qPCR stands for quantitative polymerase chain reaction and is a technology used for measuring DNA using PCR.
PCR vs. qPCR
Most applications of polymerase chain reaction focus on its utility as a way to turn a small amount of DNA into a larger amount of DNA. This is necessary for gel electrophoresis and most forms of DNA sequencing. But, it also limits how informative PCR can be. It’s often seen as a step in another measurement, rather than a tool on its own.
Quantitative PCR adds two elements to the standard PCR process:
- Fluorescent dye
These two elements turn qPCR from a processing step in another procedure – to a measurement technique in its own right.
Thermal cyclers meant for use with qPCR include a fluorometer to detect that fluorescence.
The fluorometer detects that fluorescence in real time as the thermal cycler runs, giving readings throughout the amplification process of the PCR.
As a result, quantitative PCR is also called real-time PCR or RT-PCR.
In combination with appropriate standard curves and reference values, this real-time information about reaction rates and times translates into information about relative and absolute amounts of DNA present.
Gene Expression with qPCR
One of the foremost strengths of qPCR is the ability to measure gene expression.
Gene expression, or mRNA synthesis, is a critical part of protein synthesis. Gene expression is an area of active inquiry for molecular biologists – which aids in understanding numerous biological pathways and diseases. Using reverse transcriptase, the researcher creates complementary DNA (cDNA) copies of the RNA in their sample, and then performs qPCR with that cDNA.
This process is called reverse-transcription polymerase chain reaction.
The resulting readings provide information about the amount of otherwise difficult-to-measure mRNA present in the original, un-reverse-transcribed sample.
The kind of data generated by a qPCR run depends heavily on the primers and dyes used.
Typically, a single qPCR collects data on a single gene, since the dyes used in qPCR are usually nonspecific and will merge multiple genes into a single reading if multiple primers are used in the same mix. However, with fluorescent DNA probes rather than dyes, it is possible to “multiplex” qPCR and measure multiple DNA targets, as long as each target’s corresponding probe fluoresces at a different frequency.
For more information on the applications, theory and practice of qPCR, visit our ‘Ask TaqMan’ resource center.
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