家猪Fosmid基因组文库的构建

Construction of Fosmid Library in Domestic Pig

本研究采用蛋白酶K和苯酚抽提法提取基因组DNA，通过物理方法随机剪切DNA，经T4DNA聚合酶和T4多聚核苷酸激酶末端修饰，琼脂糖凝胶电泳回收大小为25-40kb的片段，与pCC1FOS载体连接，噬菌体包装蛋白体外包装，转染大肠杆菌EP1300，构建了家猪Fosmid基因组文库。文库容量为8．50×10^9CFU／ml，平均插入片段大小约25kb，用家猪MC1卫星DNA做探针对文库进行初步筛选，获得含该卫星DNA的阳性克隆，该文库的构建为进一步筛选并研究猪卫星DNA序列奠定了基础。

Genomic DNA prepared for library construction was digested by proteinase K and extracted by phenol. The DNA was randomly sheared and end-repaired by T4 DNA Polymerase and T4 Polynucleotide Kinase. 25-40 kb fragment was recovered by 1% agarose gel electrophoresis and ligated with pCC1FOS Vector. Recombinant molecules were packaged in vitro with the Phage Packaging Extract, then transformedinto E coli EPI300. A fosmid genomic library of domestic pig was successfully constructed with the titer of 8.50 × 10^9 CFU/ml in which the insert fragment size was about 25 kb. Positive clones were achieved by in stitu hybridization with MClsatellite DNA as probe. It was indicated that the library was of high quality which can be used to screen new satellite DNA in domestic pig.