Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavage sites (original) (raw)

Nature volume 272, pages 375–377 (1978)Cite this article

Abstract

RESTRICTION endonucleases recognise specific sequences in DNA, and these endonucleases, especially those which generate cohesive ends, have been widely used to clone DNA1. However, many DNAs lack sequences which are recognised by endonucleases such as _Eco_RI, _Hin_dIII or _Bam_HI. A general method of overcoming this problem has been described recently2. This approach involves the synthesis of oligonucleotides sensitive to a specific endonuclease and the blunt end ligation of these molecules to the DNA to be cloned. In contrast, we sought a method which avoids the insertion of additional nucleotides into a DNA sequence, but depends on direct modification of DNA. If a DNA sequence differs in only one base pair from the recognition sequence of a restriction endonuclease, a particular change of this base pair will generate the proper sequence. Here we describe a way of generating restriction endonuclease cleavage sites by single base changes derived after in vitro methylation of single-stranded DNA.

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References

  1. Roberts, R. J. Crit. Rev. Biochem. 4, 123–164 (1976).
    Article CAS Google Scholar
  2. Scheller, R. H., Dickerson, R. E., Boyer, H. W., Riggs, A. D. & Itakura, K. Science 196, 177–180 (1977).
    Article ADS CAS Google Scholar
  3. Messing, J., Gronenborn, B., Muller-Hill, B. & Hofschneider, P. H. Proc. natn. Acad. Sci. U.S.A. 74, 3642–3646 (1977).
  4. Dickson, R. C., Abelson, J., Barnes, W. M. & Reznikoff, W. S. Science 187, 27–35 (1975).
    Article ADS CAS Google Scholar
  5. Messer, W. & Melchers, F. in The Lactose Operon (eds Beckwith, J. R. & Zipser, D.) 305–315 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1970).
    Google Scholar
  6. Lawley, P. D. Mutat. Res. 23, 283–295 (1974).
    Article CAS Google Scholar
  7. Gerchman, L. L. & Lundlum, D. B. Biochim. biophys. Acta 308, 310–316 (1973).
    Article CAS Google Scholar
  8. Lawley, P. D. Sha, S. A. Biochem. J. 128, 117–132 (1972).
    Article CAS Google Scholar
  9. Kirtikar, D. M. & Goldthwait, D. A. Proc. natn. Acad. Sci. U.S.A. 71, 2022–2026 (1974).
    Article ADS CAS Google Scholar
  10. Cohen, S. N., Chang, A. C. Y. & Hsu, L. Proc. natn. Acad. Sci. U.S.A. 69, 2110–2114 (1972).
    Article ADS CAS Google Scholar
  11. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. J. molec. Biol. 113, 237–251 (1977).
    Article CAS Google Scholar
  12. v.d.Hondel, C. A., Pennings, L. & Schoenmakers, J. G. G. Eur. J. Biochem. 68, 55–70 (1976).
    Article Google Scholar

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Author notes

  1. JOACHIM MESSING
    Present address: Department of Bacteriology, University of California, Davis, California, 95616

Authors and Affiliations

  1. Institut für Genetik der Universität zu Köln, Weyertal 121, 5000, Köln, 41, FRG
    BRUNO GRONENBORN
  2. Max-Planck Institut für Biochemie, 8033, Martinsried, FRG
    JOACHIM MESSING

Authors

  1. BRUNO GRONENBORN
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  2. JOACHIM MESSING
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GRONENBORN, B., MESSING, J. Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavage sites.Nature 272, 375–377 (1978). https://doi.org/10.1038/272375a0

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