基于平行模板的重叠延伸PCR优化策略
Optimization Strategy of Overlap Extension PCR Based on Parallel Template

基于平行模板的一步法重叠延伸PCR是传统基因定点突变的简化方案，其瑕疵是不完全延伸片段交叉互补会产生野生型干扰。为寻找原方案下形成交叉互补的关键因素，提高其定点突变效率，对诸多相关因素进行了分析。发现调整中介引物浓度稀释比可以增加目标突变体含量，但难以彻底消除野生型干扰；而基因长度则明显影响终产物构成。据此提出两步法重叠延伸PCR优化策略，先分别在两个小管中扩增突变位点上、下游片段，再直接混合继续PCR扩增目标基因突变体。以克隆细胞周期蛋白E的定点突变为例进行验证，得到90%阳性率。优化的两步法重叠延伸PCR方案既保持了相对简化的操作步骤，又明显提高了突变效率，是对传统重叠延伸PCR定点突变技术的重要改进。
That one-step overlap extension PCR which based on parallel template is a simplified scheme of traditional site-directed mutagenesis, however, its drawback is that some incomplete extension DNA fragments cross complement with each other lead to wild-type gene interference. In order to find out the key factors of cross complementation under the original scheme so as to improve the efficiency of site-directed mutagenesis, many related factors were checked. It was found that adjusting the concentration or dilution ratio of the intermediate primer can increase the content and percentage of the target mutant, but this treatment could not eliminate the wild type interference thoroughly; However, the gene length in the parallel template significantly affected the composition of the final product. According to the above conclusions, a two-step overlap extension PCR optimization strategy was described. First, the upstream and downstream fragments of the mutation site were amplified in two separate tubes with 1/10 diluted intermediate primers, and then they were mixed directly for another PCR reaction to amplify the mutated DNA. Taking the cloning of a mutation of cyclin E as example, 90% positive clones were obtained. The optimized two-step overlap extension PCR not only keeps the relatively simple operation steps, but also improves the mutation efficiency significantly, making it an important improvement of the traditional overlap extension PCR site-directed mutagenesis technology.