A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis
A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis
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DOI：
10.1007/s00253-006-0583-3
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发表时间：
2006-11-01
影响因子：
5
通讯作者：
Yao, Quan-Hong
中科院分区：
文献类型：
--
作者：
Peng, Ri-He;Xiong, Ai-Sheng;Yao, Quan-Hong
建立了一种简单、两步高效的细菌基因组多位点突变方法。该方法被命名为聚丙烯酰胺凝胶电泳（PAGE）介导的重叠延伸聚合酶链反应（PCR）（POEP）。第一步涉及合成包含突变位点的单个片段，两个相邻片段之间有15至25个碱基对重叠。将突变引入重叠寡核苷酸引物中，这确保了特定的引物-模板退火。PAGE用于去除污染的亲本模板、错误引发片段和剩余引物。第二步涉及突变体全长片段的合成。将来自第一步的所有纯化的PCR产物合并并用作使用高保真DNA聚合酶的第二PCR的模板，其中两个最外侧侧翼寡核苷酸作为引物。利用POEP方法，我们成功地将8个EcoRI位点导入大肠杆菌β-半乳糖苷酶（Lac Z）基因。多突变位点的总体获得率为100%。POEP方法简单，只需两步，多位点突变可靠，有望在基因修饰中得到广泛应用。
A simple, two-step efficient method to perform multiple-site mutagenesis of a gene from bacterial genome was developed. The method was named polyacrylamide gel electrophoresis (PAGE)-mediated overlap extension polymerase chain reaction (PCR) (POEP). The first step involves synthesis of individual fragments containing mutant sites with 15- to 25-bp overlap between two adjacent fragments. Mutations were introduced into the overlapping oligonucleotide primers which ensured the particular primer-template annealing. PAGE was used to remove contaminating parental templates, mispriming fragments, and leftover primers. The second step involves synthesis of the mutant full-length fragment. All purified PCR products from the first step were combined and used as the template for a second PCR using high-fidelity DNA polymerase, with the two outermost flanking oligonucleotides as primers. Using the POEP method, we have successfully introduced eight EcoRI sites into the Escherichia coli beta-galactosidase (Lac Z) gene. The overall rate of obtaining the multiple mutant sites was 100%. The POEP method is simple, involving only two steps, and reliable for multiple-site mutagenesis and is promising to be widely used in gene modification.