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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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A Simple Method for Elimination of False Positive Results in RT-PCR

  • Published : 2002.03.31
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Abstract

Discrimination between the amplification of mRNA and contaminating genomic DNA is a common problem when performing a reverse transcriptase-polymerase chain reaction (RT-PCR). Even after treatment of the samples with DNAse, it is possible that negative controls (samples in which no reverse transcriptase was added) will give positive results. This indicates that there was amplification of DNA, which was not generated during the reverse transcriptase step. The possibility exists that Taq DNA polymerase acts as a reverse transcriptase, generating cDNA from RNA during the PCR step. In order to test this hypothesis, we incubated samples with a DNAse-free RNAse after the cDNA synthesis. Comparison of the results that were obtained from these samples (incubated with or without DNAse-free RNAse) confirms that the reverse transcriptase activity of Taq DNA polymerase I is a possible source of false positive results when performing RT-PCR from intronless genes. Moreover, we describe here a simple and rapid method to overcome the false positive results that originate by this activity of Taq polymerase.

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References

  1. Chelly, J., Kaplan, J. C., Maire, P., Gautron, S. and Kahn, A. (1988) Transcription of the dystrophin gene in human muscle and non-muscle tissue. Nature 333, 858-860. https://doi.org/10.1038/333858a0
  2. Dilworth, D. D. and McCarrey, J. R. (1992) Single-step elimination of contaminating DNA prior to reverse transcriptase PCR. PCR Methods Appl. 1, 279-282. https://doi.org/10.1101/gr.1.4.279
  3. Grillo, M. and Margolis, F. L. (1990) Use of reverse transcriptase polymerase chain reaction to monitor expression of intronless genes. Biotechniques 9, 266-268.
  4. Jones, M. D. and Foulkes, N. S. (1989) Reverse transcription of mRNA by Thennus aquaticus DNA polymerase. Nucleic Acid Res. 17, 8387-8388. https://doi.org/10.1093/nar/17.20.8387
  5. Kwok, S. and Higuchi, R. (1989) Avoiding false positives with PCR. Nature 339, 237-238. https://doi.org/10.1038/339237a0
  6. Lo, Y.-M., Mehal, W. Z. and Fleming, K. A. (1988) False-positive results and the polymerase chain reaction. Lancet 2, 679.
  7. Loeb, L. A., Tartof, K. D. and Travaglini, E. C. (1973) Copying natural RNAs with E. coli DNA polymerase I. Nature New Biol. 242, 66-69. https://doi.org/10.1038/newbio242066a0
  8. Menon, R. S., Chang, Y. F., St Clair, J. and Ham, R. G. (1991) RT-PCR artifacts from processed pseudogenes. PCR Methods Appl. 1, 70-71. https://doi.org/10.1101/gr.1.1.70
  9. Sarkar, G. and Sommer, S. (1990) Shedding light on PCR contamination. Nature 343, 27. https://doi.org/10.1038/343027a0
  10. Schomig, E., Spitzenberger, F., Engelhardt, M., Martel, F., Ording, N. and Grtindemann, D. (1997) Molecular cloning and characterization of two novel transport proteins from rat kidney. FEBS Lett. 425, 79-86. https://doi.org/10.1016/S0014-5793(98)00203-8
  11. Veres, G., Gibbs, R. A., Scherer, S. E. and Caskey, C. T. (1987) The molecular basis of the sparse fur mouse mutation. Science 237, 415-417. https://doi.org/10.1126/science.3603027

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