What are the major detection chemistries used for Real-Time PCR?
There are two major detection chemistries used for Real-Time PCR: hydrolysis (TaqMan) probe-based chemistry and DNA-binding SYBR Green I dye-based chemistry. Less used detection chemistries include Molecular Beacons, Scorpion probes and LUX primers. In TaqMan probe-based chemistry, also known as the fluorogenic 5′ nuclease assay, an oligonucleotide probe anneals to a specific sequence downstream of one of the PCR primers. The oligonucleotide is labeled with a fluorescent reporter dye at the 5′ end and a quencher dye at the 3′. When the probe is intact, the reporter is in close proximity to the quencher and the fluorescent signal is low as the energy from the reporter will be transferred to the quencher through Fluorescent Resonant Energy Transfer (FRET). During PCR, as Taq DNA polymerase extends from the primers, the 5′ exonuclease activity of the enzyme cleaves the annealed probe to separate the reporter dye from the quencher dye, increasing the fluorescent signal. SYBR Green I is a d
