一种用于构建红色红曲霉基因组cosmid文库的大片段DNA制备方法

摘要

[Objective] To isolate large genomic DNA fragments from Monascus ruber for the construction of cosmid libraries. [Methods] Modified phenol-chloroform method was used to isolate genomic DNA. The isolated genomic DNA was digested by Sau3 AI to 40kb fragments on average. Then, the fragments were packaged by Stratagene's Gigapack Ⅲ XL packaging extract. A pair of degenerate primers were used to amplify a fragment of PKS ( polyketide synthase) gene from this cosmid library. [ Results] The average size of genomic DNA isolated by this method was larger than 48 kb, with a concentration of 5 μg/μl. The constructed cosmid libraries had 10 folders coverage of the Monascus spp. genome. A cosmid containing the homologue of PKS gene was obtained by PCR screening. [ Conclusion] This modified method of isolating large fragments genomic DNA of Monascus ruber was efficient and feasible.%[目的]制备用于构建红色红曲霉cosmid文库的大片段基因组DNA.[方法]采用优化的酚氯仿抽提法制备DNA,并利用Sau3AI切割至平均大小为40 kh,然后使用Stratagene包装蛋白构建cosmid文库.基于PCR法使用同源探针从该文库中进行了目的基因的筛选.[结果]制备了浓度为5μg/μL,平均片段大小大于48 kh的红色红曲霉大片段基因组DNA.利用该DNA构建的cosmid文库基因组覆盖倍数为10,并筛选到了含有目的片段的cosmid.[结论]通过该方法制备红色红曲霉大片段基因组DNA简便易行并且完全可以用于构建cosmid文库.