GC克隆载体的制备及其克隆效率研究

Preparation of GC cloning vector and its cloning efficiency of PCR products

为了研究GC克隆技术的克隆效率,从商品化T载体出发,构建了一个含有2个XcmI位点的质粒;通过PCR技术引入突变后,经XcmI酶切得到3′端各突出一个C碱基的线性化载体。随后将该线性化载体与Taq酶催化得到的PCR产物进行了连接测试。结果表明:GC克隆效率低于同等条件下的TA克隆,对不同5′端碱基引物没有表现出明显偏好性。该研究为GC克隆技术在分子生物学实验上的应用提供了一定的理论依据。

To test the cloning efficiency of GC cloning, a plasmid, which will produce a linear plasmid with a 3r terminal C-overhang residue at both ends after digestion with Xcm I, was constructed. This GC cloning vector was ligated to PCR products obtained by PCR with Taq DNA polymerase. Bacterial transformation tests showed that GC cloning efficiency was lower than TA cloning. Unlike TA cloning, GC cloning showed no preference for the base type of 5r- terminal of the PCR primer pairs, These results may be helpful in the application of GC cloning in molecular biology experiments.