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Comparison of Competitive PCR and Positive Control-Based PCR

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Quantitative PCR Protocols

Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 26))

Abstract

The exponential amplification of small amounts of nucleic acids makes polymerase chain reaction (PCR) not only powerful but also challenging as a quantitative method. Variations in nucleic acid preparation, thermal cyclic performance, the choice of the polymerase, and the amplification procedure can cause large differences in final product yield. To address the challenges of quantitative PCR, the procedure has been critically examined, leading to an understanding of the critical parameters involved in quantitative amplification. Accepted parameters can be summarized as a series of choices: external vs internal standard, exogenous vs endogenous standard, competitive vs noncompetitive amplification, and exponential vs plateau amplification (1–3).

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References

  1. Clementi, M., Menzo, S., Bagnarelli, P., Manzin, A., Valenza, A., and Varaldo, P. E. (1993) Quantitative PCR and RT-PCR in virology. PCR Methods Applic. 2, 191–196.

    CAS  Google Scholar 

  2. Ferre, F. (1992) Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Applic. 2, 1–9.

    CAS  Google Scholar 

  3. Sninsky, J. J. and Kwok, S. (1993) The application of quantitative polymerase chain reaction to therapeutic monitoring. AIDS 7(Suppl.), S29–S34.

    Article  PubMed  Google Scholar 

  4. Simmonds, P., Balfe, P., Peutherer, J. F., Ludlam, C. A., Bishop, J. O., and Leigh Brown, A. J. (1990) Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J. Virol. 64, 864–872.

    Google Scholar 

  5. Dickover, R. E., Donovan, R. M., Goldstein, E., Dandekar, S., Bush, C. E., and Carlson, J. R. (1990) Quantitation of human immunodeficiency virus DNA by using the polymerase chain reaction. J. Clin. Microbiol. 28(9), 2130–2133.

    CAS  PubMed  Google Scholar 

  6. Innocenti, P., Ottmann, M., Morand, P., Leclercq, P., and Seigneurin, J. M. (1992) HIV-1 in blood monocytes: frequency of detection of proviral DNA using PCR and comparison with the total CD4 count. AIDS Res. Hum. Retroviruses 8, 261–268.

    Article  CAS  PubMed  Google Scholar 

  7. Pannetier, C., Delassus, S., Darche, S., Saucier, C., and Kourilsky, P. (1993) Quantitative titration of nucleic acids by enzymatic amplification reactions run to saturation. Nuc. Acids Res. 21, 577–583.

    Article  CAS  Google Scholar 

  8. Piatak, M., Jr., Luk, K.-C., Williams, B., and Lifson, J. D. (1993) Quantitative competitive polymerase chain reaction for accurate quantitation of HIV DNA and RNA species. BioTechniques 14, 70–81.

    PubMed  Google Scholar 

  9. Siebert, P. D. and Larrick, J. W. (1993) PCR MIMICS: competitive DNA fragments for use as internal standards in quantitative PCR. BioTechniques 14, 244–249.

    CAS  PubMed  Google Scholar 

  10. Nedelman, J., Heagerty, P., and Lawrence, C. (1992) Quantitative PCR: procedures and precisions. Bul. Math. Biol. 54, 477–502.

    CAS  Google Scholar 

  11. Kellog, D. E., Sninsky, J. J., and Kwok, S. (1990) Quantitation of HIV-1 proviral DNA relative to cellular DNA by polymerase chain reaction. Anal. Biochem. 189, 202–208.

    Article  Google Scholar 

  12. Lee, T. H., Sunzeri, F. J., Tobler, L. H., Williams, B. G., and Busch, M. P. (1991) Quantitative assessment of HIV-1 DNA load by coamplification of HIV-1 gag and HLA-DQ-a genes. AIDS 5(6), 683–691.

    Article  CAS  PubMed  Google Scholar 

  13. Arnold, B. L., Itakura, K., and Rossi, J. J. (1992) PCR-based quantitation of low levels of HIV-1 DNA by using an external standard. Genet. Anal. Tech. Appli. 9, 113–116.

    CAS  Google Scholar 

  14. Cone, R. W., Hobson, A. C., and Huang, M. L. W. (1992) Coamplified positive control detects inhibition of polymerase chain reactions. J. Clin. Microbiol. 30(12), 3185–3189.

    CAS  PubMed  Google Scholar 

  15. Dyster, L. M., Abbott, L., Bryz-Gornia, V., Poiesz, B. J., and Papsidero, L. D. (1994) Microplate-Based DNA hybridization assays for detection of human retroviral gene sequences. J. Clin. Microbiol. 32(2), 547–550.

