Desenvolvimento de um protocolo de purificação de DNA Plasmidial pela técnica de Salting-out utilizando Sulfato de Amônio

Abstract

Development of gene transfer protocols demand high quantities of pure plasmidial DNA (pDNA). One of the purification pitfalls is the complexity of the purification protocols. The salting-out techniques allow to precipitate proteins and another biomolecules through a salt cosmotropic effect. This method can be a simple, fast and low cost system of purification. In this work we aim the establishment of a protocol for pDNA purification through ammonium sulphate treatment. Plasmids were initially produced in Escherichia coli (DH5α strain) and extracted by alkaline lysis. Later, was tested the salting-out method by treating samples with 1, 2 or 3 M ammonium sulphate. At same time, we exposed samples during different times (10, 20 e 30 minutes). Initially, to observe the behavior of the ammonium sulphate solution in the alcohol pDNA precipitation protocol, we tested 0.7 volume of isopropyl alcohol and, also, in 2 volumes of ethyl alcohol. Ethyl alcohol induce the salt precipitation, either in low concentrations. On the other hand, isopropyl alcohol formed a two phase system, without inducing salt precipitation. After, we tested the effect of isopropyl alcohol in ammonium sulphate solution from 0,25 M to 1 M; two phase system was not observed in ammonium sulfate concentrations lower than 0,5 M. Thus, we decides to treat the samples with ammonium sulphate, diluting its concentration to 0,5 M before the precipitation in isopropyl alcohol. pDNA samples were analyzed in agarose gel, spectrophotometrically, by HPLC and in gene transfer protocols. At same time, we compared the salting-out method against a commercial purification kit, analyzing the pDNA yield and purity. The better results were obtained by treating samples with 2 M ammonium sulphate. In this concentration, the product showed low RNA concentration and expected electrophoretic pattern. The obtained pDNA presented 260nm/280nm and 260nm/230nm ratios inside the ranges expected for pure DNA, we thereafter confirmed this purity by HPLC. The pDNA purified were able to transform HEK cell line, showing higher transfection efficiency than that purified by commercial kit. Taken together, our results suggest that our method could be an alternative to produce pure and low cost plasmidial DNA to be used in downstream protocols.