洋葱伯克霍尔德氏菌HMW DNA的制备及酶切研究

Study of Preparation and Restriction Endonuclease of B. cepacia HMW DNA

高质量粘粒基因组文库构建的关键是HMW DNA的长度至少为粘粒载体容量的10倍，通常粘粒载体的容量为30-50kb，因此提取的HMW DNA应不小于500kb。HMW DNA在制备时不能受到任何物理的剪切力，以免DNA断裂和损伤。利用琼脂糖凝胶包埋制备的DNA胶块经裂解和纯化后发现其DNA长度远大于500kb，明显优于商品化试剂盒提取和酚抽提法。用BamH Ⅰ、Sau3A Ⅰ、Xba Ⅰ和HindⅢ对DNA胶块进行不完全酶切研究表明，构建文库常用的Sau3A Ⅰ并不适合胶块内酶切反应，而BamHⅠ酶切B．cepacia HMW—DNA效果较好，产生的DNA片段集中在20-50kb之间，完全适合粘粒基因组文库的构建，为B．cepacia大插入片段基因组文库的构建以及功能基因组的研究奠定了良好的理论基础。

The key of the construction of high quality genornic cosmid fibrary is to prepare HMW DNA,the length of which should at least be ten times longer than of which of cosmid vector which usually ranges from 20 to 50 kb. PFGE analysis result indicated that the length of genomic DNA that was embedded and purified in agarose gel block was more than 500 kb longer,better than using genomic DNA extraction kit and phenol-chloroform extraction. Sou3A Ⅰ ,BamH Ⅰ ,HindⅢ and Xba Ⅰ were used to study partial restriction digestion of HMW DNA. The result showed that partial restriction digestion of HMW DNA with BamH Ⅰ could generate fragments with 20-50 kb size range,which were suitable for the construction of genomic cosmid library. This research would be useful for further construction of genomic cosmid library and research of functional genome.