部分扩增结合双片段连接法克隆系列截短基因突变体

Cloning of Series Truncated Gene Mutants by Partial Amplification Combining with Double Fragment Ligation

体外合成DNA伴随的随机突变是制约基因定点突变效率的重要因素。以克隆周期蛋白E(cyclin E)及其截短基因的激酶活性缺失突变体为例,对传统重叠延伸PCR(overlap extension PCR,OE-PCR)作适当改进,提出一种系列相关基因定点突变的优化方案。前期研究中已克隆了cyclin E基因及其2个截短基因T1、T2,并通过Eco RⅠ/SalⅠ双酶切插入pEGFP_C2载体。限制性酶切分析发现cyclin E基因序列中含有1个AgeⅠ位点将其分为F1、F2两段,目标突变位点KD位于F2段。对于cyclin E及其截短基因,F2段是完全一致的。因此,通过重叠延伸PCR扩增含突变位点的共有片段F2,从C2-cyclin E、C2-cyclin E_T1和C2-cyclin E_T2这3个原始质粒中切取相应的F1片段,再将F1与酶切的F2一起连接插入载体以重构完整突变体。对比检测发现OE-PCR扩增较短DNA片段更易成功。C2-cyclin E、C2-cyclin E_T1和C2-cyclin E_T2这3组均能筛选出一定数量克隆,经检测和序列鉴定,每组各得到1个序列完全准确的目标突变体。研究表明,采用部分扩增可以缩短DNA合成长度,避免了目标基因反复扩增等不利因素,从而减少随机突变;双片段连接避免了双AgeⅠ位点对常规酶切-连接的限制。两者相结合,可作为其他系列相关基因定点突变的优化方案。

The random mutation associated with DNA synthesized in vitro is an important factor that restricts the efficiency of site-directed mutation.Taking cyclin E and its truncated genes as an example,in order to clone their kinase-deficient mutants,the traditional overlap extension PCR(OE-PCR)was improved appropriately,and an improved scheme about site-directed mutagenesis for series related genes was proposed.In the previous study,cyclin E gene and its two truncated genes T1 and T2 were cloned and inserted into pEGFP_C2 vector by Eco RⅠ/SalⅠdouble digestion.Restriction analysis showed that cyclin E gene sequence contained one AgeⅠsite,which was divided into F1 and F2 segments,and the target mutation site KD was located in F2 segment.The F2 segment was identical to cyclin E and its truncated gene.Therefore,the consensus fragment F2 containing target mutation was amplified by overlap extension PCR(OE-PCR),and the corresponding F1 fragments were cut out from the three original plasmids C2-cyclin E,C2-cyclin E_T1 and C2-cyclin E_T2,and then F1 and the enzyme digested F2 were ligated into the vector to reconstruct the complete mutant.Comparative detection showed that OE-PCR was more successful in amplifying shorter DNA fragments.A certain number of clones were screened from C2-cyclin E,C2-cyclin E_T1 and C2-cyclin E_T2.After detection and DNA sequencing,one target mutant with completely accurate sequence was obtained from each group.Studies have shown that partial amplification could shorten the length of DNA synthesis,avoid the unfavorable factors such as repeated amplification of target genes,and thus reduce random mutations.And double-fragment ligation avoided the restriction of double AgeⅠsites on routine restriction-ligation.The combination of the two methods could be used as an optimization scheme for site-directed mutation of other related genes.