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. 1998 Jan 15;26(2):576–581. doi: 10.1093/nar/26.2.576

Modified base compositions at degenerate positions of a mutagenic oligonucleotide enhance randomness in site-saturation mutagenesis.

A Airaksinen 1, T Hovi 1
PMCID: PMC147293  PMID: 9421518

Abstract

Site-saturation mutagenesis, using degenerate oligonucleotide primers, is a frequently used method in introducing various mutations in a selected target codon. Oligonucleotides that are synthesized using equimolar concentrations of nucleoside phosphoramidites (dA, dC, dG, dT) in the positions to be saturated, result in a mutant population that is biased towards the original nucleotides. We found that this bias could be eliminated by modifying the concentrations of nucleoside phosphoramidites during the oligonucleotide synthesis. We synthesized eight degenerate oligonucleotides to saturate eight different codons, and sequenced a total of 344 mutagenized codons. In six of these eight oligonucleotides, we reduced to varying extents the concentrations of those nucleotides in the target positions that would form base pairs with the template. From the data, we analyzed the effects of different base compositions in the oligonucleotides when mutagenizing different codons, the influence of the positions of mismatches, and the significance of different non-Watson-Crick base pairs. Based on these results, we suggest levels to which different phosphoramidites should be reduced when synthesizing oligonucleotides for site-saturation mutagenesis.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aboul-ela F., Koh D., Tinoco I., Jr, Martin F. H. Base-base mismatches. Thermodynamics of double helix formation for dCA3XA3G + dCT3YT3G (X, Y = A,C,G,T). Nucleic Acids Res. 1985 Jul 11;13(13):4811–4824. doi: 10.1093/nar/13.13.4811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alber T., Bell J. A., Sun D. P., Nicholson H., Wozniak J. A., Cook S., Matthews B. W. Replacements of Pro86 in phage T4 lysozyme extend an alpha-helix but do not alter protein stability. Science. 1988 Feb 5;239(4840):631–635. doi: 10.1126/science.3277275. [DOI] [PubMed] [Google Scholar]
  3. Anand N. N., Brown D. M., Salisbury S. A. The stability of oligodeoxyribonucleotide duplexes containing degenerate bases. Nucleic Acids Res. 1987 Oct 26;15(20):8167–8176. doi: 10.1093/nar/15.20.8167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baumeister R., Müller G., Hecht B., Hillen W. Functional roles of amino acid residues involved in forming the alpha-helix-turn-alpha-helix operator DNA binding motif of Tet repressor from Tn10. Proteins. 1992 Oct;14(2):168–177. doi: 10.1002/prot.340140204. [DOI] [PubMed] [Google Scholar]
  5. Breslauer K. J., Frank R., Blöcker H., Marky L. A. Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3746–3750. doi: 10.1073/pnas.83.11.3746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown T., Leonard G. A., Booth E. D., Kneale G. Influence of pH on the conformation and stability of mismatch base-pairs in DNA. J Mol Biol. 1990 Apr 5;212(3):437–440. doi: 10.1016/0022-2836(90)90320-L. [DOI] [PubMed] [Google Scholar]
  7. Burstein E. S., Spalding T. A., Brann M. R. Amino acid side chains that define muscarinic receptor/G-protein coupling. Studies of the third intracellular loop. J Biol Chem. 1996 Feb 9;271(6):2882–2885. doi: 10.1074/jbc.271.6.2882. [DOI] [PubMed] [Google Scholar]
  8. Chang K. H., Auvinen P., Hyypiä T., Stanway G. The nucleotide sequence of coxsackievirus A9; implications for receptor binding and enterovirus classification. J Gen Virol. 1989 Dec;70(Pt 12):3269–3280. doi: 10.1099/0022-1317-70-12-3269. [DOI] [PubMed] [Google Scholar]
  9. Hedstrom L., Graf L., Stewart C. B., Rutter W. J., Phillips M. A. Modulation of enzyme specificity by site-directed mutagenesis. Methods Enzymol. 1991;202:671–687. doi: 10.1016/0076-6879(91)02031-4. [DOI] [PubMed] [Google Scholar]
  10. Hermes J. D., Parekh S. M., Blacklow S. C., Köster H., Knowles J. R. A reliable method for random mutagenesis: the generation of mutant libraries using spiked oligodeoxyribonucleotide primers. Gene. 1989 Dec 7;84(1):143–151. doi: 10.1016/0378-1119(89)90148-0. [DOI] [PubMed] [Google Scholar]
  11. Hughes P. J., Horsnell C., Hyypiä T., Stanway G. The coxsackievirus A9 RGD motif is not essential for virus viability. J Virol. 1995 Dec;69(12):8035–8040. doi: 10.1128/jvi.69.12.8035-8040.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Juteau J. M., Billings E., Knox J. R., Levesque R. C. Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser130 in class A beta-lactamases. Protein Eng. 1992 Oct;5(7):693–701. doi: 10.1093/protein/5.7.693. [DOI] [PubMed] [Google Scholar]
  13. Malhotra K. T., Nicholas R. A. Substitution of lysine 213 with arginine in penicillin-binding protein 5 of Escherichia coli abolishes D-alanine carboxypeptidase activity without affecting penicillin binding. J Biol Chem. 1992 Jun 5;267(16):11386–11391. [PubMed] [Google Scholar]
  14. O'Donohue M. J., Kneale G. G. Site-directed and site-saturation mutagenesis using oligonucleotide primers. Methods Mol Biol. 1994;30:211–225. doi: 10.1385/0-89603-256-6:211. [DOI] [PubMed] [Google Scholar]
  15. O'Donohue M. J., Scarlett G. P., Kneale G. G. Tyr26 and Phe73 are essential for full biological activity of the Fd gene 5 protein. FEMS Microbiol Lett. 1993 May 15;109(2-3):219–223. doi: 10.1016/0378-1097(93)90023-u. [DOI] [PubMed] [Google Scholar]
  16. Olesen K., Kielland-Brandt M. C. Altering substrate preference of carboxypeptidase Y by a novel strategy of mutagenesis eliminating wild type background. Protein Eng. 1993 Jun;6(4):409–415. doi: 10.1093/protein/6.4.409. [DOI] [PubMed] [Google Scholar]
  17. Sayers J. R., Krekel C., Eckstein F. Rapid high-efficiency site-directed mutagenesis by the phosphorothioate approach. Biotechniques. 1992 Oct;13(4):592–596. [PubMed] [Google Scholar]
  18. Werntges H., Steger G., Riesner D., Fritz H. J. Mismatches in DNA double strands: thermodynamic parameters and their correlation to repair efficiencies. Nucleic Acids Res. 1986 May 12;14(9):3773–3790. doi: 10.1093/nar/14.9.3773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Zon G., Gallo K. A., Samson C. J., Shao K. L., Summers M. F., Byrd R. A. Analytical studies of 'mixed sequence' oligodeoxyribonucleotides synthesized by competitive coupling of either methyl- or beta-cyanoethyl-N,N-diisopropylamino phosphoramidite reagents, including 2'-deoxyinosine. Nucleic Acids Res. 1985 Nov 25;13(22):8181–8196. doi: 10.1093/nar/13.22.8181. [DOI] [PMC free article] [PubMed] [Google Scholar]

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