关于细菌人工染色体(BAC)文库载体DNA制备的研究

Research on the Preparation of Bacterial Artificial Chromosome (BAC) Vector DNA

细菌人工染色体 (BAC)文库在基因组研究中起着关键作用。构建BAC文库的一个关键步骤就是BAC载体DNA的制备。制备高质量的BAC载体DNA受到包括酶切、脱磷等诸多因素的影响。以BAC载体pECBAC1为材料 ,分别采用限制性内切酶BamHⅠ和HK脱磷酶对其进行酶切和脱磷 ,并结合凝胶回收纯化技术 ,制备了可用于进一步构建BAC文库的线性载体DNA。并在此基础上 ,确定了制备BAC载体DNA的适宜条件 ,其中包括确定适宜限制内切酶用量及酶切时间 ,脱磷酶种类及浓度和凝胶回收纯化线性载体DNA等关键步骤。

Bacterial artificial chromosome (BAC) library plays a pivotal role in genomics studies. A crucial step in BAC library construction is preparation of BAC vector DNA. Preparation of highly purified vector DNA is affected by a series of factors including digestion of restriction enzyme and dephosphorylation of linearized vector DNA. In our study, the BAC vector pECBAC1 was digested by the restriction enzyme of Bam HⅠ and dephosphorylated by HK phosphatase respectively. In order to improve the ligation capability of vector DNA, gel purification of linearized vector DNA was also conducted in our study. At the same time, we did a series of experiments to get high quality of vector DNA for construction of BAC library. They included the optimal concentration of restriction enzyme, optimal digestion time, the type of phosphatase and gel purification of linearized vector DNA.