Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli
Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli
复制标题
DOI：
10.1016/j.jbiotec.2005.11.014
复制
发表时间：
2006-05-29
影响因子：
4.1
通讯作者：
Hwang, Seokhwan
中科院分区：
文献类型：
--
作者：
Lee, Changsoo;Kim, Jaai;Hwang, Seokhwan
提出了基于实时 QPCR 的重组大肠杆菌培养物中质粒拷贝数的测定方法。使用携带 pBR322（一种常见的细菌克隆载体）的重组大肠杆菌 DH5 α 测试了两种基于绝对和相对分析的兼容方法。使用两个单独的引物组实现质粒和宿主染色体 DNA 的单独检测，分别对质粒 P-内酰胺酶基因 (bla) 和染色体 D-1-脱氧木酮糖 5-磷酸合酶基因 (dxs) 具有特异性。由于bla和dxs分别是pBR322和大肠杆菌染色体DNA的单拷贝基因，因此质粒拷贝数可以通过bla与dxs的拷贝比来确定。这些方法成功地应用于测定大肠杆菌宿主细胞pBR322的质粒拷贝数。绝对分析和相对分析的结果相同且具有高度重现性，变异系数 (CV) 值分别为 2.8-3.9% 和 4.7-5.4%。结果与之前报道的大肠杆菌宿主细胞内 pBR322 拷贝数的值相对应，即 15-20。与传统使用的 Southern blot 方法相比，本研究中引入的方法操作方便且具有成本效益。本研究设计的引物组可用于测定带有bla基因质粒载体的任何重组大肠杆菌的质粒拷贝数。 (c) 2005 Elsevier B.V. 保留所有权利。
Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5 alpha harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid P-lactamase gene (bla) and for the chromosomal D-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs.These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8-3.9% and 4.7-5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15-20.The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E. coli with a plasmid vector having bla gene. (c) 2005 Elsevier B.V. All rights reserved.