An Yang, Yang Jing, Xu Xinxin, Liu Gang
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Wei Sheng Wu Xue Bao. 2009 Oct;49(10):1385-8.
To isolate large genomic DNA fragments from Monascus ruber for the construction of cosmid libraries.
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.
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.
This modified method of isolating large fragments genomic DNA of Monascus ruber was efficient and feasible.
从红曲霉中分离大基因组DNA片段用于构建黏粒文库。
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。
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。
这种改良的红曲霉大片段基因组DNA分离方法高效可行。
