Hecker R, Colpan M, Riesner D
J Chromatogr. 1985 Jun 19;326:251-61. doi: 10.1016/s0021-9673(01)87451-3.
High-performance liquid chromatography on Nucleogen-DEAE 4000-10 has been applied to several problems of the isolation of DNA restriction fragments. Large amounts of DNA fragments of high purity are necessary for biophysical studies and for molecular hybridization in basic research, as well as in medical diagnosis. The influence of various parameters, such as buffer, pH, eluting salt, gradient slope, flow-rate and the addition of urea on the resolution of fragments by high-performance liquid chromatography were studied on an analytical scale, and the optimal conditions were then used for the large-scale preparation of milligram amounts. The best resolution of fragments between 25 and 1500 base pairs was obtained with a linear gradient from 500 mM to 1200 mM sodium chloride in 6 M urea -30 mM sodium phosphate (pH 6.0). Quantitative data are given for the purity and recovery of the sample, and the capacity and lifetime of the column. The following applications of high-performance liquid chromatography of restriction fragments are described: preparation of 2 mg of fragments, separation of 1 mg of DNA insert from 7 mg of its plasmid vector, and analysis of DNA-RNA hybrids.
利用Nucleogen-DEAE 4000-10进行高效液相色谱分析已应用于DNA限制性片段分离的若干问题。在基础研究以及医学诊断中，生物物理研究和分子杂交需要大量高纯度的DNA片段。在分析规模上研究了各种参数（如缓冲液、pH值、洗脱盐、梯度斜率、流速和尿素添加量）对高效液相色谱分离片段分辨率的影响，然后将最佳条件用于毫克量的大规模制备。在6M尿素-30mM磷酸钠（pH 6.0）中，采用500mM至1200mM氯化钠的线性梯度，可获得25至1500个碱基对片段的最佳分辨率。给出了样品纯度、回收率、柱容量和使用寿命的定量数据。本文还描述了限制性片段高效液相色谱的以下应用：制备2mg片段、从7mg质粒载体中分离1mg DNA插入片段以及分析DNA-RNA杂交体。
