基于PCR的质粒快速定点诱变方法建立与评价

基因突变对生命的进化具有重要意义，针对质粒的DNA定点诱变技术也是基因工程、蛋白质工程研究中的重要手段之一。为了提高质粒定点诱变的效率，本研究利用引物部分重叠的设计方案，使用Tm值相对固定的引物设计模式，将同一引物分为重叠区（Tm=50±2℃）和非重叠区（Tm=60±2℃），并在严格控制模板用量（2 pg/kb）的基础上，通过20个PCR循环对目标质粒进行定点诱变扩增，随后取0.5 μL产物直接用于转化。在FastPfu Fly酶系中，利用此法构建了6个含碱基替换、缺失和插入的质粒，均获得成功，突变效率可达96%以上，阳性克隆获得数达70个以上。此外，利用4种不同PCR酶系对该法的适用性进行了评价，结果表明突变效率均可达93%以上，阳性克隆获得数均在10个以上。通过适当增加PCR模板用量（10 pg/kb）并使用纯化后的PCR产物进行转化，该法可适用于转化效率大于106（cfu/μg）的任意感受态细胞，对应的突变效率可大于91%，阳性克隆获得数大于20。根据本法的作用原理，该方案适合质粒中10～20 bp（因重叠区GC含量及碱基序列的不同而改变）以内的任意碱基替换和插入，以及任意长度的DNA片段缺失。且具有通用性强、耗时少、诱变成功率高、成本低、对感受态及转化效率无特殊要求等优点，适合各实验室的日常研究使用。

Development and Evaluation of a Rapid PCR Based Site-directed Mutagenesis Protocol for Plasmids

Mutagenesis plays an important role during evolution of life. The technique of site-directed mutagenesis within plasmid molecule is one of the key methods in genetic and protein engineering. In order to improve the efficiency of plasmid mutagenesis process, we have developed a rapid PCR based site directed mutagenesis protocol in this study. In this protocol, the primers were designed as partial overlapping pairs with relative fixed Tm, and each one of the primers can be divided into an overlapping region (Tm=50±2 ℃) and a non overlapping region (Tm=60±2 ℃). When the amount of template was strictly controlled (2 pg/kb plasmid size), the site directed mutagenesis of target plasmid could be achieved by 20 PCR cycles, followed by 0.5 μL of the PCR product directly used for transformation. Using this protocol, 6 plasmids containing base substitutions, deletions and insertions were successfully constructed by FastPfu Fly system, with more than 96% mutation efficiency and greater than 70 positive clones. Additionally, the applicability of this protocol was evaluated with 4 different PCR systems, and the result showed that the mutation efficiency could be 93% or more, the number of positive clones was more than 10. Furthermore, by increasing the amount of PCR template (to 10 pg/kb plasmid size) and purifing PCR products for transformation, the method can be applied to any competent cells with the transformation efficiency above 106 (cfu/μg), for the mutation efficiency greater than 91%, and the positive clones obtained were more than 20. According to the principle of this technique, it is suitable for the substitution and insertion of DNA fragments of 10-20 bp (depends on GC content and the sequence of overlapping region) or deletion of any length in a plasmid. The method is highly versatile, time saving, robust and economic. Without special requirements for competent cells and transformation efficiency, the protocol suits the needs of site directed mutagenesis in most laboratory.