
Quantitative PCR - qPCR
Quantitative PCR or real-time
qPCR is a particular method of polymerase chain reaction to measure the initial
amount of DNA. In fact, quantitative PCR measures the number of amplicons (DNA
portion defined by a pair of primers).
It enables continuous
monitoring ("in real time") of the PCR amplification process by
detecting the fluorescence emitted by the neo-formed PCR products.
The profile of a conventional
quantitative PCR reaction can be broken down into 3 steps:
- A first step known as
background noise: the amount of amplified fragment is insufficient to
generate a fluorescent signal greater than the background noise (and
therefore the fluorescence generated).
- A second step of
exponential phase of growth: the quantity of amplified fragment generates
a fluorescent signal higher than the detection threshold of the apparatus,
and then the number of amplified products doubles on each cycle. In
logarithmic coordinates, this phase is represented by a line.
- A final stage of the
plateau phase: certain components of the reaction (and in particular the
number of available Taq molecules) become limiting. The system no longer allows
exponential amplification.
The detection can be carried
out using SYBR green, a fluorescent organic compound which binds to nucleic
acids, or probes, Taqman or FRET.