Impact of intrinsic DNA structure on processing of plasmids for gene therapy and DNA vaccines

Several non-Watson Crick DNA structures have been discovered to date, which may be incorporated into future plasmid constructs for gene therapy and DNA vaccine products. In this study, intrinsic DNA structures were included at a defined point in a 2.9 kb plasmid, and their effects on cell growth rat...

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Bibliographic Details
Published in:Journal of biotechnology 2004-11, Vol.114 (3), p.239-254
Main Authors: Cooke, James R., McKie, Elizabeth A., Ward, John M., Keshavarz-Moore, Eli
Format: Article
Language:English
Subjects:
Base Sequence
Biological and medical sciences
Bioreactors
Biotechnology
Cloning, Molecular
DNA - chemistry
DNA vaccines
DNA, Z-Form - chemistry
Escherichia coli - genetics
Escherichia coli - growth & development
Fundamental and applied biological sciences. Psychology
Gene therapy
Genetic Therapy - methods
Kanamycin Resistance - genetics
Molecular Sequence Data
Nucleic Acid Conformation
Plasmid production
Plasmid stability
Plasmids - chemistry
Plasmids - genetics
Platelet-Derived Growth Factor - genetics
Triplex-DNA
Vaccines, DNA - genetics
Z-DNA
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Summary:Several non-Watson Crick DNA structures have been discovered to date, which may be incorporated into future plasmid constructs for gene therapy and DNA vaccine products. In this study, intrinsic DNA structures were included at a defined point in a 2.9 kb plasmid, and their effects on cell growth rate, total plasmid yield, and topology (i.e. the relative proportions of supercoiled plasmid, open circular and linear forms), were determined. The stability of the inserted sequences were assessed using gel electrophoresis. Z-DNA was shown to be unstable in a batch Escherichia coli DH1 production system grown in complex medium. Encouragingly other sequences studied (triplex, bend and quadruplex) did not cause spontaneous deletions, and no detrimental effect was found on growth rate or on total plasmid yield; indicating that such sequences could be included in future DNA products without any detrimental effect on plasmid yields; although the intra molecular triplex studied significantly decreased the proportion of supercoiled species.
ISSN:0168-1656
1873-4863
DOI:10.1016/j.jbiotec.2004.06.011