Abstract
The polymerase chain reaction (PCR) is an extremely sensitive method owing to the repetitive multiplication of template molecules. This property is a drawback for quantitative measurements because small differences in the multiplication factor lead to large differences in the amount of product. Two methods can be used to solve the problem of quantification; kinetic methods based on the determination or comparison of the amplification factor; and coamplification methods, which compare the amount of product to that of a simultaneously amplified standard template. An overview of the theoretical background of both methods is presented. For selection of a suitable method, both theoretical and practical considerations are important. Kinetic methods are the most convenient if PCR can be performed without opening the tubes, as in some apparatus using fluorescence detection. Coamplification methods can be done without expensive equipment but requires the parallel running of several PCR tubes. When the number of mitial template molecules is close to one, as in the limiting dilution technique, statistical considerations become important.
References
Schnell, S. and Mendoza, C. (1997) Enzymological considerations for a theoretical description of the quantitative competitive polymerase chain reaction (QC-PCR). J. Theor. Biol. 184, 433–440.
Schnell, S. and Mendoza, C. (1997) Theoretical description of the polymerase chain reaction. J. Theor. Biol. 188, 313–318.
Wiesner, R. J. (1992) Direct quantification of picomolar concentrations of mRNAs by mathematical analysis of a reverse transcription/exponential polymerase chain reaction assay. Nucleic Acids Res. 20, 5863–5864.
Ferre, F. (1992) Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Appl. 2, 1–9.
Cross, N. C. (1995) Quantitative PCR techniques and applications. Br. J. Haematol. 89, 693–697.
Reischl, U. and Kochanowski, B. (1995) Quantitative PCR. A survey of the present technology. Mol. Biotechnol. 3, 55–71.
Raeymaekers, L. (1995) A commentary on the practical applications of competitive PCR. Genome Res, 5, 91–94.
Kwok, S., Kellogg, D. E., McKinney, N., Spasic, D., Goda, L., Levenson, C., et al., (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res. 18, 999–1005.
Wang, A. M., Doyle, M. V., and Mark, D. F. (1989) Quantitation of mRNA by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA. 86, 9717–9721.
Gilliland, G., Perrin, S., Blanchard, K., and Bunn, H. F. (1990) Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc. Natl. Acad. Sci. USA 87, 2725–2729.
Siebert, P. D. and Larrick, J. W. (1992) Competitive PCR. Nature. 359, 557,558.
Mathieu Daude, F., Welsh, J., Vogt, T. and McClelland, M. (1996) DNA rehybridization during PCR: the “Cot effect” and its consequences. Nucleic Acids Res. 24, 2080–2086.
Bouaboula, M., Legoux, P., Pessegue, B., Delpech, B., Dumont, X., Piechaczyk, M., et al., (1992) Standardization of mRNA titration using a polymerase chain reaction method involving co-amplification with a multispecific internal control. J. Biol. Chem. 267, 21,830–21,838.
Raeymaekers, L. (1993) Quantitative PCR: theoretical considerations with practical implications. Anal. Biochem. 214, 582–585.
Nedelman, J., Heagerty, P., and Lawrence, C. (1992) Quantitative PCR with internal controls. Comput. Appl. Biosci. 8, 65–70.
Peccoud, J. and Jacob, C. (1996) Theoretical uncertainty of measurements using quantitative polymerase chain reaction. Biophys. J. 71, 101–108.
Villarreal, X. C., Grant, B. W., and Long, G. L. (1991) Demonstration of osteonectin mRNA in megakaryocytes: the use of the polymerase chain reaction. Blood. 78, 1216–1222.
Sykes, P. J., Neoh, S. H., Brisco, M. J., Hughes, E., Condon, J., and Morley, A. A. (1992) Quantitation of targets for PCR by use of limiting dilution. Biotechniques. 13, 444–449.
Zachar, V., Thomas, R. A., and Goustin, A. S. (1993) Absolute quantification of target DNA: a simple competitive PCR for efficient analysis of multiple samples. Nucleic Acids Res. 21, 2017–2018.
Santagati, S., Bettini, E., Asdente, M., Muramatsu, M., and Maggi, A. (1993) Theoretical considerations for the application of competitive polymerase chain reaction to the quantitation of a low abundance mRNA: estrogen receptor. Biochem. Pharmacol. 46, 1797–1803.
Nicoletti, A. and Sassy-Prigent, C. (1996) An alternative quantitative polymerase chain reaction method. Anal. Biochem. 236, 229–241.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Raeymaekers, L. Basic principles of quantitative PCR. Mol Biotechnol 15, 115–122 (2000). https://doi.org/10.1385/MB:15:2:115
Issue Date:
DOI: https://doi.org/10.1385/MB:15:2:115