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 Neuroscience
Unit Number:  Unit 4.10
DOI:  10.1002/0471142301.ns0410s03
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 unit presents a protocol which 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. The length of the oligonucleotide primer is highly variable and depends on the nature of the change being made.

     
 
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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 (see recipe) containing 0.25 µg/ml uridine
  • E. coli CJ236 or alternative dut ung F′ strain (Invitrogen and CPMB 97.80.4711)
  • 5× PEG/NaCl solution (see recipe)
  • TE buffer ( appendix 2A)
  • T4 polynucleotide kinase and 10× kinase buffer (see recipe)
  • 10 mM ATP (neutralize pH with 1 M NaOH; ɛ 259 = 15.4 mM−1 cm−1 at pH 7.0)
  • Mutagenic oligonucleotide primer
  • 100 and 500 mM EDTA, pH 8.0( appendix 2A)
  • 20× SSC ( appendix 2A)
  • 5× polymerase mix (see recipe)
  • T4 or T7 DNA polymerase (not Sequenase; see step annotation)
  • T4 DNA ligase
  • Additional reagents and equipment for phage titering (see CPMB UNIT and appendix 1A in this manual), phenol extraction (CPMB UNIT ), ethanol precipitation (CPMB UNIT ), agarose gel electrophoresis (CPMB UNIT ), preparation and transfection of competent cells (CPMB UNIT ), and DNA sequence analysis (CPMB UNIT )
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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. Ann. Rev. Genet. 19:423‐463.
Key Reference
   Kunkel, 1985. See above.
  Presents the original description of this technique.
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