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Real Time PCR [TaqMan] - Technology

Overview of Technology:

Real Time PCR is an extremely sensitive PCR technology utilized to quantitate gene expression levels of targets in as little as 2 pg of total RNA. Measurement of PCR products is monitored during the exponential phase of the PCR reaction, where precision is high, compared to traditional PCR methods which measure products at the end point or plateau phase.

Methodology:

TaqMan assays, also referred to as 5 Nuclease assays, exploit the 5'-->3' exonuclease activity of Taq DNA polymerase. Each assay contains a gene specific primer and a fluorescently labeled TaqMan probe. The probe contains a 5 reporter dye and a 3 quencher dye. The 3 end is also blocked to prevent extension during PCR. The probe is designed to anneal the target sequence between the forward and reverse PCR primers. While the probe is intact, the quencher suppresses the fluorescence of the reporter dye. During amplification, Taq DNA polymerase cleaves the probe and displaces it from the target, allowing extension to continue. Cleavage of the probe separates the reporter dye from the quencher dye, resulting in an increase in fluorescence. The increased fluorescence only occurs if the target sequence is amplified and is complimentary to the probe, thus preventing detection of nonspecific amplification. For any given cycle within the exponential phase, the amount of product, and hence fluorescence signal, is directly proportional to the initial copy number. Thus, higher copy number templates will cross a fluorescence detection threshold before lower copy templates.

Instrumentation & Software:

The ABI PRISM 7900HT Sequence Detection System is designed for high throughput screening. The Sequence Detection Software, a proprietary software from ABI, runs the Sequence Detection System. The software utilizes the collected data to calculate the cycle threshold (CT), ie [the PCR cycle at which the flourescent signal crosses the threshold for each gene being interrogated]. The baseline and threshold settings are adjustable components of the algorithm. There are two methods available to analyze quantitative PCR data: absolute or relative quantitation. Absolute quantitation relates the fluorescence signal [or CT]to a standard curve, whereas relative quantitation relates the signal [or CT] to another sample such as an untreated control.