Advances in quantitative PCR technology: 5' nuclease assays

Abstract

The development of 5' nuclease assays represents a significant advance in nucleic acid quantitation. This approach utilizes the 5'-3' exonuclease activity of Thermus aquaticus (Taq) polymerase to cleave a dual-labelled probe annealed to a target sequence during amplification. The release of a fluorogenic tag from the 5' end of the probe is proportional to the target sequence concentration (copy number), and can be measured either at endpoint (post-amplification), or in real time', where the increase in emission intensity is followed on a per-cycle basis.