利用优化的反向PCR策略高效构建多位点突变体

Optimized inverse PCR strategy for constructing multilocus mutants efficiently

摘要:

蛋白质的突变体是研究其结构和功能的基础，文中旨在建立一种高效、快捷的多位点突变体构建方法。当要突变4个及以上相邻的氨基酸残基时，设计两长两短 (长引物Ⅰ/Ⅲ、短引物Ⅱ/Ⅳ) 4条引物：长引物包含突变位点，且突变碱基数≤20 bp，短引物不包含突变位点；两条引物的GC含量≤80%、退火温度之差≤40 ℃，分别以Ⅰ/Ⅱ和Ⅲ/Ⅳ两对引物和模板进行两组反向PCR扩增。扩增后各体系均可得到含有突变位点的非甲基化线性质粒，且以Ⅰ/Ⅱ和Ⅲ/Ⅳ为引物扩增得到的两组线性质粒的断开位点分布在突变位点两侧。用DpnⅠ酶切回收后等摩尔比混合的PCR产物除去甲基化模板，再进行一轮变性和退火处理，两组线性质粒在95 ℃变性后互相以来自对方的单链DNA为模板退火形成开环质粒，转化大肠杆菌感受态细胞即可得到包含突变位点的转化子。结果表明，该方法可同时突变4–11个连续氨基酸残基 (8–20 bp，将大幅简化多位点突变体的构建，从而进一步提高蛋白质结构和功能研究的效率。

关键词:引物，反向PCR，定点突变，多位点突变体

Abstract:

Mutants of proteins are the basis for studying their structure and function, this work aimed to establish an efficient and rapid method for constructing multi-site mutants. When four or more adjacent amino acid residues need to be mutated, firstly, two long and two short primers (long primersⅠ/Ⅲ, short primersⅡ/Ⅳ) were designed: the long primers contain mutated sites, and the number of mutant bases is ≤20 bp, the short primers do not contain mutated sites; GC contents of the long and short primers are ≤80%, and the difference of annealing temperature is ≤40 °C. Then two sets of reverse PCR amplifications were performed using primer pairs (Ⅰ/Ⅱand Ⅲ/Ⅳ) and templates, respectively. After amplification, each system can obtain non-methylated linear plasmids which contain mutated sites, and the breakpoints of the two sets of linear plasmids amplified by primers Ⅰ/Ⅱ and Ⅲ/Ⅳ were distributed on both sides of the mutated sites. Followed by digested by DpnⅠ to remove the methylated templates, the recovered PCR products, which were mixed in an equimolar ratio, were performed another round of denaturation and annealing: the two sets of linear plasmids were denatured at 95 °C and then annealed with each other’s single-stranded DNA as templates to form open-loop plasmids, and then the transformants containing the mutations will be obtained after transformed the open-loop plasmids into Escherichia coli competent cells. Results showed that, this method can mutate 4 to 11 consecutive amino acid residues (8–20 bp) simultaneously, which will greatly simplify the construction of multi-site mutants, Thereby improve the efficiency of protein structure and function research further.

Key words:primer, reverse PCR, site-directed mutagenesis, multilocus mutant