双片段连接法克隆融合基因及其突变体

Cloning of fusion gene and its mutants by double fragments ligation

为降低体外扩增DNA而引入随机突变的风险,仅通过PCR在CDK2序列5′端接入连接序列。由于双Bam HⅠ位点的干扰,cyclin E与CDK2序列无法依次插入载体,故采用双片段连接法将两者一起连接插入质粒载体以构建融合基因;采用相似策略,用CDK2突变体序列替换融合基因中对应野生型序列以构建融合基因突变体。两组双片段连接物转化感受态大肠杆菌后均能筛选出若干菌落,并成功鉴定出目标克隆。双片段连接法突破了严格的酶切位点要求对单片段顺序连接的限制,拓宽了酶切-连接法拼接DNA片段的适用范围。据此可充分利用现有条件,通过灵活置换DNA片段简化融合基因及其突变体克隆流程。

In order to reduce random mutation that was accompanied by DNA amplification in vitro,PCR was only used to attach a short linker sequence to the 5′end of CDK2(cyclin dependent kinase 2).Because of the interference of double Bam HⅠsites,cyclin E and CDK2 sequences could not be inserted into vector in an orderly way.Therefore,two gene fragments were linked together into vector,so as to construct the fusion gene.Employing similar strategy,CDK2 mutant sequence was used to displace the corresponding wild type sequence of the fusion gene in order to rebuild its mutant.After transformation of competent Escherichia coli with both double fragments ligation product,several colonies were screened out and some target clones were identified successfully.Double fragments ligation broke the rigorous demand of restriction sites of step by step ligation,and expanded the application of enzyme cleave-ligation in DNA assembly.On these grounds,it was convenient to clone fusion gene and its mutant by replacement of DNA elements with the assistance of the identified recombinant plasmid.