Frontiers of Agriculture in China >

2009 , Vol. 3 >Issue 1: 43 - 46

DOI: https://doi.org/10.1007/s11703-009-0010-5

RESEARCH ARTICLE

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

  • Haiqiang LU 1 ,
  • Hongwei YU 1 ,
  • Runfang GUO 1 ,
  • Yingmin JIA , 1,2
Expand
  • 1. Food Science and Technology College Agricultural University of Hebei, Baoding 071000, China
  • 2. Hebei University of Science and Technology, Shijiazhuang 05000,China

Received date: 27 Sep 2008

Accepted date: 09 Oct 2008

Published date: 05 Mar 2009

Copyright

2014 Higher Education Press and Springer-Verlag Berlin Heidelberg
Fold

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.

Key words： site-directed mutagenesis; phytase; the single-stranded product; PCR

Cite this article

Haiqiang LU , Hongwei YU , Runfang GUO , Yingmin JIA . Improvement of megaprimer method for site-directed mutagenesis and its application to phytase[J]. Frontiers of Agriculture in China, 2009 , 3(1) : 43 -46 . DOI: 10.1007/s11703-009-0010-5

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).

References

Publishing order | Descend order by publishing year | Descend order by cited within
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

DOI

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

DOI

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

DOI

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

DOI

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

DOI

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

DOI

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

Options
Outlines

/

〈 〉