寡核苷酸片段载体的酶切连接和无缝克隆构建方法比较

Comparison of enzyme digestion and ligation and seamless cloning methods for the construction of oligonucleotide fragment vectors

目的比较酶切连接和无缝克隆方法在构建pCGS3-CK载体上的优缺点,为寡核苷酸片段载体的构建提供参考。方法酶切连接是需要合成两条互补的寡聚核苷酸单链,然后经过退火形成具有黏性末端的双链结构片段,在DNA连接酶的作用下与具有相同酶切的线性化载体连接,形成环状质粒。无缝克隆是在目标序列两端各加上15~20个与载体序列同源性的碱基,然后经过退火形成双链结构的目的片段,在重组酶的作用下与线性化的载体连接,形成环状质粒。结果从阳性率角度进行比较,酶切连接的阳性率为95.65%,而无缝克隆的阳性率为89.13%。结论与无缝克隆方法相比,用酶切连接方法构建寡核苷酸片段载体阳性率高,实验成本低。

Objective To compare the advantages and disadvantages of enzyme digestion and ligation and seamless cloning methods for the construction of pCGS3-CK vector,so as to provide reference for the construction of oligonucleotide fragment vectors.Methods Enzyme digestion and ligation was to synthesize two complementary oligonucleotide single strands,and then anneal to form a double-stranded structure fragment with a viscous terminal.Under the action of DNA ligase,it was connected with a linearized vector with the same enzyme digestion to form a circular plasmid.Seamless cloning was to add 15-20 bases homologous to the vector sequence at both ends of the target sequence,and then anneal to form the target fragment with double-stranded structure.Under the action of the recombinant enzyme,it was connected to the linearized vector to form a circular plasmid.Results Comparing from the perspective of positive rate,the positive rate of enzyme digestion and ligation was 95.65%,while the positive rate of seamless cloning was 89.13%.Conclusion Compared with the seamless cloning method,the positive rate of the oligonucleotide fragment vector constructed by the enzyme digestion and ligation method is high with low experimental costs.