Taqman® assay

D.A. Kulesh, R.O. Baker, B.M. Loveless, D. Norwood, S.H. Zwiers, E. Mucker, C. Hartmann, R. Herrera, D. Miller, D. Christensen, L.P. Wasieloski Jr., J. Huggins and P.B. Jahrling, Smallpox and pan-orthopox virus detection by real-time 3 -minor groove binder TaqMan assays on the Roche lightcycler and the Cepheid smart cycler platforms, J. Clin. Microbiol., 42 (2004) 601-609. 

Fig. 5. Illustration of the 5 -nucleotidase (TaqMan) assay for allele discrimination. (A) The allele discrimination assay employs two unlabeled PCR primers and two doubly fluorescent labeled PCR probes for visuaUzation of a mutant allele. The target sequence is initially denatured and amplified in the presence of each of the primers and probes. Increasing polymerization in the presence of a thermostable polymerase which contains a 5 proofreading function allows cleavage of one fluorescent indicator from an appropriate probe during the cycling reaction. (B) Probes are designed with a fluorescent reporter and a quencher moiety. AmpUfication reactions are spiked with additional fluorescent quenchers in order to render the reaction initially dark to the photomultipUer mbe or diode. The probes are designed...

Because post-PCR processing or manipulation is not required, the Taqman assay is simple to perform once the assay has been optimized. Unfortunately, the assay, which is... 

There are direct measures of gene expression, but these tend to be limited by expense. They include TaqMan assays, which can measure the expression of several hundred genes directly, massively parallel signature sequencing (MPSS), expressed sequence tag (EST) counts, and serial analysis of gene expression (SAGE). Microarrays measure gene expression indirectly, and dye effects such as the dye bias of Cy3/Cy5 require lowess correction. 

As an alternative to the allele-specific PCR and the restriction digest of PCR products (often called PCR-RFLP), several commercial platforms are available to genotype individual SNPs in large numbers of samples. In our discussion here, we will focus on three commonly used platforms the 5 -exonuclease TaqMan assay. 

TaqMan assays are commercially available for many SNPs and can be designed on demand by Applied Biosystems. The method requires a real-time PCR instrument or a fluorescent plate reader to measure the fluorescence of the two dyes at the end of the PCR reaction. 

The concept of the threshold cycle (Ct) is the most important principle in TaqMan assays. Values are calculated for the amount of reporter dye released during each PCR cycle, and this is representative of the amount of the product amplified. The Ct is the cycle number at which the fluorescent signal from the reporter dye is first detected at a statistically significant level above the background [13]. The more template mRNA present at the beginning of the reaction, the lower the Ct will be needed to reach this threshold level of fluorescence. 

When a TaqMan assay is validated (the efficiency of replication is close to 1), the relative quantity of the target gene can be calculated using a ACt method using the relationship fold of induction=2 < " > = "" " >. 

E. Application of TaqMan Assay in Quantification of Phytochemicals and Gene Expression... 

Nordfors, L., Jansson, M., Sandberg, G., et al. (2000) Large-scale genotyping of single nucleotide polymorphisms by Pyrosequencing and validation against the 5 nuclease (Taqman assay. Hum. Mutat. 19, 395 01. 

However, real-time PCR is not a quantitative method per se. As a starting point for real-time PCR, the following description provides suggestions for PCR conditions and a temperature-time profile that should give reasonable results. Specifically, for TaqMan assays the following procedure is recommended to optimize the primer-probe concentration it is known as a primer-probe matrix. At the beginning the probe... 

Jothikumar, N., Kang, G., and Hill, V.R., Broadly reactive TaqMan assay for real-time RT-PCR detection of rotavirus in clinical and environmental samples, J. Virol Methods, 155, 126, 2009. 

Ponchel, F, Toomes, C., Bransfield, K., et al., 2003. Real-time PCR based on SYBR-Green I fluorescence an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and miao gene deletions. BMC Biotechnol. 3, 18. 

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