1887

Abstract

The genetic diversity of a vesicular stomatitis virus population was analysed by RT-PCR, cloning and sequencing of two ~ 500 nucleotide regions of the virus genome. PCR amplifications were performed in parallel experiments with both and DNA polymerases, and important differences were observed. Between 10 and 22 mutations were detected when virus populations were analysed by amplification (20 clones from each region), whereas amplification of the same samples with revealed between 0 and 5 mutations. PCR fidelity assays, performed under the same PCR conditions as those used in the population analysis, showed that the error-rate estimate of 0·27 × 10 misincorpor- ations per bp per cycle was within the range estimated elsewhere from PCR amplification of recombinant plasmids (0·27–0·85 × 10 errors per bp per cycle) or from functional assays (0·2–2 × 10 errors per bp per cycle). The error rate of was found to be 9·3 times higher than the error rate of with DNA as a template, and about 10 times higher with cDNAs obtained by reverse transcription of viral RNA templates from natural populations. In the present study, we discuss (i) the implications of errors on the analysis of genetic variability, based on both the frequency and nature (replacement vs synonymous) of the observed substitutions and (ii) the sample size required to assess the genetic variability in a virus population generated by a single infection.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-79-12-2921
1998-12-01
2024-12-13
Loading full text...

Full text loading...

/deliver/fulltext/jgv/79/12/9880005.html?itemId=/content/journal/jgv/10.1099/0022-1317-79-12-2921&mimeType=html&fmt=ahah

