TaqMan Real Time PCR technology basics

The TaqMan Real Time PCR method was named on the enzyme Taq Polymerase and the famous videogame PackMan which resembles the mechanism of the method (Taq Polymerase + PacMan = TaqMan).
The TaqMan Real Time PCR assay is based on Taq Polymerase 5´–3´ nuclease activity. During the process of hybridization, the enzyme cleaves a dual-labeled probe to the complementary target sequence and fluorophore-based detection.
In real-time PCR methods, the fluorescence signal allows quantification of the accumulated product during the exponential stages of the PCR. The TaqMan Real Time PCR significantly increases the specificity of the detection.

TaqMan Real Time PCR Principle

The TaqMan Real Time PCR probe, which is labeled with two fluorescent dyes, is created within the amplicon defined by a gene-specific PCR primer pair. The 5’ end is labeled with a reporter dye (usually 6-carboxy-fluorescein, FAM), while the 3’ end is labeled with a second fluorescent dye (6-carboxy-tetramethyl-rhodamine, TAMRA). As long as the probe is intact, the emission of the reporter dye is quenched by the second fluorescence dye. With the beginning of the extension phase of PCR, the polymerase enzyme starts to cleave the TaqMan probe, which results in a release of reporter dye. An automated sequence detector, equipped with specific software, is used to monitor the increasing amount of reporter florescent dye.

In Taqman  Real Time PCR assays, the usage of specific software plays very important role in the normalization of the raw data. The algorithm normalizes the reporter signal (Rn) to a passive reference. The standard deviation of the background Rn in the first few cycles then is multiplied by a default factor of 10 to determine a threshold. This calculation is made in order to define the threshold cycle (Ct) – the cycle at which this baseline level is exceeded (see the figure below).
The absolute value of threshold cycle (Ct) depends on the initial template copy number, the efficiency of both DNA amplification and cleavage of the TaqMan Real Time PCR probe. The Ct values of the samples are interpolated to an external reference curve constructed by plotting the relative or absolute amounts of a serial dilution of a known template vs the corresponding Ct values. The oligos of each target of interest can be designed specific software using uniform selection parameters.

Reliability of TaqMan Real Time PCR methods

The use of TaqMan Real Time PCR for the quantification of gene expression has been shown to be at least as reliable as the application other quantitative PCR techniques (e.g. competitive PCR assays). In comparison to the Northern blot assessment, only a minimal fraction of mRNA is necessary to quantify gene expression by TaqMan Real Time PCR.

Scientific Applications of TaqMan probes

The method of TaqMan Real Time PCR is widely used in:

• Bacterial Identification assays
• Gene expression assays and verification of microarray results
• Discriminate alleles
• Determination of viral sequences in clinical samples
• DNA quantification
• SNP genotyping