title: Plasmid DNA affinity partitioning using polyethylene glycol - sodium sulfate aqueous two-phase systems.
abstract: Extraction of plasmid DNA (pDNA) from an alkaline bacterial cell lysate has been investigated using partitioning and affinity partitioning in aqueous two-phase systems (ATPSs) containing 12% (w/w) polyethylene glycol and 12% (w/w) sodium sulfate. In this work, a modified 20bp pyrimidine oligonucleotide acting as a triplex-forming oligonucleotide (TFO) was employed as an affinity ligand to improve the partition behavior. The pUC118 plasmid was chosen to be extracted because of its specific oligonucleotide recognition sequence. In order to find the proper conditions, operational parameters such as PEG molecular weight, pH and lysate load were also studied. The results showed that adding the affinity ligand strongly changed the partitioning behavior of pDNA without any considerable impact on the partitioning of RNA and protein content. The best achieved result was at pH=6 and PEG MW of 600Da and 60% of lysate load. In these conditions, 67% of pDNA was recovered in the top phase while the contaminants mainly accumulated in the bottom phase.
authors.affiliation: School of Chemical Engineering, Iran University of Science and Technology, Narmak, Tehran, Iran.
authors.forename: Behzad
authors.initials: B
authors.lastname: Nazer
authors.affiliation: School of Chemical Engineering, Iran University of Science and Technology, Narmak, Tehran, Iran. Electronic address: m_dehghani@iust.ac.ir.
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Dehghani
authors.affiliation: Laboratory of Biophysics and Molecular Biology, Departments of Biophysics and Bioinformatics, Institute of Biochemistry and Biophysics, University of Tehran, Enghelab, Tehran, Iran.
authors.forename: Bahram
authors.initials: B
authors.lastname: Goliaei
mesh_terms: D005591:Chemical Fractionation / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D010957:Plasmids / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D011092:Polyethylene Glycols / Q000737:chemistry*; D013431:Sulfates / Q000737:chemistry*
chemical_list: D013431:Sulfates; C012036:sodium sulfate; D011092:Polyethylene Glycols; D004247:DNA
keywords: Aqueous two-phase; Bio-separations; DNA; Partitioning; Purification