PCR – diagnostics. Aizhan Zhussupova

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СКАЧАТЬ a threshold level set above background fluorescence and starts to increase exponentially (exponential phase). Eventually the fluorescence signal levels off because the fluorescence saturates the detector of the real time PCR machine (plateau phase) and any changes in DNA concentration can no longer be recognized.

      Thus, main difference is that the amplified DNA is detected as the reaction progresses in «real time», while in the standard PCR, the product of the reaction is detected at its end.

      Two common methods for the detection of products in qPCR are: (1) non-specific fluorescent dyes that intercalate with any doublestranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides, which are labeled with a fluorescent reporter, permitting detection only after hybridization of the probe with its complementary sequence to quantify mRNA and non-coding RNA in cells or tissues.

      Quantification cycle (C_q) is the metric used for analyzing qPCR results, where: background fluorescence is subtracted from raw data; a fluorescence threshold value is chosen, either manually or using an instrument-specific algorithm; data analysis searches data curves for each sample and estimates a C_q value that represents where that sample crossed the threshold.

      The exact level used for this threshold should be chosen so that it captures data during the exponential phase, when reaction efficiency is still stable and hence the results are more reliable. The threshold value should be the same for all samples analyzed in a run.

      qPCR is carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of a specified wavelength and detect the fluorescence emitted by the excited fluorophore. The thermal cycler is also able to rapidly heat and chill samples, thereby taking advantage of the physicochemical properties of the nucleic acids and DNA polymerase.

      Real-time PCR thermal cycler systems consist of three basic parts: a thermal system to perform temperature cycling; an optical system to emit light necessary for activation of the fluorophore(s) combined with a system to capture the generated fluorescence; software to control the instrument operation, and collect and analyze the data generated. For each of these points, there are several technical solutions available:

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