Improvement of megaprimer method for site-directed mutagenesis and its application to phytase

Abstract

Site-directed mutagenesis is used extensively for probing gene function. In this paper we describe an improved megaprimer method to the site-directed mutagenesis of phytase from Aspergillus niger, which allowed the mutations to be performed more efficiently in less time than other traditional methods. Three rounds of PCR and two pairs of primers were required in this method, and additionally, the restriction enzyme Dpn I was used for the elimination of template instead of the gel purification in this process. The entire procedure was performed in one tube. Moreover, this method was easier for obtaining large mutant genes than other methods. We successfully carried out the site-directed mutagenesis of phytase by adopting this method.

Keywords

site-directed mutagenesis / phytase / the single-stranded product / PCR

References

[1] Barettino D, Feigenbutz M, Valcárcel R, Stunnenberg H G (1994). Improved method for PCR-mediated site-directed mutagenesis. Nucleic Acids Research, 22(3): 541-542

[2] Gyllensten U B, Erlich H A (1988). Generation of single-stranded DNA by the Polymerase Chain Reaction and its application to direct sequencing of the HLA-DQA. Proc Natl Acad Sci, U S A, 85(20): 7652-7656

[3] Kuipers O P, Boot H J, de Vos W M (1991). Improved site-directed mutagenesis method using PCR Nucleic Acids Research, 19(16): 4558

[4] Ling M M, Robinson B H (1997). Approaches to DNA mutagenesis: An Overview. Analytical Biochemistry, 254(2): 157-178

[5] Nabavi S, Nazar R N (2005). Simplified one-tube “megaprimer” polymerase chain reaction mutagenesis. Analytical Biochemistry, 345: 346-348

[6] Shenoy A R, Visweswariah S S (2003). Site-directed mutagenesis using a single mutagenic oligonucleotide and DpnI digestion of template DNA. Analytical Biochemistry, 319(2): 335-336

[7] Tomschy A, Tessier M, Wyss M, Brugger R, Broger C, Schnoebelen L, van Loon A P, Pasamontes L (2000). Optimization of the catalytic properties of Aspergillus fumigatus phytase based on the three-dimensional structure. Protein Science, 9(7): 1304-1311

[8] Zhao X, Huo K K, Li Y Y (2000). Synonymous codon usage in Pichia pastoris. Chinese Journal of Biotechnology16(3): 308-311

Acknowledgements

This work was a part of the research program financed by the Key Science and Technology Project of the 11th 5-year-plan of Hebei Province, China (No. 06220106D).

RIGHTS & PERMISSIONS

2014 Higher Education Press and Springer-Verlag Berlin Heidelberg