Kapp U, Langowski J
EMBL, Grenoble, France.
Anal Biochem. 1992 Nov 1;206(2):293-9. doi: 10.1016/0003-2697(92)90369-i.
A method for the separation of superhelical DNA on the basis of superhelical density by reverse-phase HPLC on RP-18 columns is described. The technique can be used to prepare superhelical DNA in milligram amounts and narrow topoisomer distributions in 0.1 mg amounts. We show example separations of the plasmids pUC18 (2687 bp) and pi AN13 (895 bp). While the best separation for pUC18 yields topoisomer distributions of two or three major components, the small plasmid can be separated into single topoisomer fractions. The basis of the separation is probably an interaction of partially opened bases with the hydrophobic column matrix. This hypothesis is supported by the elution behavior of DNA fragments on this column: DNA fragments with sticky ends, even at a length of several hundred base pairs, elute at much higher methanol concentrations than blunt-ended fragments.
本文描述了一种基于超螺旋密度，利用反相高效液相色谱在RP - 18柱上分离超螺旋DNA的方法。该技术可用于制备毫克量的超螺旋DNA，并能以0.1毫克量获得窄的拓扑异构体分布。我们展示了质粒pUC18（2687 bp）和pi AN13（895 bp）的分离实例。对于pUC18，最佳分离效果产生两到三个主要组分的拓扑异构体分布，而小质粒可分离成单个拓扑异构体组分。分离的基础可能是部分打开的碱基与疏水柱基质之间的相互作用。该假设得到了DNA片段在此柱上洗脱行为的支持：具有粘性末端的DNA片段，即使长度为几百个碱基对，在比平端片段高得多的甲醇浓度下洗脱。