    CAS  PubMed  Google Scholar 

  16. Kohsaka, H., Taniguchi, A., Richman, D. D., and Carson, D. A. (1993) Microtiter format gene quantification by covalent capture of competitive PCR products: application to HIV-1 detection. Nuc. Acids Res. 21(15), 3469–3472.

    Article  CAS  Google Scholar 

  17. Mallet, F., Hebrard, C., Brand, D., Chapuis, E., Cros, P., Allibert, P., Besnier, J.-M., Barin, F., and Mandrand, B. (1993) Enzyme-linked oligosorbent assay for detection of polymerase chain reaction-amplified human immunodeficiency virus type 1. J. Clin. Microbiol. 31, 1444–1449.

    CAS  PubMed  Google Scholar 

  18. Mallet, F., Hebrard, C., Livrozet, J. M., Lees, O., Tron, F., Touraine, J. L., and Mandrand, B. (1995) Quantitation of human immunodeficiency virus type 1 DNA by two PCR procedures coupled with enzyme-linked oligosorbent assay. J. Clin. Microbiol. 33(12), 3201–3208.

    CAS  PubMed  Google Scholar 

  19. Yerly, S., Chamot, E., Hirschel, B., and Perrin, L. H. (1992) Quantitation of human immunodeficiency virus provirus and circulating virus: Relationship with immunologic parameters. J. Infect. Dis. 166, 269–276.

    CAS  PubMed  Google Scholar 

  20. Aoki, S. A., Yarchoan, R., Thomas, R. V., Pluda, J. M., Marczyk, K., Broder, S., and Mitsuya, H. (1990) Quantitative analysis of HIV-1 proviral DNA in pheripheral blood mononuclear cells from patients with AIDS or ARC: decrease of proviral DNA content following treatment with 2′, 3′-dideoxyinosine (ddI). AIDS Res. Hum. Retroviruses 6(11), 1331–1339.

    CAS  PubMed  Google Scholar 

  21. Mallet, F., Cros, P., and Mandrand, B. (1995) Enzyme-linked oligosorbent assay for detection of PCR-amplified HIV-1, in PCR: Protocol for Diagnosis of Human and Animals Virus Diseases (Becker, Y. and Darai, G., eds.), Springer-Verlag, Heidelberg, pp. 19–28.

    Google Scholar 

  22. Allibert, P., Cros, P., and Mandrand, B. (1992) Automated detection of nucleic acid sequences of HPV 16, 18 and 6/11. Eur. J. Biomed. Tech. 3(14), 152–155.

    Google Scholar 

  23. Mabilat, C., Desvarenne, S., Panteix, G., Machabert, N., Bernillon, M.H., Guardiola, G., and Cros, P. (1994) Routine automated identification of Mycobacterium tuberculosis complex isolates with a DNA probe. J. Clin. Microbiol. 32(11), 2702–2705.

    CAS  PubMed  Google Scholar 

  24. Cros, P., Allibert, P., Mandrand, B., Tiercy, J.M., and Mach, B. (1992) Oligonucleotide genotyping of HLA polymorphism on microtitre plates. Lancet 340, 870–873.

    Article  CAS  PubMed  Google Scholar 

  25. Clouse, K. A., Powell, D., Washington, I., Poli, G., Strebel, K., Farrar, W., Barstad, P., Kovacs, J., Fauci, A. S., and Folks, T. M. (1989) Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. J. Immunol. 142, 431–438.

    CAS  PubMed  Google Scholar 

  26. Souazé, F., Ntodou-Thomé, A., Tran, C. Y., Rostène, W., and Forgez, P. (1996) Quantitative RT-PCR: Limits and Accuracy. BioTechniques 21, 280–285.

    PubMed  Google Scholar 

  27. Coutlée, F., He, Y., Saint-Antoine, P., Olivier, C., and Kessous, A. (1995) Coamplification of HIV type 1 and b-globin gene DNA sequences in a nonisotopic polymerase chain reaction assay to control for amplification efficiency. AIDS Res. Hum. Retroviruses 11, 363–371.

    Article  PubMed  Google Scholar 

  28. Chelly, J., Kaplan, J. C., Maire, P., Gautron, S., and Kahn, A. (1988) Transcription of the dystrophin gene in muscle and non-muscle tissues. Nature 333, 858–860.

