How does the 5 nuclease assay work?
In the 5′ nuclease assay (also known as the TaqMan assay), allelic discrimination is based on the characteristic 5′ to 3′ exonuclease activity of Taq DNA polymerase. PCR using flanking primers is performed including fluorescent oligonucleotide probes in a homogeneous assay. The probes consist of a 5′ reporter dye and a 3′ quencher dye, and are specific to the region containing the base change in the region to be amplified. The 5′ nuclease activity cleaves the probe if hybridisation occurs, releasing the reporter from the quencher. Two different probes with different fluorogenic reporters are put in the reaction for allele discrimination, one specific to complement each of the variant alleles to be typed. If there is a mismatch between the probe and target DNA sequence, the hybridisation is significantly reduced, therefore stopping the cleavage of reporter from quencher, and release of fluorescent signal. The amounts of each signal released indicate which allele(s) of the target region
