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

Construct cosmid librariesby isolating large genomic DNA fragments from Monascus ruber

摘要:

摘要：【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 kb，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5 μg/μL，平均片段大小大于48 kb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D

Abstract:

Abstract: [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 Sau3AI 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 48kb, with a concentration of 5μg/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.