Taqman Based Real Time PCR

TaqMan PCR is another target specific real-time PCR technique. The technique takes use of the Taq polymerase's 5′→3′ nuclease activity to identify and quantify specific PCR products as the reaction progresses. A nucleic-acid probe complementary to an internal region of the target DNA is used in TaqMan PCR. The internal TaqMan probe is coupled with a reporter fluorochrome and a quencher fluorochrome. The fluorescence generated by the reporter fluorochrome is absorbed by the quencher fluorochrome as long as the two fluorochromes are in close proximity. Thus, two fluorescent moieties are attached to the probe and the emission spectrum of one fluorophore overlaps the excitation spectrum of the other, causing the first fluorophore to be “quenched” by the second. If product is produced, the probe is destroyed by Taq polymerase's 5′-nuclease activity (Tyagi and Kramer 1996), which is specific for DNA hybridized to template (=TaqMan activity). The probe's deterioration allows the two fluorophores to separate, reducing quenching and increasing light intensity. The likelihood of contamination is significantly decreased because this test uses fluorescence measurements that may be conducted without exposing the PCR tube. Additionally, because no electrophoresis is needed, labor expenses are minimized (Kalinina et al. 1997, Sambrook and Russell 2001, Baker 2011).

Figure 1 Taqman Based Real Time PCR showing amplification of specific target sequence

References

Baker, M., (2011). qPCR: quicker and easier but don’t be sloppy. Nat. Methods 8: 207–212.

Kalinina, O., Lebedeva, I., Brown, J., and Silver, J., (1997). Nanoliter scale PCR with TaqMan detection. Nucleic Acids Res. 25: 1999–2004.

Sambrook, J., and Russell, D.W., (2001)., 3rd Ed. Molecular Cloning: A Laboratory Manual, Vol. 2 Cold Spring Harbor Laboratory Press, Cold Sprig Harbor, NY.

https://www.sciencedirect.com/science/article/pii/B0122270800017262