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Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
25602 43584 2006 8 PDF Available
Title
Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli
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.

Keywords
Plasmid copy number; Real-time quantitative PCR (QPCR); Absolute quantification; Relative quantification; pBR322; Escherichia coli
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Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 123, Issue 3, 29 May 2006, Pages 273–280
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us