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Quantitative polymerase chain reaction
Quantitative polymerase chain reaction (qPCR) is a modification of the polymerase chain reaction used to rapidly measure the quantity of DNA, complementary DNA or ribonucleic acid present in a sample. Like other forms of polymerase chain reaction, the process is used to amplify DNA samples, via the temperature-mediated enzyme DNA polymerase.
PCR theoretically amplifies DNA exponentially, doubling the number of molecules present with each amplification cycle. The number of amplification cycles and the amount of PCR end-product should allow one to calculate the initial quantity of genetic material, but numerous factors complicate this calculation. The ethidium bromide staining typically used to assess a successful PCR prevents further amplification, and is only semi-quantitative. The polymerase chain reaction may not be exponential for the first several cycles, and furthermore, plateaus eventually, so care must be taken to measure the final amount of DNA while the reaction is still in the exponential growth phase. To overcome these difficulties, several different quantitative methods have been developed.
The most sensitive quantification methods are done by the real-time polymerase chain reaction, where the amount of DNA is measured after each cycle of PCR by use of fluorescent markers. Other end-point methods measure DNA after PCR is completed. These methods depend on addition of a competitor RNA (for reverse-transcriptase PCR) or DNA in serial dilutions or co-amplification of an internal control to ensure that the amplification is stopped while in the exponential growth phase.
Although real-time quantitative polymerase chain reaction is often marketed as RT-PCR, it should not to be confused with reverse transcription polymerase chain reaction, which is also referred to as RT-PCR, but is used to amplify RNA samples. The two methods may be used in concert to reverse transcribe RNA and then quantitate the resulting cDNA using real-time PCR (often referred to as real-time RT-PCR).
End-point Quantitation Methods
Quantitative Competitive PCR
This method has a low dynamic range compared to real-time PCR, and care must be taken to use appropriate competitor DNA.
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Quantitative_polymerase_chain_reaction". A list of authors is available in Wikipedia.|