多片段扩增结合Gibson组装法克隆基因定点突变

Using multiple-fragment amplification combined with Gibson assembly to clone genes with site-directed mutations

摘要:

为寻找一种简洁高效的基因定点突变方案，利用Gibson组装技术对重叠延伸PCR法进行简化，并以克隆细胞周期蛋白依赖性激酶4基因单位点与双位点突变为例进行验证。采用与重叠延伸PCR相似的策略扩增含点突变的基因片段，同时采用双酶切制备线性质粒载体，保证质粒载体与基因片段含有一小段重叠序列作为接头。直接将基因片段与线性载体通过Gibson组装法拼接成完整质粒。经转化、筛选与检测，成功得到数个单位点与双位点目标突变体克隆，且阳性率均为100%。由于没有繁琐的多轮PCR扩增和频繁的DNA回收操作，也无需消化原始质粒，该方案避免了很多干扰定点突变的因素，能简便、高效地克隆基因单位点与多位点突变。对比而言，该方案规避了重叠延伸PCR与滚环扩增法的主要缺陷，是一种基因定点突变的良好解决方案。

Abstract:

In order to develop a simple and efficient site-directed mutagenesis solution,the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations,with the aim to simplify the overlap extension PCR.The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR.Meanwhile,an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments.The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal,single-reaction,creating a recombinant plasmid.After the recombinant plasmids were transformed into competent Escherichia coli DH5α,several clones were screened from each group.Through restriction analysis and DNA sequencing,it was found that the randomly selected clones were 100% target mutants.Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation,and there was no need to digest the original plasmid,this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis.Hence,genes with single or multiple mutations could be cloned easily and efficiently.In summary,the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol,making it a good solution for site-directed mutagenesis.