title: Comparison of alkaline lysis with electroextraction and optimization of electric pulses to extract plasmid DNA from Escherichia coli.
abstract: The use of plasmid DNA (pDNA) as a pharmaceutical tool has increased since it represents a safer vector for gene transfer compared to viral vectors. Different pDNA extraction methods have been described; among them is alkaline lysis, currently the most commonly used. Although alkaline lysis represents an established method for isolation of pDNA, some drawbacks are recognized, such as entrapment of pDNA in cell debris, leading to lower pDNA recovery; the time-consuming process; and increase of the volume due to the buffers used, all leading to increased cost of production. We compared the concentration of extracted pDNA when two methods for extracting pDNA from Escherichia coli were used: alkaline lysis and a method based on membrane electroporation, electroextraction. At the same time, we also studied the effect of different pulse protocols on bacterial inactivation. The concentration of pDNA was assayed with anion exchange chromatography. When alkaline lysis was used, two incubations of lysis time (5 and 10 min) were compared in terms of the amount of isolated pDNA. We did not observe any difference in pDNA concentration regardless of incubation time used. In electroextraction, different pulse protocols were used in order to exceed the pDNA concentration obtained by alkaline lysis. We show that electroextraction gives a higher concentration of extracted pDNA than alkaline lysis, suggesting the use of electroporation as a potentially superior method for extracting pDNA from E. coli. In addition, electroextraction represents a quicker alternative to alkaline lysis for extracting pDNA.
authors.affiliation: Faculty of Electrical Engineering, Laboratory of Biocybernetics, University of Ljubljana, Trzaska 25, 1000, Ljubljana, Slovenia.
authors.forename: Saša
authors.initials: S
authors.lastname: Haberl
authors.forename: Marko
authors.initials: M
authors.lastname: Jarc
authors.forename: Aleš
authors.initials: A
authors.lastname: Strancar
authors.forename: Matjaž
authors.initials: M
authors.lastname: Peterka
authors.forename: Duša
authors.initials: D
authors.lastname: Hodžić
authors.forename: Damijan
authors.initials: D
authors.lastname: Miklavčič
mesh_terms: D002458:Cell Fractionation / Q000379:methods*; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics*; D050296:Microbial Viability; D010539:Permeability; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012972:Sodium Hydroxide / Q000737:chemistry
chemical_list: D012972:Sodium Hydroxide