[通过从红曲霉中分离大基因组DNA片段构建黏粒文库]
[Construct cosmid libraries by isolating large genomic DNA fragments from Monascus ruber].
作者信息
An Yang, Yang Jing, Xu Xinxin, Liu Gang
机构信息
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
出版信息
Wei Sheng Wu Xue Bao. 2009 Oct;49(10):1385-8.
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 microg/microl. 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
从红曲霉中分离大基因组DNA片段用于构建黏粒文库。
方法
采用改良的酚-氯仿法分离基因组DNA。将分离得到的基因组DNA用Sau3AI平均酶切至40kb片段。然后，用Stratagene公司的Gigapack III XL包装提取物对片段进行包装。使用一对简并引物从该黏粒文库中扩增聚酮合酶（PKS）基因片段。
结果
用该方法分离得到的基因组DNA平均大小大于48kb，浓度为5μg/μl。构建的黏粒文库对红曲霉菌种基因组有10倍覆盖。通过PCR筛选获得了一个含有PKS基因同源物的黏粒。
结论
这种改良的红曲霉大片段基因组DNA分离方法高效可行。
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