A rapid and efficient method for multiple-site mutagenesis with a modified overlap extension PCR |
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Authors: | Yingfeng An Jianfei Ji Wenfang Wu Anguo Lv Ribo Huang Yutuo Wei |
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Institution: | (1) Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China;(2) Graduate School of Chinese Academy of Sciences, Beijing, China;(3) Beckman Research Institute, City of Hope National Medical Center, Duarte, USA;(4) Laboratory of Protein Engineering, College of Life Science & Technology, Guangxi University, Guangxi, China |
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Abstract: | A rapid and efficient method to perform site-directed mutagenesis based on an improved version of overlap extension by polymerase
chain reaction (OE-PCR) is demonstrated in this paper. For this method, which we name modified (M)OE-PCR, there are five steps:
(1) synthesis of individual DNA fragments of interest (with average 20-bp overlap between adjacent fragments) by PCR with
high-fidelity pfu DNA polymerase, (2) double-mixing (every two adjacent fragments are mixed to implement OE-PCR without primers),
(3) pre-extension (the teams above are mixed to obtain full-length reassembled DNA by OE-PCR without primers), (4) synthesis
of the entire DNA of interest by PCR with outermost primers and template DNA from step 3, (5) post-extension (ten cycles of
PCR at 72°C for annealing and extension are implemented). The method is rapid, simple and error-free. It provides an efficient
choice, especially for multiple-site mutagenesis of DNAs; and it can theoretically be applied to the modification of any DNA
fragment. Using the MOE-PCR method, we have successfully obtained a modified sam1 gene with eight rare codons optimized simultaneously.
Electronic Supplementary Material Supplementary material is available for this article at . |
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