What is Quantitative Real-Time PCR (qPCR)?

Quantitative real-time PCR is also known as qPCR. It is a Significant development of PCR technology that is can reliably detect and measure the products generated during each cycle of the PCR process. This technique is used to detect, characterize and quantify nucleic acids for numerous applications. (NCBI)

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In qPCR assays, reaction progression is monitored by an increase in fluorescence signal – the fluorophore can be a double-stranded DNA binding dye (most commonly SYBR Green I) 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 stranded DNA. The results in an increased fluorescence signal as the reaction progresses and double stranded DNA is formed. The most common probe-based assay is a 5-prime nuclease assay, known as TaqMan.


(NCBI: https://www.ncbi.nlm.nih.gov/probe/docs/techqpcr/ )

 

In TaqMan assays, the 5-prime flap endonuclease domain of Taq DNA polymerase cleaves a target-specific probe, separating a fluorophore on the 5-prime end from a quencher on the 3-prime end, resulting in an amplification-dependent increase in fluorescence.

A thermocycler, coupled with a fluorometer, modulates the reaction temperature during DNA amplification, while also monitoring the fluorescence signal at each PCR cycle, generating a real-time qPCR 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 fluorescent signal measureable over background and contains an abundance of reaction components, permitting a detectable doubling event at every PCR cycle. When the fluorescent 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


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