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| Simultaneous Introduction of Double-Site Mutations by Improved SOE-PCR |
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Abstract [Objective] In order to improve the processing of construction the mutant with double mutations at two different residue sites. [Methods] According to the method for construction of the full-length DNA fragment by DNA shuffling, the primer pairs were designed at the mutation site, three small DNA fragments were PCR amplified using wild type gene as template, respectively, then separately mixed three DNA fragments and used as templates to carry out PCR without primer. The PCR product was subjected to the last-step PCR with primers to amplify the full-length gene AamanA with double-site mutations. [Results] The DNA sequenced results indicates the mutant with double mutations at residues E151 and E231 was succeed to create. The Thin-layer chromatography and enzyme activity assay clearly shown the catalytic activity of the mutant with double mutations was lost. [Conclusion] This is a simple, economic, rapid and effective method to construct two mutations in the DNA fragment. It has the potential application value in the field of molecular biology, such as characterization the reaction mechanism of enzyme and modification the structure of the proteins, and so on.
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Received: 28 April 2010
Published: 19 October 2010
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Corresponding Authors:
Jian-Song JU
E-mail: jujiansong@126.com
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| 1.何震宇，李月琴，林元藻. 重叠延伸PCR对DNA片段进行定点双突变. 氨基酸和生物资源, 2007, 29 (3) : 78-82.
He Z Y, Li Y Q, Lin Y Z. Simultaneous introduction of two separate site-specific mutations in a DNA fragment using Overlap Extension PCR. Amino acids & Biotic Resources, 2007, 29(3): 78-82.
2.张浩，毛秉智. 定点突变技术的研究进展. 免疫学杂志，2000，16（4）：108-110.
Zhang H, Mao B Z. Advance in technology of site-directed mutagenesis. Immunological Journal, 2000, 16(4): 108-110.
3.Horton R M, Cai Z L, Ho S N, et al. Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. BioTechniques, 1990, 8(5): 528-535.
4.Hechman K L, Pease L R. Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc, 2007, 2(4): 924-932.
5.Senanayake S D, and Brian D A. Precise large deletions by the PCR-based overlap extension method. Mol Biotechnol, 1995, 4(1): 13-15.
6.Zhang Y, Ju J, Ma Y，et al. Biochemical and structural characterization of the intracellular Mananase AaManA of Alicycolbacillus acidocaldarius reveals a novel Glycoside Hydrolase Family belonging to Clan GH-A. J Biol Chem, 2008, 283 (46): 31551-31558.
7.Stemmer W.: Rapid evolution of a protein in vitro by DNA shuffling. Nature, 1994, 370: 389-391.
8.Ohnishi K, Okuta A, Ju J, et al. Molecular breeding of 2, 3-dihydroxybiphenyl 1, 2-dioxygenase for enhanced resistance to 3-chlorocatechol. J Biochem (Tokyo), 2004, 135: 305-317.
9.Ju J, Misono H, Ohnishi K. Directed evolution of bacterial alanine racemases with higher expression level. J Biosci Bioeng, 2005, 100 (3): 246-254.
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