    Article  CAS  PubMed  Google Scholar 

  29. Yamamura, M., Uyemura, K., Deans, R. J., Weinberg, K., Rea, T. H., Bloom, B. R., and Modlin, R. L. (1991) Defining protective responses to pathogens: cytokine profiles in leprosy lesions. Science 254, 277–279.

    Article  CAS  PubMed  Google Scholar 

  30. Alard, P., Lantz, O., Sebagh, M., Calvo, C. F., Weill, D., Chavanel, G., Senik, A., and Charpentier, B. (1993) A versatile ELISA-PCR assay for mRNA quantitation from a few cells. BioTechniques 15, 730–737.

    CAS  PubMed  Google Scholar 

  31. Mallet, F., Oriol, G., and Mandrand, B. (1998) Characterization of RNA using Continuous RT-PCR coupled with ELOSA in Methods in Molecular Biology, Vol. 86: RNA Isolation and Characterization Protocols (Rapley, R. and Manning, D. L., eds.), Humana Press, Totowa, NJ, pp. 161–172.

    Chapter  Google Scholar 

  32. Morrison, F. and Gannon, F. (1994) The impact of the PCR plateau phase on quantitative PCR. Biochim. Biophys. Acta 1219, 493–498.

    CAS  PubMed  Google Scholar 

  33. Siebert, P. D. and Larrick, J. W. (1992) Competitive PCR. Nature 359, 557–558.

    Article  CAS  PubMed  Google Scholar 

  34. Mallet, F., Oriol, G., Mary, C., Verrier, B., and Mandrand, B. (1995) RT-PCR using AMV-RT and Taq DNA polymerase: characterization and comparaison to uncoupled procedures. BioTechniques 18, 678–687.

    CAS  PubMed  Google Scholar 

  35. Mallet, F. (1996) Continuous RT-PCR using AMV-RT and Taq in PCR: Essential Techniques (Burke, J. F., ed.), BIOS Scientific Publishers Ltd., Oxford, pp. 82–85.

    Google Scholar 

  36. Diviacco, S., Norio, P., Zentilin, L., Menzo, S., Clementi, M., Biamonti, G., Riva, S., Falaschi, A., and Giacca, M. (1992) A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates. Gene 122, 3013–3020.

    Article  Google Scholar 

  37. Sholler, R., Gervasi, G., Avigdor, R., and Castanier, M. (1987) Comparaison de deux méthodes de dosage in Colloque sur les actualités en immunoanalyse, CORATA, Université de Bordeaux II, Bergeret, Bordeaux, 2–4 Dec 1987, pp. 225–233.

    Google Scholar 

  38. Natarajan, V., Plishka, R. J., Scott, E. W., Lane, H. C., and Salzman, N. P. (1994) An internally controlled virion PCR for the measurement of HIV-1 RNA in plasma. PCR Methods Appl. 3, 346–350.

    CAS  PubMed  Google Scholar 

  39. Van Gemen, B., Van Beuningen, R., Nabbe, A., Van Strijp, D., Jurriaans, S., Lens, P., and Kievits, T. (1994) A one-tube quantitative HIV-1 RNA NASBA nucleic acid amplification assay using electrochemiluminescent (ECL) labelled probes. J. Virol. Methods 49, 157–168.

    Article  PubMed  Google Scholar 

  40. Vener, T., Axelsson, M., Albert, J., Uhlén, M., and Lundeberg, J. (1996) Quantitation of HIV-1 using multiple competitiors in a single-tube assay. BioTechniques 21, 248–255.

    CAS  PubMed  Google Scholar 

  41. Zimmermann, K., Schögl, D., Plaimauer, B., and Mannhalter, J. W. (1996) Quantitative multiple competitive PCR of HIV-1 DNA in a single reaction tube. BioTechniques 21, 480–484.

    CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Unité Mixte de Recherche, CNRS-BioMérieux, Ecole Normale Supérieure de Lyon, France

    François Mallet

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  1. François Mallet
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Editors and Affiliations

  1. University of Regensburg, Germany

    Bernd Kochanowski  & Udo Reischl  & 

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© 1999 Humana Press Inc., Totowa, NJ

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Mallet, F. (1999). Comparison of Competitive PCR and Positive Control-Based PCR. In: Kochanowski, B., Reischl, U. (eds) Quantitative PCR Protocols. Methods in Molecular Medicine™, vol 26. Humana Press. https://doi.org/10.1385/0-89603-518-2:103

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  • DOI: https://doi.org/10.1385/0-89603-518-2:103

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-518-8

  • Online ISBN: 978-1-59259-262-3

  • eBook Packages: Springer Protocols

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