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. 1996 Jul 15;24(14):2627–2631. doi: 10.1093/nar/24.14.2627

Hypermutagenic PCR involving all four transitions and a sizeable proportion of transversions.

J P Vartanian 1, M Henry 1, S Wain-Hobson 1
PMCID: PMC145995  PMID: 8758987

Abstract

Very complex mutant libraries of the dihydrofolate reductase (DHFR) gene encoded by the Escherichia coli plasmid R67 were created using hypermutagenic PCR with biased deoxynucleotide triphosphate (dNTP) concentrations. Exploiting the particular stability of the G:T mismatch, the DHFR gene could be enriched in A+T by employing biased deoxypyrimidine triphosphate concentrations, i.e. [dTTP] > [dCTP]. A sizeable fraction of hypermutants were functional. A combination of [dTTP] > [dCTP] and [dGTP] > [dATP] biases generated mutations at unexpectedly low frequencies. This could be overcome by the addition of Mn2+ cations. Overall mutation frequencies of 10% per amplification (range 4-18% per clone) could be attained. All four transitions and a smaller number of transversions were produced throughout the gene. PCR mutagenesis could be so extensive as to inactivate all amplified versions of the gene.

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Selected References

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  1. Battula N., Loeb L. A. The infidelity of avian myeloblastosis virus deoxyribonucleic acid polymerase in polynucleotide replication. J Biol Chem. 1974 Jul 10;249(13):4086–4093. [PubMed] [Google Scholar]
  2. Beckman R. A., Mildvan A. S., Loeb L. A. On the fidelity of DNA replication: manganese mutagenesis in vitro. Biochemistry. 1985 Oct 8;24(21):5810–5817. doi: 10.1021/bi00342a019. [DOI] [PubMed] [Google Scholar]
  3. Cadwell R. C., Joyce G. F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 1992 Aug;2(1):28–33. doi: 10.1101/gr.2.1.28. [DOI] [PubMed] [Google Scholar]
  4. Chen K., Arnold F. H. Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5618–5622. doi: 10.1073/pnas.90.12.5618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Drake J. W. Rates of spontaneous mutation among RNA viruses. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4171–4175. doi: 10.1073/pnas.90.9.4171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fromant M., Blanquet S., Plateau P. Direct random mutagenesis of gene-sized DNA fragments using polymerase chain reaction. Anal Biochem. 1995 Jan 1;224(1):347–353. doi: 10.1006/abio.1995.1050. [DOI] [PubMed] [Google Scholar]
  7. Goodman M. F., Keener S., Guidotti S., Branscomb E. W. On the enzymatic basis for mutagenesis by manganese. J Biol Chem. 1983 Mar 25;258(6):3469–3475. [PubMed] [Google Scholar]
  8. Holland J. J., Domingo E., de la Torre J. C., Steinhauer D. A. Mutation frequencies at defined single codon sites in vesicular stomatitis virus and poliovirus can be increased only slightly by chemical mutagenesis. J Virol. 1990 Aug;64(8):3960–3962. doi: 10.1128/jvi.64.8.3960-3962.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Huang M. M., Arnheim N., Goodman M. F. Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR. Nucleic Acids Res. 1992 Sep 11;20(17):4567–4573. doi: 10.1093/nar/20.17.4567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kunz B. A., Kohalmi S. E. Modulation of mutagenesis by deoxyribonucleotide levels. Annu Rev Genet. 1991;25:339–359. doi: 10.1146/annurev.ge.25.120191.002011. [DOI] [PubMed] [Google Scholar]
  11. Kwok S., Kellogg D. E., McKinney N., Spasic D., Goda L., Levenson C., Sninsky J. J. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res. 1990 Feb 25;18(4):999–1005. doi: 10.1093/nar/18.4.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Martinez M. A., Pezo V., Marlière P., Wain-Hobson S. Exploring the functional robustness of an enzyme by in vitro evolution. EMBO J. 1996 Mar 15;15(6):1203–1210. [PMC free article] [PubMed] [Google Scholar]
  13. Martinez M. A., Vartanian J. P., Wain-Hobson S. Hypermutagenesis of RNA using human immunodeficiency virus type 1 reverse transcriptase and biased dNTP concentrations. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11787–11791. doi: 10.1073/pnas.91.25.11787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Martínez M. A., Sala M., Vartanian J. P., Wain-Hobson S. Reverse transcriptase and substrate dependence of the RNA hypermutagenesis reaction. Nucleic Acids Res. 1995 Jul 25;23(14):2573–2578. doi: 10.1093/nar/23.14.2573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mathews C. K., Ji J. DNA precursor asymmetries, replication fidelity, and variable genome evolution. Bioessays. 1992 May;14(5):295–301. doi: 10.1002/bies.950140502. [DOI] [PubMed] [Google Scholar]
  16. Meuth M. The molecular basis of mutations induced by deoxyribonucleoside triphosphate pool imbalances in mammalian cells. Exp Cell Res. 1989 Apr;181(2):305–316. doi: 10.1016/0014-4827(89)90090-6. [DOI] [PubMed] [Google Scholar]
  17. Modrich P. DNA mismatch correction. Annu Rev Biochem. 1987;56:435–466. doi: 10.1146/annurev.bi.56.070187.002251. [DOI] [PubMed] [Google Scholar]
  18. Pathak V. K., Temin H. M. Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: substitutions, frameshifts, and hypermutations. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6019–6023. doi: 10.1073/pnas.87.16.6019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pezo V., Martinez M. A., Wain-Hobson S. Fate of direct and inverted repeats in the RNA hypermutagenesis reaction. Nucleic Acids Res. 1996 Jan 15;24(2):253–256. doi: 10.1093/nar/24.2.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Phear G., Nalbantoglu J., Meuth M. Next-nucleotide effects in mutations driven by DNA precursor pool imbalances at the aprt locus of Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4450–4454. doi: 10.1073/pnas.84.13.4450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rellos P., Scopes R. K. Polymerase chain reaction-based random mutagenesis: production and characterization of thermostable mutants of Zymomonas mobilis alcohol dehydrogenase-2. Protein Expr Purif. 1994 Jun;5(3):270–277. doi: 10.1006/prep.1994.1041. [DOI] [PubMed] [Google Scholar]
  22. Rueda P., García-Barreno B., Melero J. A. Loss of conserved cysteine residues in the attachment (G) glycoprotein of two human respiratory syncytial virus escape mutants that contain multiple A-G substitutions (hypermutations). Virology. 1994 Feb;198(2):653–662. doi: 10.1006/viro.1994.1077. [DOI] [PubMed] [Google Scholar]
  23. Vartanian J. P., Meyerhans A., Asjö B., Wain-Hobson S. Selection, recombination, and G----A hypermutation of human immunodeficiency virus type 1 genomes. J Virol. 1991 Apr;65(4):1779–1788. doi: 10.1128/jvi.65.4.1779-1788.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Vartanian J. P., Meyerhans A., Sala M., Wain-Hobson S. G-->A hypermutation of the human immunodeficiency virus type 1 genome: evidence for dCTP pool imbalance during reverse transcription. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3092–3096. doi: 10.1073/pnas.91.8.3092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Zaccolo M., Williams D. M., Brown D. M., Gherardi E. An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues. J Mol Biol. 1996 Feb 2;255(4):589–603. doi: 10.1006/jmbi.1996.0049. [DOI] [PubMed] [Google Scholar]

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