downstream during amplification (extended green arrow), the exonuclease activity of the DNA polymerase cleaves away the hybridized probe and removes the connection between the fluorescent dye (black box) and the quencher (Q). An increase in fluorescence signal ensues as more amplicons are generated.