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Oligonucleotide‐Directed Mutagenesis Without Phenotypic Selection

Thomas A. Kunkel¹

¹National 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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Basic Protocol
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol

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 1.4.5)
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; 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 1.11 and appendix 1A in this manual), phenol extraction (cpmb unit 2.1A), ethanol precipitation (cpmb unit 2.1A), agarose gel electrophoresis (cpmb unit 2.5A), preparation and transfection of competent cells (cpmb unit 1.8), and DNA sequence analysis (cpmb unit 7.4A)

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Figures

Figure 4.10.1

Oligonucleotide-directed mutagenesis without phenotypic selection. Single-stranded DNA containing a small number of uracil (U) residues in place of thymine is prepared from adut^– ung^– strain. A synthetic oligonucleotide containing the mutation of interest is annealed to the template (the mismatch is shown as a discontinuity in the oligonucleotide) and treated with T4 DNA polymerase and T4 DNA ligase to produce a double-stranded circular molecule. Introduction of this heteroduplex molecule into a wild-type (dut⁺ ung⁺) strain allows for the efficient recovery of mutant DNA.

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Literature Cited

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.