The investigation of plasmid purification by using polymeric surfactants

The development of molecular therapies such as non-viral gene therapy and DNA vaccination for infectious diseases have increased the need for high quantities of highly purified plazmid DNA (pDNA). Although standard molecular biology protocols are available ,these are not suitable. In addition they frequently use toxic reagents that should not be used in the purification of therapeutic products. In this study, the polymer compounds were used to develop purification methods for pDNA. In the fırst method N,N?,N?- Polyoksietylen (10)-N tallow- 1,3-di amine propane ( Peg-10 tallow) surfactant was used as a precipitation agent . Stuctural analysis of Peg-10 tallow was determined by Elementar analysis, FT-IR, UV-VIS, NMR (1H, 13C ).The conductivity of conditions of precipitation method was determined. A modified alkaline lysis method was used for cell lysis to obtain pDNA and RNA solution. Precipitaion of the pDNA by Peg-10 tallow cationic surfactant was determinated in different acid, different salt and different lysate concentrations. The results were realized by 1% agarose gel analysis. Aqueous two phase systems (ATPS) is used as a system for seperation of biomolecules and particles. In the second method was presented partitioning of the plasmid DNA in polyetylene glycol(PEG) / salt (NH4)2SO4 aqueous two phase systems (ATPS). %35 PEG 600 - %6 (NH4)2SO4 ATPS system was selected .A modified alkaline lysis method was used for cell lysis to obtain pDNA and RNA solution.100% mass of lysate was loaded to the system. In addition the bottom phase of ATPS systems was integrated with isopropanol precipitation step. The results were realized by %1 agarose gel analysis. Finally PEG 600 system provides plazmid purification better than peg-10 tallow precipitation system. Key Words:Gene Therapy, DNA Vaccination, p(DNA) purification, Peg-10 tallow, PEG 600