Molecular Biotechnology

, Volume 15, Issue 2, pp 115–122 | Cite as

Basic principles of quantitative PCR

  • Luc Raeymaekers
Review

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.

Index Entries

PCR quantitative PCR competitive kinetics theory 

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References

  1. 1.
    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.PubMedCrossRefGoogle Scholar
  2. 2.
    Schnell, S. and Mendoza, C. (1997) Theoretical description of the polymerase chain reaction. J. Theor. Biol. 188, 313–318.PubMedCrossRefGoogle Scholar
  3. 3.
    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.PubMedCrossRefGoogle Scholar
  4. 4.
    Ferre, F. (1992) Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Appl. 2, 1–9.PubMedGoogle Scholar
  5. 5.
    Cross, N. C. (1995) Quantitative PCR techniques and applications. Br. J. Haematol. 89, 693–697.PubMedCrossRefGoogle Scholar
  6. 6.
    Reischl, U. and Kochanowski, B. (1995) Quantitative PCR. A survey of the present technology. Mol. Biotechnol. 3, 55–71.PubMedGoogle Scholar
  7. 7.
    Raeymaekers, L. (1995) A commentary on the practical applications of competitive PCR. Genome Res, 5, 91–94.PubMedCrossRefGoogle Scholar
  8. 8.
    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.PubMedCrossRefGoogle Scholar
  9. 9.
    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.PubMedCrossRefGoogle Scholar
  10. 10.
    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.PubMedCrossRefGoogle Scholar
  11. 11.
    Siebert, P. D. and Larrick, J. W. (1992) Competitive PCR. Nature. 359, 557,558.PubMedCrossRefGoogle Scholar
  12. 12.
    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.PubMedCrossRefGoogle Scholar
  13. 13.
    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.Google Scholar
  14. 14.
    Raeymaekers, L. (1993) Quantitative PCR: theoretical considerations with practical implications. Anal. Biochem. 214, 582–585.PubMedCrossRefGoogle Scholar
  15. 15.
    Nedelman, J., Heagerty, P., and Lawrence, C. (1992) Quantitative PCR with internal controls. Comput. Appl. Biosci. 8, 65–70.PubMedGoogle Scholar
  16. 16.
    Peccoud, J. and Jacob, C. (1996) Theoretical uncertainty of measurements using quantitative polymerase chain reaction. Biophys. J. 71, 101–108.PubMedCrossRefGoogle Scholar
  17. 17.
    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.PubMedGoogle Scholar
  18. 18.
    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.PubMedGoogle Scholar
  19. 19.
    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.PubMedCrossRefGoogle Scholar
  20. 20.
    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.PubMedCrossRefGoogle Scholar
  21. 21.
    Nicoletti, A. and Sassy-Prigent, C. (1996) An alternative quantitative polymerase chain reaction method. Anal. Biochem. 236, 229–241.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2000

Authors and Affiliations

  • Luc Raeymaekers
    • 1
  1. 1.Laboratorium voor FysiologieKULeuven, Campus Gasthuisberg O/NLeuvenBelgium

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