References

  1. Andre P., Kim A., Khrapko K., Thilly W. G. 1997; Fidelity and mutational spectrum of Pfu DNA polymerase on a human mitochondrial DNA sequence. Genome Research 7:843–852
    [Google Scholar]
  2. Clark A. G., Whittam T. S. 1992; Sequencing errors and molecular evolutionary analysis. Molecular Biology and Evolution 9:744–752
    [Google Scholar]
  3. Cline J., Barman J. C., Hogrefe H. H. 1996; PCR fidelity of Pfu polymerase and other thermostable DNA polymerases. Nucleic Acids Research 24:3546–3551
    [Google Scholar]
  4. Dear S., Staden R. 1992; A standard file format for data from DNA sequencing instruments. DNA Sequence 3:107–110
    [Google Scholar]
  5. Devereux J., Haeberli P., Smithies O. 1984; A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12:387–395
    [Google Scholar]
  6. Devereux H. L., Brown D., Dusheiko G. M., Emery V. C., Lee C. A. 1997; Long-term evolution of the 5′UTR and a region of NS4 containing a CTL epitope of hepatitis C virus in two haemophilic patients. Journal of General Virology 78:583–590
    [Google Scholar]
  7. Domingo E., Holland J. J. 1994; Mutation rates and rapid evolution of RNA viruses. In The Evolutionary Biology of Viruses pp. 161–184 Morse S. S. Edited by New York: Raven Press;
    [Google Scholar]
  8. Dunning A. M., Talmud P., Humphries S. E. 1988; Errors in the polymerase chain reaction. Nucleic Acids Research 16:10393
    [Google Scholar]
  9. Eckert K. A., Kunkel T. A. 1990; High fidelity by the Thermus aquaticus DNA polymerase. Nucleic Acids Research 18:3737–3744
    [Google Scholar]
  10. Elena S. F., Davila M., Novella I. S., Holland J. J., Domingo E., Moya A. 1998; Evolutionary dynamics of fitness recovery from the debilitating effects of Muller’s ratchet. Evolution 52:351–356
    [Google Scholar]
  11. Endo T., Ikeo K., Gojobori T. 1996; Large-scale search for genes on which positive selection may operate. Molecular Biology and Evolution 13:685–690
    [Google Scholar]
  12. Ennis P. D., Zemmour J., Russell D. S., Parham P. 1990; Rapid cloning of HLA-A,B cDNA by using the polymerase chain reaction: frequency and nature of errors produced in amplification. Proceedings of the National Academy of Sciences, USA 87:2833–2837
    [Google Scholar]
  13. Fucharoen S., Fucharoen G., Fucharoen P., Fukamaki Y. 1989; A novel ochre mutation in the β-thalassemia gene of a Thai. Identification by direct cloning of the entire β-globin gene amplified using polymerase chain reactions. Journal of Biological Chemistry 264:7780–7783
    [Google Scholar]
  14. Holland J. J., de la Torre J. C., Clarke D. K., Duarte E. A. 1991; Quantitation of the relative fitness and great adaptability of clonal populations of RNA viruses. Journal of Virology 65:2960–2967
    [Google Scholar]
  15. Ji J. P., Loeb L. A. 1992; Fidelity of HIV-1 reverse transcriptase copying RNA in vitro. Biochemistry 31:954–958
    [Google Scholar]
  16. Keohavong P., Thilly W. G. 1989; Fidelity of DNA polymerases in DNA amplification. Proceedings of the National Academy of Sciences, USA 86:9253–9257
    [Google Scholar]
  17. Kwiatowski J., Skarecky D., Hernandez S., Pham D., Quijas F., Ayala F. J. 1991; High fidelity of the polymerase chain reaction. Molecular Biology and Evolution 8:884–887
    [Google Scholar]
  18. Li W. H., Graur D. 1991 Fundamentals of Molecular Evolution Sunderland, MA: Sinauer;
    [Google Scholar]
  19. Liu S. L., Rodrigo A. G., Shankarappa R., Learn G. H., Hsu L., Davidov O., Zhao L. P., Mullins J. I. 1996; HIV quasispecies and resampling. Science 273:415–416
    [Google Scholar]
  20. Logemann J., Schell J., Willmitzer L. 1987; Improved method for the isolation of RNA from plant tissues. Analytical Biochemistry 163:16–20
    [Google Scholar]
  21. Lu M., Funsch B., Wiese M., Roggendorf M. 1995; Analysis of hepatitis C virus quasispecies populations by temperature gradient gel electrophoresis. Journal of General Virology 76:881–887
    [Google Scholar]
  22. Lundberg K. S., Shoemaker D. D., Adams M. W., Short J. M., Sorge J. A., Mathur E. J. 1991; High-fidelity amplification using a thermostable DNA polymerase isolated from Pirococcus furiosus. Gene 108:1–6
    [Google Scholar]
  23. Marchuk D., Drumm M., Saulino A., Collins F. S. 1990; Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Research 19:1154
    [Google Scholar]
  24. Martell M., Esteban J. I., Quer J., Genesca J., Weiner A., Esteban R., Guardia J., Gomez J. 1992; Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. Journal of Virology 66:3225–3229
    [Google Scholar]
  25. Meyerhans A., Vartanian J. P., Wain-Hobson S. 1990; DNA recombination during PCR. Nucleic Acids Research 18:1687–1691
    [Google Scholar]
  26. Nei M. 1987 Molecular Evolutionary Genetics New York: Columbia University Press;
    [Google Scholar]
  27. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. 1988; Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491
    [Google Scholar]
  28. Smith D. B., McAllister J., Casino C., Simmonds P. 1997; Virus ‘quasispecies’: making a mountain out of a molehill?. Journal of General Virology 78:1511–1519
    [Google Scholar]
  29. Sokal R. R., Rohlf F. J. 1995 Biometry, 3rd edn. New York: W. H. Freeman;
    [Google Scholar]
  30. Steinhauer D. A., de la Torre J. C., Holland J. J. 1989; High nucleotide substitution error frequencies in clonal pools of vesicular stomatitis virus. Journal of Virology 63:2063–2071
    [Google Scholar]
  31. Viazov S., Riffelmann M., Khoudyakov Y., Fields H., Varenholz C., Roggerndorf M. 1997; Genetic heterogeneity of hepatitis G virus isolates from different parts of the world. Journal of General Virology 78:577–581
    [Google Scholar]
/content/journal/jgv/10.1099/0022-1317-79-12-2921
Loading
/content/journal/jgv/10.1099/0022-1317-79-12-2921
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error