Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1994 Jul 11;22(13):2587–2591. doi: 10.1093/nar/22.13.2587

A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase.

V Picard 1, E Ersdal-Badju 1, A Lu 1, S C Bock 1
PMCID: PMC308213  PMID: 8041621

Abstract

A rapid method for efficiently generating site-directed mutations on a clean sequence background is described. This modification of the megaprimer PCR mutagenesis approach can be performed in one tube in less than 4.5 hours, and does not require purification of intermediate products. High fidelity of DNA sequence replication is obtained by employing Pfu DNA polymerase and limiting the total number of amplification cycles to 30. The mutagenesis efficiency of the procedure is high enough to allow rapid, direct identification of mutants by restriction digest or sequencing techniques.

Full text

PDF
2588

Images in this article

2589Image
on p.2589
2590Image
on p.2590

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Atreya C. D., Atreya P. L., Pirone T. P. A strategy for rapid identification and selection of site-directed low-frequency point mutations. Biotechniques. 1990 Dec;9(6):702–703. [PubMed] [Google Scholar]
  2. Barettino D., Feigenbutz M., Valcárcel R., Stunnenberg H. G. Improved method for PCR-mediated site-directed mutagenesis. Nucleic Acids Res. 1994 Feb 11;22(3):541–542. doi: 10.1093/nar/22.3.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Hu G. DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3' end of a DNA fragment. DNA Cell Biol. 1993 Oct;12(8):763–770. doi: 10.1089/dna.1993.12.763. [DOI] [PubMed] [Google Scholar]
  6. Kuipers O. P., Boot H. J., de Vos W. M. Improved site-directed mutagenesis method using PCR. Nucleic Acids Res. 1991 Aug 25;19(16):4558–4558. doi: 10.1093/nar/19.16.4558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Landt O., Grunert H. P., Hahn U. A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene. 1990 Nov 30;96(1):125–128. doi: 10.1016/0378-1119(90)90351-q. [DOI] [PubMed] [Google Scholar]
  8. Lundberg K. S., Shoemaker D. D., Adams M. W., Short J. M., Sorge J. A., Mathur E. J. High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene. 1991 Dec 1;108(1):1–6. doi: 10.1016/0378-1119(91)90480-y. [DOI] [PubMed] [Google Scholar]
  9. Marini F., 3rd, Naeem A., Lapeyre J. N. An efficient 1-tube PCR method for internal site-directed mutagenesis of large amplified molecules. Nucleic Acids Res. 1993 May 11;21(9):2277–2278. doi: 10.1093/nar/21.9.2277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Sarkar G., Sommer S. S. The "megaprimer" method of site-directed mutagenesis. Biotechniques. 1990 Apr;8(4):404–407. [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES