Asymmetric overlap extension PCR method bypassing intermediate purification and the amplification of wild-type template in site-directed mutagenesis |
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Authors: | Yue-Hua Xiao Meng-Hui Yin Lei Hou Ming Luo Yan Pei |
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Affiliation: | (1) Key Laboratory of Biotechnology and Crop Quality Improvement of Ministry of Agriculture of China, Biotechnology Research Center, Southwest University, Chongqing, 400716, P.R. China |
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Abstract: | By combining asymmetric PCR and overlap extension, we developed a novel asymmetric overlap extension PCR (AOE-PCR) method for site-directed mutagenesis which bypassed the need for intermediate purification and excluded the amplification of a wild-type template. This method was used to introduce single base mutations into a small GTPase gene from cotton and to simultaneously introduce two mutations just by repeating this method using the first round AOE-PCR products as template. Our results suggested that the AOE-PCR method represents a valuable improvement of the original overlap extension PCR for site-directed mutagenesis. |
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Keywords: | Asymmetric overlap extension PCR Asymmetric PCR Overlap extension PCR Site-directed mutagenesis |
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