Giovannini R, Freitag R, Tennikova T B
Laboratoire de Biotechnologie Cellulaire, Ecole Polytechnique Federale de Lausanne, Switzerland.
Anal Chem. 1998 Aug 15;70(16):3348-54. doi: 10.1021/ac980390w.
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 mm, 10-micron 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.
膜吸附剂在蛋白质色谱中已得到广泛应用。本文首次以一个7.2 kb的主要为超螺旋形式的质粒为例，研究了多核苷酸在这些固定相上的行为。研究了梯度洗脱和等度洗脱。与蛋白质高效膜色谱（HPMC）不同，DNA色谱中可以进行等度洗脱。在梯度洗脱的情况下，起始缓冲液中可以使用更高的盐浓度。在优化条件下，两种方法都导致单质粒峰分裂为三个最大值，这与琼脂糖凝胶中的三条带（尽管是分离的）相对应。推测这三个部分分别是超螺旋、切口和开环质粒DNA。质粒的线性化降低了吸附能，线性化质粒比未线性化的质粒洗脱得更早。将HPMC实验与使用传统填充床阴离子交换柱（BioScale Q2，7×52 mm，10微米多孔颗粒）和新型整体式阴离子交换柱（UNO Q1，7×35 mm）进行的类似实验进行了比较。根据新发展的HPMC理论对这些实验中观察到的结果和特征差异进行了解释。
