Peng Ri-He, Xiong Ai-Sheng, Yao Quan-Hong
Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotechnology Research Center, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, Shanghai, People's Republic of China.
Appl Microbiol Biotechnol. 2006 Nov;73(1):234-40. doi: 10.1007/s00253-006-0583-3. Epub 2006 Oct 5.
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.
开发了一种简单的两步高效方法，用于对细菌基因组中的基因进行多位点诱变。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个碱基对的重叠。将突变引入重叠的寡核苷酸引物中，以确保特定的引物-模板退火。使用PAGE去除污染的亲本模板、错误引物片段和残留引物。第二步涉及合成突变全长片段。第一步中所有纯化的PCR产物合并，并用作使用高保真DNA聚合酶进行第二次PCR的模板，以两个最外侧的侧翼寡核苷酸作为引物。使用POEP方法，我们已成功地将八个EcoRI位点引入大肠杆菌β-半乳糖苷酶（Lac Z）基因。获得多个突变位点的总体成功率为100%。POEP方法简单，仅涉及两步，对于多位点诱变可靠，有望在基因修饰中广泛应用。
