In contrast to traditional PCR where the presence or absence of a DNA sequence is analysed upon reaction completion, quantitative PCR or qPCR, monitors DNA amplification as the reaction progresses. In qPCR assays, progression is monitored by an increase in fluorescence signal. The fluorophore can be a double stranded DNA binding dye, most commonly SYBR Green 1, or covalently attached to a target specific probe as in TaqMan assays.
In SYBR assays the dye fluoresces when bound to double stranded DNA, but displays weak fluorescence in the presence of single standed DNA. This results in an increase in fluorescent signal as the reaction progresses and dsDNA is formed.
The most common probe based assay is a 5' nuclease based assay known as TaqMan. In TaqMan assays the 5' flap endonuclease domain of Taq DNA polymerase cleaves a target specific probe, separating a fluorophore on the 5' end from a quencher on the 3' end resulting in an amplification dependent increase in fluorescence.
A thermocycler coupled with a fluorimeter modulates the reaction temperature during DNA amplification, while also monitoring the fluorescence signal at each PCR cycle, generating a real time PCR curve.
There are four distinct phases within the amplification curve; LAG, EXPONENTIAL, LINEAR and PLATEAU.
During the LAG phase amplification occurs but the signal is not strong enough to be detected above background. The EXPONENTIAL phase is used for quantification because it produces a fluorescence signal measurable over background and contains an abundance of reaction components permitting a detectable doubling event at every PCR cycle. When the fluorescence qPCR signal is detectable over the background fluorescence a Quantification Cycle, or Cq value, can be determined. Cq values can be used to evaluate relative target abundance between two or more samples. Or to calculate absolute target quantities in reference to an appropriate standard curve.
qPCR can also be modified to detect and quantify RNA.
Interpretation of qPCR data requires careful experimental design and analysis. For parameters on designing and reporting qPCR experiments please read the MIQE guidelines, which have been established to ensure consistent experimental design and data analysis appropriate for peer review publication.
For more information, visit LUNAqPCR.com
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