Fan X, Liu J, Tang H, Jin Y, Wang D B
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai, 200031, People's Republic of China.
Anal Biochem. 2000 Dec 1;287(1):95-101. doi: 10.1006/abio.2000.4817.
The PyPuPu and PyPuPy intermolecular triple-stranded DNA (tsDNA) can be determined more easily by capillary electrophoresis (CE) than by traditional methods. The tsDNA and its component compounds can be well separated by using a sieving matrix of 1.0% hydroxypropylmethylcellulose (HPMC) containing 2.5 mM magnesium ions. Such factors as buffer pH, the concentration of triplex-forming oligonucleotide (TFO), temperature, and the concentration of magnesium cation in the formation and stabilization of triple-stranded helices have been studied with capillary electrophoresis. The triplex cannot be formed when the buffer pH is lower than 4.0. When the concentration of TFO is four times higher than that of dsDNA, all of the dsDNA molecules can be associated. The limit of capillary electrophoresis detection with good reproducibility is 0.5-1 nM (S/N = 3). The CE analysis of short tsDNA takes only 40 min, whereas gel electrophoresis needs at least 5 h.
与传统方法相比，通过毛细管电泳（CE）可以更轻松地测定PyPuPu和PyPuPy分子间三链DNA（tsDNA）。使用含有2.5 mM镁离子的1.0%羟丙基甲基纤维素（HPMC）筛分基质，可以很好地分离tsDNA及其组成化合物。利用毛细管电泳研究了缓冲液pH值、三链形成寡核苷酸（TFO）浓度、温度以及镁阳离子浓度等因素在三链螺旋形成和稳定过程中的作用。当缓冲液pH值低于4.0时，无法形成三链体。当TFO浓度比双链DNA高四倍时，所有双链DNA分子都可以缔合。具有良好重现性的毛细管电泳检测限为0.5-1 nM（信噪比=3）。短tsDNA的CE分析仅需40分钟，而凝胶电泳至少需要5小时。
