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Kim, Jaai
School of Urban and Environmental Engineering
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Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Cited 164 times inthomson ciCited 155 times inthomson ci
Title
Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli
Author
Lee, ChangsooKim, JaaiShin, Seung GuHwang, Seokhwan
Keywords
Absolute quantification; Escherichia coli; pBR322; Plasmid copy number; Real-time quantitative PCR (QPCR); Relative quantification
Issue Date
2006-05
Publisher
ELSEVIER SCIENCE BV
Citation
JOURNAL OF BIOTECHNOLOGY, v.123, no.3, pp.273 - 280
Abstract
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 DH5α 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 β-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.
URI
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DOI
10.1016/j.jbiotec.2005.11.014
ISSN
0168-1656
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