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. 1993 Aug 25;21(17):3977–3980. doi: 10.1093/nar/21.17.3977

A simple and efficient method for site-directed mutagenesis with double-stranded plasmid DNA.

D Lai 1, X Zhu 1, S Pestka 1
PMCID: PMC309981  PMID: 8371973

Abstract

A general, simple and efficient method for preparing site-specific mutations in double-stranded plasmid DNA without the need for special plasmids, bacterial strains or reagents is described. Only one synthetic oligonucleotide for each mutation is required, subcloning is unnecessary and a high efficiency of mutation (58-97%) was obtained. If two synthetic oligonucleotide primers are used, two separate mutations can be simultaneously created in a single reaction tube.

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Selected References

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  1. Cordell B., Bell G., Tischer E., DeNoto F. M., Ullrich A., Pictet R., Rutter W. J., Goodman H. M. Isolation and characterization of a cloned rat insulin gene. Cell. 1979 Oct;18(2):533–543. doi: 10.1016/0092-8674(79)90070-9. [DOI] [PubMed] [Google Scholar]
  2. DeChiara T. M., Erlitz F., Tarnowski S. J. Procedures for in vitro DNA mutagenesis of human leukocyte interferon sequences. Methods Enzymol. 1986;119:403–415. doi: 10.1016/0076-6879(86)19060-4. [DOI] [PubMed] [Google Scholar]
  3. Derbyshire K. M., Salvo J. J., Grindley N. D. A simple and efficient procedure for saturation mutagenesis using mixed oligodeoxynucleotides. Gene. 1986;46(2-3):145–152. doi: 10.1016/0378-1119(86)90398-7. [DOI] [PubMed] [Google Scholar]
  4. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  6. Jones D. H., Howard B. H. A rapid method for site-specific mutagenesis and directional subcloning by using the polymerase chain reaction to generate recombinant circles. Biotechniques. 1990 Feb;8(2):178–183. [PubMed] [Google Scholar]
  7. Kadowaki H., Kadowaki T., Wondisford F. E., Taylor S. I. Use of polymerase chain reaction catalyzed by Taq DNA polymerase for site-specific mutagenesis. Gene. 1989 Mar 15;76(1):161–166. doi: 10.1016/0378-1119(89)90018-8. [DOI] [PubMed] [Google Scholar]
  8. Kramer B., Kramer W., Fritz H. J. Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli. Cell. 1984 Oct;38(3):879–887. doi: 10.1016/0092-8674(84)90283-6. [DOI] [PubMed] [Google Scholar]
  9. Li B. L., Langer J. A., Schwartz B., Pestka S. Creation of phosphorylation sites in proteins: construction of a phosphorylatable human interferon alpha. Proc Natl Acad Sci U S A. 1989 Jan;86(2):558–562. doi: 10.1073/pnas.86.2.558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Newton C. R., Graham A., Heptinstall L. E., Powell S. J., Summers C., Kalsheker N., Smith J. C., Markham A. F. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 1989 Apr 11;17(7):2503–2516. doi: 10.1093/nar/17.7.2503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nishimura A., Morita M., Nishimura Y., Sugino Y. A rapid and highly efficient method for preparation of competent Escherichia coli cells. Nucleic Acids Res. 1990 Oct 25;18(20):6169–6169. doi: 10.1093/nar/18.20.6169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Sommer S. S., Cassady J. D., Sobell J. L., Bottema C. D. A novel method for detecting point mutations or polymorphisms and its application to population screening for carriers of phenylketonuria. Mayo Clin Proc. 1989 Nov;64(11):1361–1372. doi: 10.1016/s0025-6196(12)65378-6. [DOI] [PubMed] [Google Scholar]
  13. Vallette F., Mege E., Reiss A., Adesnik M. Construction of mutant and chimeric genes using the polymerase chain reaction. Nucleic Acids Res. 1989 Jan 25;17(2):723–733. doi: 10.1093/nar/17.2.723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Zell R., Fritz H. J. DNA mismatch-repair in Escherichia coli counteracting the hydrolytic deamination of 5-methyl-cytosine residues. EMBO J. 1987 Jun;6(6):1809–1815. doi: 10.1002/j.1460-2075.1987.tb02435.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]

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