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. 1992 Apr 11;20(7):1487–1490. doi: 10.1093/nar/20.7.1487

Reverse transcriptase inhibits Taq polymerase activity.

L N Sellner 1, R J Coelen 1, J S Mackenzie 1
PMCID: PMC312227  PMID: 1374554

Abstract

Detection of viral RNA by polymerase chain reaction (PCR) requires the prior reverse transcription of the viral RNA. In order to minimise the number of manual manipulations required for processing large numbers of samples, we attempted to design a system whereby all the reagents required for both reverse transcription and amplification can be added to one tube and a single, non-interrupted thermal cycling program performed. Whilst attempting to set up such a one-tube system with Taq polymerase (Taq; Biotech International) and avian myoblastosis virus (AMV) reverse transcriptase (RT), we noticed a substantial decrease in the sensitivity of detection of viral RNA. Investigation of this phenomenon has revealed direct interference of RT with Taq polymerase. Evidence supporting this conclusion includes the following observations: (1) increasing the ratio of Taq to RT improves sensitivity; (2) adding non-homologous RNA improves sensitivity; (3) RT that has been heat inactivated prior to Taq addition does not exert this effect; (4) the effect is not sequence restricted; (5) the Mg2+ ions are not sequestered by RT. In addition, the effect is not limited to AMV RT, Moloney murine leukaemia virus RT also affects Taq activity.

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Selected References

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  1. Baker B. L., Di Bisceglie A. M., Kaneko S., Miller R., Feinstone S. M., Waggoner J. G., Hoofnagle J. H. Determination of hepatitis B virus DNA in serum using the polymerase chain reaction: clinical significance and correlation with serological and biochemical markers. Hepatology. 1991 Apr;13(4):632–636. [PubMed] [Google Scholar]
  2. Carman W. F., Williamson C., Cunliffe B. A., Kidd A. H. Reverse transcription and subsequent DNA amplification of rubella virus RNA. J Virol Methods. 1989 Jul;25(1):21–29. doi: 10.1016/0166-0934(89)90097-9. [DOI] [PubMed] [Google Scholar]
  3. Deubel V., Laille M., Hugnot J. P., Chungue E., Guesdon J. L., Drouet M. T., Bassot S., Chevrier D. Identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood. J Virol Methods. 1990 Oct;30(1):41–54. doi: 10.1016/0166-0934(90)90042-e. [DOI] [PubMed] [Google Scholar]
  4. Godec M. S., Asher D. M., Swoveland P. T., Eldadah Z. A., Feinstone S. M., Goldfarb L. G., Gibbs C. J., Jr, Gajdusek D. C. Detection of measles virus genomic sequences in SSPE brain tissue by the polymerase chain reaction. J Med Virol. 1990 Apr;30(4):237–244. doi: 10.1002/jmv.1890300402. [DOI] [PubMed] [Google Scholar]
  5. Hart C., Schochetman G., Spira T., Lifson A., Moore J., Galphin J., Sninsky J., Ou C. Y. Direct detection of HIV RNA expression in seropositive subjects. Lancet. 1988 Sep 10;2(8611):596–599. doi: 10.1016/s0140-6736(88)90639-3. [DOI] [PubMed] [Google Scholar]
  6. Larzul D., Guigue F., Sninsky J. J., Mack D. H., Bréchot C., Guesdon J. L. Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification. J Virol Methods. 1988 Jul;20(3):227–237. doi: 10.1016/0166-0934(88)90126-7. [DOI] [PubMed] [Google Scholar]
  7. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  8. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  9. Shibata D. K., Arnheim N., Martin W. J. Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J Exp Med. 1988 Jan 1;167(1):225–230. doi: 10.1084/jem.167.1.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Xu L., Harbour D., McCrae M. A. The application of polymerase chain reaction to the detection of rotaviruses in faeces. J Virol Methods. 1990 Jan;27(1):29–37. doi: 10.1016/0166-0934(90)90143-4. [DOI] [PubMed] [Google Scholar]
  11. Zhang W. D., Evans D. H. Detection and identification of human influenza viruses by the polymerase chain reaction. J Virol Methods. 1991 Jun;33(1-2):165–189. doi: 10.1016/0166-0934(91)90017-t. [DOI] [PubMed] [Google Scholar]

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