Hines R N, O'Connor K C, Vella G, Warren W
Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201.
Biotechniques. 1992 Mar;12(3):430-4.
Numerous methods have previously been reported for the final steps in the large-scale purification of plasmid DNA. Although gel permeation and reverse-phase high-performance liquid chromatography have been utilized for this procedure in the past, the limited capacity of these systems often necessitated multiple rounds of chromatography, especially with the high copy number plasmids commonly in use today. In this paper, the use of the high-capacity, high-resolution Protein-Pak DEAE 8HR column is presented for the large-scale isolation of highly purified plasmid DNA from crude E. coli cell lysates. Up to 5 mg of plasmid DNA have been purified in a single 50-minute chromatography run. The purified DNA demonstrated excellent biological activity as demonstrated by restriction endonuclease digestion, E. coli transformation and DNA-mediated gene transfection of eukaryotic cells.
此前已有许多方法报道用于质粒DNA大规模纯化的最后步骤。尽管过去曾使用凝胶渗透和反相高效液相色谱法进行此操作，但这些系统的容量有限，通常需要多轮色谱分离，尤其是对于当今常用的高拷贝数质粒。本文介绍了使用高容量、高分辨率的Protein-Pak DEAE 8HR柱从大肠杆菌细胞粗裂解物中大规模分离高纯度质粒DNA。在单次50分钟的色谱运行中可纯化多达5毫克的质粒DNA。经限制性内切酶消化、大肠杆菌转化以及真核细胞的DNA介导基因转染证明，纯化后的DNA具有出色的生物活性。
