High-performance membrane chromatography of supercoiled plasmid DNA
High-performance membrane chromatography of supercoiled plasmid DNA
复制标题
DOI：
10.1021/ac980390w
复制
发表时间：
1998-08-15
影响因子：
7.4
通讯作者：
Tennikova, TB
中科院分区：
文献类型：
--
作者：
Giovannini, R;Freitag, R;Tennikova, TB
膜吸附剂在蛋白质色谱中得到了很好的应用。本文首次以7.2 kb超旋质粒为例，研究了多核苷酸在这些固定相上的行为。研究了梯度和等温洗脱。与蛋白质高效膜层析（HPMC）相反，DNA层析可以进行等温洗脱。在梯度洗脱的情况下，可以在起始缓冲液中使用高得多的盐浓度。在优化条件下，两种方法都将单个质粒峰分裂成三个最大值，这与琼脂糖凝胶中的三个（尽管是分离的）条带相对应，推测这三个部分是超螺旋、缺口和开放的环状质粒DNA。质粒的线性化降低了吸附能，线性化质粒比非线性化质粒更早被洗脱。将HPMC实验与使用传统填充床阴离子交换柱（BioScale Q2, 7 × 52 mn， 10 μ m多孔颗粒）和新型整体型阴离子交换柱（UNO Q1, 7 × 35 mm）进行的类似实验进行了比较，并根据新发展的HPMC理论解释了这些实验中观察到的结果和特征差异。
Membrane adsorbers are well established in protein chromatography. The present paper investigated for the first time the behavior of polynucleotides on these stationary phases, taking a 7.2-kb predominantly supercoiled plasmid as example. Gradient and isocratic elution was studied. In contrast to protein high-performance membrane chromatography (HPMC), isocratic elution is possible in DNA chromatography. In the case of gradient elution, much higher salt concentrations can be used in the starting buffer. Under optimized conditions, both approaches led to a splitting of the single plasmid peak into three maximums, which corresponded to the three-albeit isolated-bands in the agarose gel, Presumably the three fractions were supercoiled, nicked, and open circular plasmid DNA. Linearization of the plasmid lowered the adsorption energy, and the linearized plasmid eluted earlier than the nonlinearized one. The HPMC experiments were compared to similar ones performed using a conventional packed-bed anion-exchange column (BioScale Q2, 7 x 52 mn, 10-mu m porous particles) and a novel monolithic-type anion-exchange column (UNO Q1, 7 x 35 mm), The results and characteristic differences observed in these experiments were interpreted in the light of the newly developed theory of HPMC.