LC hybridization probes, also called FRET probes, are labelled with a single fluorescent reporter. Two LC hybridization probes specific for two adjacent sequences in the target DNA are used for quantitative Real-Time PCR. One probe is labelled with a donor fluorophore and the other labelled with an acceptor (or reporter) fluorophore. Both LC hybridization probes are designed to anneal between the upstream and downstream PCR primers. When the probes are not bound to the target sequence, the fluorescent signal from the reporter dye is not detected. However, when the probes hybridize to the target sequence during the PCR annealing step, the close proximity of the two fluorophores allows energy transfer from the donor to the acceptor dye, resulting in a fluorescent signal that is detected. The amount of signal is proportional to the amount of target sequence, and is measured in real time to allow quantification of the amount of target sequence.
LC hybridization probes are usually constructed with FAM as the 3’ donor fluorophore, and ROX or Cy® 5 as the 5’ acceptor fluorophore. A 3’ phosphate group is also added to prevent extension of the reporter probe by Taq DNA polymerase during the PCR cycles. All probes are HPLC purified and controlled by MALDI-TOF Mass Spectrometry. LC hybridization probes are provided lyophilized in individual tubes.
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