Quantitative real-time polymerase chain reaction for determination of plasmid copy number in bacteria

Abstract

A method for determination of plasmid copy number (PCN) in bacteria by real-time quantitative polymerase chain reaction (QPCR) was developed as an alternative to current PCN assays. Conventional methods for PCN estimation are generally not of high throughput, laborious, have low reproducibility, require large amounts of biological samples and are applicable only for a narrow dynamic range. Real-time QPCR, using the ABI Prism 7000, was able to sensitively detect the quantity of the pUC ori based plasmid, NS3, transformed into Escherichia coli host, DH5alpha, to be 411+/-6.1. The PCN of pBR322 plasmid DNA in DH5alpha was estimated to be 40+/-0.6 which is within its previously reported PCN range of approximately 30 to 70. QPCR was found to show good reproducibility and high sensitivity in detecting a two fold difference in template concentration, and a wide linear dynamic range covering 0.5 pg to 50 ng of DNA. PCNs of DH5alpha bearing plasmids pBR322 and NS3 computed from real-time QPCR assay were validated by that of agarose gel assay, and a marginal difference of only 13.0% and 10.7% was found for the two plasmids respectively. The QPCR assay was able to detect changes in PCN of plasmid producing DH5alpha during the course of a 2 l batch fermentation.