Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

An automated process to extract plasmid DNA by alkaline lysis

  • 539 Accesses

  • 4 Citations

Abstract

With advances in the development of DNA vaccines and gene therapy, there is a growing need for plasmid DNA with high quality for fundamental research and clinical trials. In this report, a scalable automated process for large-scale preparation of plasmid is described. This process is based on alkaline lysis and can be easily scaled up to meet demands for larger quantities. In the process, harvested bacteria are passed through two mixing chambers at controlled speeds to affect lysis and control alkalinity. The resulting solution is passed through a series of filters to remove contaminants, and ethanol precipitated. System parameters are examined to maximize the quantity and quality of the prepared plasmid. Using this procedure, plasmid can be extracted and purified from 1 l of Escherichia coli cultures at an OD600 nm of 50 in less than 45 min. The plasmid yields are approximately 90 mg/l culture.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Clemson M, Kelly WJ (2003) Optimizing alkaline lysis for DNA plasmid recovery. Biotechnol Appl Biochem 37:235–244

  2. Crystal RG (1995) The gene as the drug. Nat Med 1:15–17

  3. Eastman EM, Durland RH (1998) Manufacturing and quality control of plasmid-based gene expression systems. Adv Drug Deliv Rev 30:33–48

  4. Ferreira GN, Monteiro GA, Prazeres DM, Cabral JM (2000) Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications. Trends Biotechnol 18:380–388

  5. Friedmann T (1997) The road toward human gene therapy—a 25-year perspective. Ann Med. 29:575–577

  6. Jin H, Li Y, Ma Z, Zhang F, Xie Q, Gu D, Wang B (2004) Effect of chemical adjuvants on DNA vaccination. Vaccine 22:2925–2935

  7. Kelly B, Hatton T (1991) The fermentation/downstream processing interface. Bioseparation 1:333–349

  8. Lahijani R, Hulley G, Soriano G, Horn NA, Marquet M (1996) High-yield production of pBR322-derived plasmids intended for human gene therapy by employing a temperature-controllable point mutation. Hum Gene Ther 7:1971–1980

  9. Levy MS, O’Kennedy RD, Ayazi-Shamlou P, Dunnill P (2000) Biochemical engineering approaches to the challenges of producing pure plasmid DNA. Trends Biotechnol 18:296–305

  10. Liljeqvist S, Stahl S (1999) Production of recombinant subunit vaccines: protein immunogens, live delivery systems and nucleic acid vaccines. J Biotechnol 73:1–33

  11. Prather KJ, Sagar S, Murphy J, Chartrain M (2003) Industrial scale production of plasmid DNA for vaccine and gene therapy: plasmid design, production, and purification. Enzyme Microb Technol 33:865–883

  12. Prazeres DM, Ferreira GN, Monteiro GA, Cooney CL, Cabral JM (1999) Large-scale production of pharmaceutical-grade plasmid DNA for gene therapy: problems and bottlenecks. Trends Biotechnol 17:169–174

  13. Prazeres DM, Schluep T, Cooney C (1998) Preparative purification of supercoiled plasmid DNA using anion-exchange chromatography. J Chromatogr A 806:31–45

  14. Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

  15. Theodossiou I, Collins IJ, Ward JM, Thomas ORT, Dunnill P (1997) The processing of a plasmid-based gene from E. Coli. Primary recovery by filtration. Bioprocess Eng 16:175–183

  16. Wang B, Merva M, Williams WV, Weiner DB (1995) Large-scale preparation of plasmid DNA by microwave lysis. Biotechniques 18:554–555

  17. Zhu K, Jin H, Ma Y, Ren Z, Xiao C, He Z, Zhang F, Zhu Q, Wang B (2005) A continuous thermal lysis procedure for the large-scale preparation of plasmid DNA. J Biotechnol 118: 257–264

Download references

Acknowledgment

This work was supported in part by the China Key Technologies R&D programme (2004AA213102 and 2003AA241110) and a research initiative fund provided to B.W. by CAU. We would also like to thank Dr. QL Yu for her assistance with the work.

Author information

Correspondence to Bin Wang.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Li, X., Jin, H., Wu, Z. et al. An automated process to extract plasmid DNA by alkaline lysis. Appl Microbiol Biotechnol 75, 1217–1223 (2007). https://doi.org/10.1007/s00253-007-0925-9

Download citation

Keywords

  • Plasmid
  • Large-scale
  • Alkaline lysis
  • Automated process