Oligonucleotide‐Directed Mutagenesis without Phenotypic Selection

Thomas A. Kunkel1

1 National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 8.1
DOI:  10.1002/0471142727.mb0801s13
Online Posting Date:  May, 2001
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Abstract

A DNA sequence can be specifically altered by synthesizing the desired sequence change within an oligonucleotide, and then converting this into a biologically active circular DNA strand by using the oligonucleotide to prime in vitro synthesis on a single‐stranded circular template. This protocol uses a DNA template containing a small number of uracil residues in place of thymine. Use of the uracil‐containing template allows rapid and efficient recovery of mutants; in principle this same template can be applied to most of the other mutagenesis protocols in use.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Single‐stranded bacteriophage vector with insert
  • TY medium containing 0.25 µg/ml uridine (unit 1.5)
  • E. coli CJ236 or alternative dutung F′ strain ( Invitrogen and 97.80.4711Table 1.4.5)
  • recipe5× PEG/NaCl solution
  • TE buffer ( appendix 22)
  • T4 polynucleotide kinase (unit 3.10) and 10× kinase buffer (unit 3.4)
  • 10 mM ATP (unit 3.4)
  • Mutagenic oligonucleotide primer
  • 100 and 500 mM EDTA, pH 8.0 ( appendix 22)
  • 20× SSC ( appendix 22)
  • recipe5× polymerase mix
  • T4 or T7 DNA polymerase (not Sequenase; see unit 3.5)
  • T4 DNA ligase (measured in Weiss units; unit 3.14)
  • Additional reagents and equipment for phage titering (unit 1.11), phenol extraction (unit 2.1), ethanol precipitation (unit 2.1), agarose gel electrophoresis (unit 2.5), preparation and transfection of competent cells (unit 1.8), and DNA sequence analysis (unit 7.4)
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Figures

Videos

Literature Cited

   Kunkel, T.A. 1985. Rapid and efficient site‐specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. U.S.A. 82:488‐492.
   Kunkel, T.A., Roberts, J.D., and Zakour, R.A. 1987. Rapid and efficient site‐specific mutagenesis without phenotypic selection. Meth. Enzymol. 154:367‐382.
   Smith, M. 1985. In vitro mutagenesis. Annu. Rev. Genet. 19:423‐463.
Key Reference
   Kunkel, 1985. See above.
  Presents the original description of this technique.
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