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. 1996 Jun 1;24(11):2080–2086. doi: 10.1093/nar/24.11.2080

DNA rehybridization during PCR: the 'Cot effect' and its consequences.

F Mathieu-Daudé 1, J Welsh 1, T Vogt 1, M McClelland 1
PMCID: PMC145907  PMID: 8668539

Abstract

The rate of amplification of abundant PCR products generally declines faster than that of less abundant products in the same tube in the later cycles of PCR. As a consequence, differences in product abundance diminish as the number of PCR cycles increases. Rehybridization of PCR products which may interfere with primer binding or extension can explain this significant feature in late cycles. Rehybridization occurs with a half-time dependent on the reciprocal of the DNA concentration. Thus, if multiple PCR products are amplified in the same tube, reannealing occurs faster for the more abundant PCR products. In RT-PCR using an internal control, this results in a systematic bias against the more abundant of the two PCR products. In RNA fingerprinting by arbitrarily primed PCR (or differentially display of cDNAs), very large or absolute differences in the expression of a transcript between samples are preserved but smaller real differences may be gradually erased as the PCR reaction proceeds. Thus, this 'Cot effect' may systematically cause an underestimate of the true difference in starting template concentrations. However, differences in starting template concentrations will be better preserved in the less abundant PCR products. Furthermore, the slow down in amplification of abundant products will allow these rarer products to become more visible in the fingerprint which may, in turn, allow rarer cDNAs to be sampled more efficiently. In some applications, where the object is to stochiometrically amplify a mixture of nucleic acids, the bias against abundant PCR products can be partly overcome by limiting the number of PCR cycles and, thus, the concentration of the products. In other cases, abundance normalization at later cycles may be useful, such as in the production of normalized libraries.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Borriello F., Weinberg D. S., Mutter G. L. Evaluation of gene deletions by quantitative polymerase chain reaction. Experience with the alpha-thalassemia model. Diagn Mol Pathol. 1994 Dec;3(4):246–254. doi: 10.1097/00019606-199412000-00006. [DOI] [PubMed] [Google Scholar]
  2. Bouaboula M., Legoux P., Pességué B., Delpech B., Dumont X., Piechaczyk M., Casellas P., Shire D. Standardization of mRNA titration using a polymerase chain reaction method involving co-amplification with a multispecific internal control. J Biol Chem. 1992 Oct 25;267(30):21830–21838. [PubMed] [Google Scholar]
  3. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  4. Cunningham I. New culture medium for maintenance of tsetse tissues and growth of trypanosomatids. J Protozool. 1977 May;24(2):325–329. doi: 10.1111/j.1550-7408.1977.tb00987.x. [DOI] [PubMed] [Google Scholar]
  5. Gilliland G., Perrin S., Blanchard K., Bunn H. F. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2725–2729. doi: 10.1073/pnas.87.7.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kohno T., Morishita K., Takano H., Shapiro D. N., Yokota J. Homozygous deletion at chromosome 2q33 in human small-cell lung carcinoma identified by arbitrarily primed PCR genomic fingerprinting. Oncogene. 1994 Jan;9(1):103–108. [PubMed] [Google Scholar]
  7. Lanham S. M., Godfrey D. G. Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp Parasitol. 1970 Dec;28(3):521–534. doi: 10.1016/0014-4894(70)90120-7. [DOI] [PubMed] [Google Scholar]
  8. Liang P., Pardee A. B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992 Aug 14;257(5072):967–971. doi: 10.1126/science.1354393. [DOI] [PubMed] [Google Scholar]
  9. Mathieu-Daudé F., Cheng R., Welsh J., McClelland M. Screening of differentially amplified cDNA products from RNA arbitrarily primed PCR fingerprints using single strand conformation polymorphism (SSCP) gels. Nucleic Acids Res. 1996 Apr 15;24(8):1504–1507. doi: 10.1093/nar/24.8.1504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. McClelland M., Mathieu-Daude F., Welsh J. RNA fingerprinting and differential display using arbitrarily primed PCR. Trends Genet. 1995 Jun;11(6):242–246. doi: 10.1016/s0168-9525(00)89058-7. [DOI] [PubMed] [Google Scholar]
  11. McClelland M., Ralph D., Cheng R., Welsh J. Interactions among regulators of RNA abundance characterized using RNA fingerprinting by arbitrarily primed PCR. Nucleic Acids Res. 1994 Oct 25;22(21):4419–4431. doi: 10.1093/nar/22.21.4419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Morrison C., Gannon F. The impact of the PCR plateau phase on quantitative PCR. Biochim Biophys Acta. 1994 Oct 18;1219(2):493–498. doi: 10.1016/0167-4781(94)90076-0. [DOI] [PubMed] [Google Scholar]
  13. Murphy N. B., Pellé R. The use of arbitrary primers and the RADES method for the rapid identification of developmentally regulated genes in trypanosomes. Gene. 1994 Apr 8;141(1):53–61. doi: 10.1016/0378-1119(94)90127-9. [DOI] [PubMed] [Google Scholar]
  14. Peinado M. A., Malkhosyan S., Velazquez A., Perucho M. Isolation and characterization of allelic losses and gains in colorectal tumors by arbitrarily primed polymerase chain reaction. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10065–10069. doi: 10.1073/pnas.89.21.10065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ralph D., McClelland M., Welsh J. RNA fingerprinting using arbitrarily primed PCR identifies differentially regulated RNAs in mink lung (Mv1Lu) cells growth arrested by transforming growth factor beta 1. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10710–10714. doi: 10.1073/pnas.90.22.10710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wang A. M., Doyle M. V., Mark D. F. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9717–9721. doi: 10.1073/pnas.86.24.9717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Welsh J., Chada K., Dalal S. S., Cheng R., Ralph D., McClelland M. Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res. 1992 Oct 11;20(19):4965–4970. doi: 10.1093/nar/20.19.4965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Welsh J., McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 1990 Dec 25;18(24):7213–7218. doi: 10.1093/nar/18.24.7213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Welsh J., Petersen C., McClelland M. Polymorphisms generated by arbitrarily primed PCR in the mouse: application to strain identification and genetic mapping. Nucleic Acids Res. 1991 Jan 25;19(2):303–306. doi: 10.1093/nar/19.2.303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wetmur J. G. DNA probes: applications of the principles of nucleic acid hybridization. Crit Rev Biochem Mol Biol. 1991;26(3-4):227–259. doi: 10.3109/10409239109114069. [DOI] [PubMed] [Google Scholar]
  21. Williams J. G., Kubelik A. R., Livak K. J., Rafalski J. A., Tingey S. V. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 1990 Nov 25;18(22):6531–6535. doi: 10.1093/nar/18.22.6531. [DOI] [PMC free article] [PubMed] [Google Scholar]

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