Microfluidics Application: Real time PCR (Polymerase Chain Reaction)
Real time quantification during simultaneous amplification(PCR – Polymerase Chain Reaction) is enabled by gene-specific probes, that is, relatively short DNA strands binding specifically to the target sequence during the annealing step.
When the polymerase elongates the target sequence, it comes across the probe and digests it. Digestion of the probe disassociates two additional molecules that are bound to the probe. These molecules are the reporter dye and a quencher molecule. The reporter dye generates a fluorescence signal when it is stimulated by a light of a certain wavelength.
However, as long as it is bound to the primer together with the quencher molecule, the fluorescence signal is suppressed due to fluorescence energy transfer (FRET).
When the enzyme disassociates the reporter dye and quencher from the probe, the reporter dye is free to generate fluorescence signals that can be measured by photomultiplier diode. The digestion effect is irreversible thus leading to an increasing fluorescence signal the more probes are digested and amplicons are generated. The reaction mechanism for the real time PCR is shown in the following scheme:
The reporter dye is equipped with a fluorescence label that has a specific absorption and emission wavelength. By combination of labels with different emission spectra, real time multiplex PCR is enabled.
Fluorescence signals are measured as relative fluorescence units that can be normalized to a standard so that the relative change of fluorescence can be determined. This is how deviations due to variations in reporter dye concentration in the mixture or bleaching effects can be equilibrated.
Signal detection in a thermocycling device starts with a constant low fluorescent noise during the first thermocycles. At a certain amplification level, the fluorescence signal starts to increase exponentially. After a few further PCR cycles, the signal increases linearly and finally comes into saturation because the probes that are contained in the reaction mix are used up.
A high concentration of target DNA leads to an early signal amplification.
In contrast, little concentrations require more thermocycles to reach a certain threshold until their fluorescence signal is sufficiently detectable. Therefore, the sample concentrations can be discriminated by comparing the time points at which a defined fluorescence level is reached.
This threshold is plotted as a horizontal line in real-time graphs and this way, the respective threshold cycles (cT) can be easily compared and related to the initial concentrations at the sample.