Digestion of DNA with Restriction Endonucleases

Kenneth D. Bloch1, Barbara Grossmann2

1 Massachusetts General Hospital, Boston, Massachusetts, 2 Amersham Life Science, Inc, Cleveland, OH
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.1
DOI:  10.1002/0471142727.mb0301s31
Online Posting Date:  May, 2001
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Abstract

Restriction endonucleases recognize short DNA sequences and cleave double‐stranded DNA at specific sites within or adjacent to the recognition sequences. Restriction endonuclease cleavage of DNA into discrete fragments is one of the most basic procedures in molecular biology. The first method presented in this unit is the cleavage of a single DNA sample with a single restriction endonuclease. A number of common applications of this technique are also described. These include digesting a given DNA sample with more than one endonuclease, digesting multiple DNA samples with the same endonuclease, and partially digesting DNA such that cleavage only occurs at a subset of the restriction sites. A protocol for methylating specific DNA sequences and protecting them from restriction endonuclease cleavage is also presented. A collection of tables describing restriction endonucleases and their properties (including information about recognition sequences, types of termini produced, buffer conditions, and conditions for thermal inactivation) is given at the end of the unit.

     
 
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Table of Contents

  • Restriction Endonucleases
  • Basic Protocol 1: Digesting a Single DNA Sample with a Single Restriction Endonuclease
  • Alternate Protocol 1: Digesting DNA with Multiple Restriction Endonucleases
  • Alternate Protocol 2: Digesting Multiple Samples of DNA
  • Alternate Protocol 3: Partial Digestion of DNA with Restriction Endonucleases
  • Support Protocol 1: Methylation of DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Digesting a Single DNA Sample with a Single Restriction Endonuclease

  Materials
  • DNA sample in H 2O or TE buffer ( appendix 22)
  • recipe10× restriction endonuclease buffers (see recipe)
  • Restriction endonucleases (Table 3.1.1 and Table 3.1.3)
  • 10× loading buffer (unit 2.5)
  • 0.5 M EDTA, pH 8.0 (optional; appendix 22)
  • Additional reagents and equipment for agarose or polyacrylamide gelelectrophoresis (unit 2.5 or unit 2.7), DNA extraction (optional; unit 2.1), andethanol precipitation (optional; unit 2.1)

Alternate Protocol 1: Digesting DNA with Multiple Restriction Endonucleases

  • recipe10× methylase buffer (see recipe)
  • S‐adenosylmethionine (SAM)
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Figures

Videos

Literature Cited

Literature Cited
   ACS (American Chemical Society). 1995. Biotech Buyers' Guide 1995. ACS, Washington, D.C.
   Fuchs, R. and Blakesley, R. 1983. Guide to the use of type II restriction endonucleases. Methods Enzymol. 100:3‐38.
   McClelland, M., Hanish, J., Nelson, M., and Patel, Y. 1988. KGB: A single buffer for all restriction endonucleases. Nucl. Acids Res. 16:364.
   O'Farrell, P.H., Kutter, E., and Nakanishe, M. 1980. Mol. Gen. Genet. 179:411‐435.
   Roberts, R.J. 1994. Restriction enzymes and their isoschizomers. Nucl. Acids Res. 20 (Supp.#1):2167‐2180.
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