Abstract
A method for the intermediate recovery of plasmid DNA (pDNA) from alkaline lysates is described that comprises differential isopropanol precipitation steps. In a first low-cut precipitation, a smaller amount of isopropanol (20% v/v) is used so that only high molecular weight RNA precipitates. After solid liquid separation, a high-cut precipitation is performed by bringing isopropanol concentration to 70% v/v to precipitate pDNA. Tests made with lysates show that the differential precipitation increases purity threefold compared to the conventional one-step precipitation at 70% v/v without affecting pDNA recovery (>80%).
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References
Prather KJ, Sagar S, Murphy J et al (2003) Industrial scale production of plasmid DNA for vaccine and gene therapy: plasmid design, production, and purification. Enzym Microb Technol 33:865–883
Mairhofer J, Grabherr R (2008) Rational vector design for efficient non-viral gene delivery: challenges facing the use of plasmid DNA. Mol Biotechnol 39:97–104
Prazeres DMF, Monteiro GA (2014) Plasmid biopharmaceuticals. Microbiol Spectr 2. https://doi.org/10.1128/microbiolspec.PLAS-0022-2014
Prazeres DMF (2011) Plasmid biopharmaceuticals: basics, applications and manufacturing. John Wiley & Sons, Hoboken, NJ. https://doi.org/10.1002/9780470939918.ch12
Flock S, Labarbe R, Houssier C (1996) Dielectric constant and ionic strength effects on DNA precipitation. Biophys J 70:1456–1465
Freitas SS, Santos JAL, Prazeres DMF (2006) Optimization of isopropanol and ammonium sulfate precipitation steps in the purification of plasmid DNA. Biotechnol Prog 22:1179–1186
Silva-Santos AR, Alves CPA, Prazeres DMF et al (2017) A process for supercoiled plasmid DNA purification based on multimodal chromatography. Sep Purif Technol 182:94–100
Azzoni AR, Ribeiro SC, Monteiro GA et al (2007) The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression. J Gene Med 9:392–402
Sambrook J, Russell DW (2006) Neutral polyacrylamide gel electrophoresis. CSH Protoc 2006. https://doi.org/10.1101/pdb.prot4028
Singer A, Eiteman MA, Altman E (2009) DNA plasmid production in different host strains of Escherichia coli. J Ind Microbiol Biotechnol 36:521–530
Diogo MM, Queiroz JA, Prazeres DMF (2003) Assessment of purity and quantification of plasmid DNA in process solutions using high-performance hydrophobic interaction chromatography. J Chromatogr A 998:109–117
Acknowledgments
Funding received by iBB-Institute for Bioengineering and Biosciences from FCT-Portuguese Foundation for Science and Technology, Portugal (grant UID/BIO/04565/2019), the research project PureMab (PTDC/QEQ-PRS/0286/2014), and from Programa Operacional Regional de Lisboa 2020, Portugal (Project N. 007317) is acknowledged. A. Rita Silva-Santos acknowledges FCT for the PhD grant PD/BD/135138/2017 (BIOTECnico program).
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Wagner, A., Silva-Santos, A.R., Rosa, S.S., Gierak, S., Azevedo, A.M., Prazeres, D.M.F. (2021). Primary Purification of Plasmid DNA Using Differential Isopropanol Precipitation. In: Sousa, Â. (eds) DNA Vaccines. Methods in Molecular Biology, vol 2197. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0872-2_8
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DOI: https://doi.org/10.1007/978-1-0716-0872-2_8
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