Quantitative polymerase chain reaction

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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 will eventually reach a plateau, 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).

Contents

[edit] End-point Quantitation Methods

1- Relative.

2- Competitive.

Relative quantitation

Compares transcript abundance across multiple samples, using a co-amplified internal control for sample normalization. Results are expressed as ratios of the gene specific expression / the internal control expression. This result is a corrected relative value for the gene specific product in each sample. These values may be compared between samples for an estimate of the relative expression of target in the samples; e.g, 2.5-fold more NOS2 gene in sample 2 than in sample 1. Common internal controls include GAPDH and ß-actin mRNAs and 18S rRNA.

[edit] Quantitative Competitive PCR

#The unknown sample and a known sample are prepared with a known concentration of a similarly sized section of target DNA for amplification.
  1. Both reactions are run for the same length of time in identical conditions (preferably using the same primers, or at least primers of similar annealing temperatures).
  2. Using agarose gel electrophoresis separate the products of the reaction from their original DNA and spare primers.
  3. The relative quantities of the known and unknown samples are measured to determine the quantity of the unknown.

[edit] Disadvantage of End-Point PCR

Agarose gel results are obtained from the end point of the reaction; time consuming; results are based on size-based discrimination only; agarose gel resolution is very poor, about 10 fold. Real-Time PCR can detect as little as a two-fold change; Poor Precision, low sensitivity, low dynamic range compared to real-time PCR, and care must be taken to use appropriate competitor DNA and ethidium bromide for staining is not very quantitative.

[edit] References

  • Ding C, Cantor CR (2004). Quantitative analysis of nucleic acids--the last few years of progress., Journal of Biochemistry and Molecular Biology, 37:1, pp. 1–10. Abstract at PMID 14761298.
  • www.gene-quantification.info all about real-time qPCR and qRT-PCR
  • www.realtimepcr.dk Experience with QPCR from the University of Copenhagen
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Polymerase chain reaction techniques