Restriction Endonuclease Digestion of DNA

  • Duncan R. Smith
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 58)

Abstract

The ability to cleave DNA at specific sites is one of the cornerstones of today’s methods of DNA manipulation. Restriction endonucleases are bacterial enzymes that cleave duplex DNA at specific target sequences with the production of defined fragments. These enzymes can be purchased from the many manufacturers of biotechnology products. The nomenclature of enzymes is based on a simple system, proposed by Smith and Nathans (1). The name of the enzyme (such as BamHI, EcoRI, and so on) tells us about the origin of the enzyme, but does not give us any information about the specificity of cleavage (see Note 1). This has to be determined for each individual enzyme. The recognition site for most of the commonly used enzymes is a short palindromic sequence, usually either 4, 5, or 6 bp in length, such as AGCT (for AluI), GAATTC (for EcoRI), and so on. Each enzyme cuts the palindrome at a particular site, and two different enzymes may have the same recognition sequence, but cleave the DNA at different points within that sequence. The cleavage sites fall into three different categories, either flush (or blunt) in which the recognition site is cut in the middle, or with either 5′- or 3′-over-hangs, in which case unpaired bases will be produced on both ends of the fragment. For a comprehensive review of restriction endonucleases, see Fuchs and Blakesley (2).

Keywords

Glycerol Phenol Albumin EDTA Agarose 

References

  1. 1.
    Smith, H. O. and Nathans, D. (1973) A suggested nomenclature for bacterial host modification and restriction systems and then enzymes. J. Mol. Biol. 81, 419–423.PubMedCrossRefGoogle Scholar
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    Fuchs, R. and Blakesley, R. (1983) Guide to the use of Type II restriction endonucleases. Methods Enzymol. 100, 3–38.PubMedCrossRefGoogle Scholar
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    McClelland, M., Hanish, J., Nelson, M., and Patel, Y. (1988) KGB: a single buffer for all restriction endonucleases. Nucleic Acids Res.. 16, 364.PubMedCrossRefGoogle Scholar
  4. 4.
    O’Farrell, P. H., Kutter, E., and Nakanishe, M. (1980) A restriction map of the bacteriophage T4 genome. Mol. Gen. Genet. 170, 411–435.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Duncan R. Smith
    • 1
  1. 1.Department of Colorectal SurgerySingapore General HospitalRepublic of Singapore

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