Determination of plasmid content in eukaryotic and prokaryotic cells using Real-Time PCR
© Azzoni et al; licensee BioMed Central Ltd. 2006
Published: 10 October 2006
Determination of the plasmid content in prokaryotic cells during plasmid DNA (pDNA) production and in eukaryotic cells after transfection is crucial for DNA vaccine development. In Escherichia coli, pDNA is usually determined after plasmid extraction, either by UV absorbance or by densitometry of ethidium bromide-stained agarose gels. Fluorescence microscopy techniques are mainly used in eukaryotic cells. These techniques are time-consuming and labour-intensive and can not be used for process control. Thus, a Real-Time PCR method was developed to monitor the plasmid content of E. coli and Chinese Hamster Ovary (CHO) cells.
Monitoring the pDNA content on producing and recipient cells is crucial for DNA vaccine development. The Real-Time PCR method developed on this work provides quasi-online results and is suitable for process control and optimisation. The procedure was first developed for E. coli and was quickly adapted to CHO cells. It is therefore likely that it can be modified for application with other prokaryotic and eukaryotic systems.
This article is published under license to BioMed Central Ltd.