Abstract
Rapid advances in the fields of DNA vaccines and gene therapy have produced increased demands for large quantities of recombinant plasmid DNA. The protocol presented here extracts plasmid DNA in a scalable continuous process based on an alkaline lysis protocol. In the process, harvested bacteria are passed through two mixing chambers at controlled speeds to effect lysis and control alkalinity. The resulting solution is passed through a series of filters to remove contaminants and then ethanol precipitated. This process replaces all the centrifugation steps before obtaining crude plasmid and can be easily scaled up to meet demands for larger quantities. Using this procedure, plasmid can be extracted and purified from 4 l of Escherichia coli culture at an OD 600 nm of 50 in <90 min. The plasmid yields are ā¼80ā90 mg lā1 culture.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Friedmann, T. The road toward human gene therapyāa 25-year perspective. Ann. Med. 29, 575ā577 (1997).
Liljeqvist, S. & Stahl, S. Production of recombinant subunit vaccines: protein immunogens, live delivery systems and nucleic acid vaccines. J. Biotechnol. 73, 1ā33 (1999).
Crystal, R.G. The gene as the drug. Nat. Med. 1, 15ā17 (1995).
Ferreira, G.N., Monteiro, G.A., Prazeres, D.M. & Cabral, J.M. Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications. Trends Biotechnol. 18, 380ā388 (2000).
Levy, M.S., O'Kennedy, R.D., Ayazi-Shamlou, P. & Dunnill, P. Biochemical engineering approaches to the challenges of producing pure plasmid DNA. Trends Biotechnol. 18, 296ā305 (2000).
Eastman, E.M. & Durland, R.H. Manufacturing and quality control of plasmid-based gene expression systems. Adv. Drug Deliv. Rev. 30, 33ā48 (1998).
Prather, K.J., Sagar, S., Murphy, J. & Chartrain, M. Industrial scale production of plasmid DNA for vaccine and gene therapy: plasmid design, production, and purification. Enzyme Microb. Technol. 33, 865ā883 (2003).
Prazeres, D.M., Ferreira, G.N., Monteiro, G.A., Cooney, C.L. & Cabral, J.M. Large-scale production of pharmaceutical-grade plasmid DNA for gene therapy: problems and bottlenecks. Trends Biotechnol. 17, 169ā174 (1999).
Birnboim, H.C. & Doly, J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513ā1523 (1979).
Kelly, B. & Hatton, T. The fermentation/downstream processing interface. Bioseparation 1, 333ā349 (1991).
Prazeres, D.M., Schluep, T. & Cooney, C. Preparative purification of supercoiled plasmid DNA using anion-exchange chromatography. J. Chromatogr. A 806, 31ā45 (1998).
Theodossiou, I., Collins, I.J., Ward, J.M., Thomas, O.R.T. & Dunnill, P. The processing of a plasmid-based gene from E. coli. Primary recovery by filtration. Bioprocess Eng. 16, 175ā183 (1997).
Sambrook, J. & Russell, D.W. Molecular Cloning: A Laboratory Manual 3rd edn. (Cold Spring Press, New York, 2001).
Wang, B., Merva, M., Williams, W.V. & Weiner, D.B. Large-scale preparation of plasmid DNA by microwave lysis. Biotechniques 18, 554ā555 (1995).
Zhu, K., Jin, H., He, Z., Zhu, Q. & Wang, B. A continuous method for the large-scale extraction of plasmid DNA by modified boiling lysis. Nat. Protoc. 1, 3088ā3093 (2006).
Zhu, K. et al. A continuous thermal lysis procedure for the large-scale preparation of plasmid DNA. J. Biotechnol. 118, 257ā264 (2005).
Li, X. et al. An automated process to extract plasmid DNA by alkaline lysis. Appl. Microbiol Biotechnol. 75, 1217ā1223 (2007).
Hoopes, B.C. & McClure, W.R. Studies on the selectivity of DNA precipitation by spermine. Nucleic Acids Res. 9, 5493ā5504 (1981).
Gustincich, S., Manfioletti, G., Del Sal, G., Schneider, C. & Carninci, P. A fast method for high-quality genomic DNA extraction from whole human blood. Biotechniques 11, 298ā300, 302 (1991).
Ishaq, M., Wolf, B. & Ritter, C. Large-scale isolation of plasmid DNA using cetyltrimethylammonium bromide. Biotechniques 9, 19ā20, 22, 24 (1990).
Davies, M.J. et al. Improved manufacture and application of an agarose magnetizable solid-phase support. Appl. Biochem. Biotechnol. 68, 95ā112 (1997).
Jin, H. et al. Effect of chemical adjuvants on DNA vaccination. Vaccine 22, 2925ā2935 (2004).
Sayers, J.R., Evans, D. & Thomson, J.B. Identification and eradication of a denatured DNA isolated during alkaline lysis-based plasmid purification procedures. Anal. Biochem. 241, 186ā189 (1996).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Li, X., Jin, H., Wu, Z. et al. A continuous process to extract plasmid DNA based on alkaline lysis. Nat Protoc 3, 176ā180 (2008). https://doi.org/10.1038/nprot.2007.526
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2007.526
This article is cited by
-
Facile Alkaline Lysis of Escherichia coli Cells in High-Throughput Mode for Screening Enzyme Mutants: Arylsulfatase as an Example
Applied Biochemistry and Biotechnology (2016)
-
Effects and applications of sub-lethal ultrasound, electroporation and UV radiations in bioprocessing
Annals of Microbiology (2013)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
