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 libraries by isolating large genomic DNA fragments from Monascus ruber

作　　者：安洋[1,2] 杨晶[1,2] 徐欣欣[1,2] 刘钢[1] 

机构地区：[1]中国科学院微生物研究所真菌地衣实验室,北京100101 [2]中国科学院研究生院,北京100049

出　　处：《微生物学报》2009年第10期1385-1388,共4页Acta Microbiologica Sinica

基　　金：国家科技支撑计划(2007BAI26B01)~~

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5μg/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DNA简便易行并且完全可以用于构建cosmid文库。[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 48 kb, with a concentration of 5/μg/μl. Theconstructed eosmid 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.

关 键 词：红色红曲霉 大片段基因组DNA cosmid文库 

分 类 号：Q78[生物学—分子生物学]