Asymmetric overlap extension PCR method bypassing intermediate purification and the amplification of wild-type template in site-directed mutagenesis
Asymmetric overlap extension PCR method bypassing intermediate purification and the amplification of wild-type template in site-directed mutagenesis
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DOI：
10.1007/s10529-007-9327-4
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发表时间：
2007-06-01
影响因子：
2.7
通讯作者：
Pei, Yan
中科院分区：
文献类型：
--
作者：
Xiao, Yue-Hua;Yin, Meng-Hui;Pei, Yan
通过不对称PCR和重叠延伸的结合，我们开发了一种新的位点定向诱变的不对称重叠延伸PCR （AOE-PCR）方法，该方法绕过了中间纯化的需要，并且排除了野生型模板的扩增。该方法将单个碱基突变导入棉花GTPase小基因，并以第一轮AOE-PCR产物为模板，重复该方法同时导入两个突变。我们的研究结果表明，AOE-PCR方法代表了原始重叠延伸PCR的有价值的改进，用于定点诱变。
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.