TaqMan® probes

Kuboniwa, M. Amano, A. Kimura, K. R. Sekine, S. Kato, S. Yamamoto, Y. Okahashi, N. Iida, T. Shizukuishi, S. Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes. Oral Microbiol. Immunol. 2004,19,168-176. 

Reischer, G. H., Lemmens, M., Farnleitner, A., Adler, A., and Mach, R. L. (2004). Quantification of Fusarium graminearum in infected wheat by species specific real-time PCR applying a TaqMan probe. J. Microbiol. Methods 59,141-146. 

High-Resolution Melting (HRM) is a different technique also based on PCR but excluding the use of TaqMan probes and the requirement to define... 

TaqMan probe that is annealed to the PCR target, and thus the luminescence from the cleaved luminophore comes to be observed. SNP typing is also possible using TaqMan probes which are designed to contain the SNP site of interest. Some modifications using luminescent lanthanide complexes have been reported by groups at the University of Turku and PerkinElmer, Inc as described below. 

Taqman exploits the 5 nuclease activity of the DNA-Polymerase to cleave Taqman probes during PCR. This cleavage results in the liberation of a quencher dye and a subsequent fluorescence of a reporter dye. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. 

III) Allele specific hybridisation (Figure 2) Fragments amplified in an allele unspecific PCR are measured with allele specific probes (Molecular Beacons, TaqMan probes or Scorpion), which can either be used in a single PCR or in multiplex reactions for detection of both alleles in one reaction [13]. This method has minor background problems, however the dynamic range may be limited due to competition of the probes. The Molecular Beacon proach has been developed for B. graminis and M. fijiensis [13]. 

Real-time PCR probe lpM TaqMan probe stock (5 -FAM-TGTACGTGAGTGGGCATGTAGCAAGAGG-BHQ, where FAM is 6-carboxyfluorescein and BFIQ the Black FI ole Quencher-1 IDT, Coralville, IA). 

A real-time PCR master-mix was made consisting of 1.25 pL of the forward and reverse primers, 5pL Amplitaq buffer, 4pL MgCl2,2.5 pL of dNTPs, 3.75 pL of TaqMan probe, 1 pL of the ROX reference dye, 26pL of ddH20, and 0.3 pL of Amplitaq gold polymerase. 

Multiplex PCR can be used in relative quantification of a target and an endogenous reference in the same tube. The availability of multiple reporter dyes for TaqMan probes (FAM, VIC, JOE) makes it possible to detect amplification of more than one target in the same tube. Two important factors for multiplex PCR are (1) to limit the concentration of primers for the most abundant gene and (2) the deconvolution of the collected spectra to distinguish between reporter dyes. Multiplex real-time RT-PCR has been demonstrated in detection of single-copy mRNA of Xist gene in mouse embryos [12]. 

Hydrolysis probes (e.g.,TaqMan probes) are the most widely used and pubhshed in detection chemistry hterature for probe-based real-time PCR apphcations. In addition to the primers, it includes a third oligonucleotide, 20 to 26 bases in length, in the reaction known as the probe. A fluorescent reporter dye, most frequendy... 

Reaction with SybrGreen I Reaction with TaqMan Probe Reaction with Hybridization Probes... 

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