Monodisperse DNA restriction fragments - II. Sedimentation velocity and equilibrium experiments
Monodisperse DNA restriction fragments - II. Sedimentation velocity and equilibrium experiments
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DOI：
10.1016/j.jcis.2005.04.114
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发表时间：
2005-11-01
影响因子：
9.9
通讯作者：
Philipse, AP
中科院分区：
文献类型：
--
作者：
Koenderink, GH;Planken, KL;Philipse, AP
我们报道了用分析性超速离心机进行的沉降速度和平衡测量，以阐明有限的灵活性对半柔性、单分散、双链、钝端DNA限制片段的运输特性的影响。我们研究了一系列具有400、800和1600个碱基对(3到11个持续长度)的同源片段，这些片段是为此目的专门设计和合成的(第1部分)。从沉淀测量得到的分子量与根据碱基对数量计算的预期值很好地吻合。无限稀释时的沉降系数与蠕虫状圆柱体的理论预测符合得很好。一阶体积分数系数(Phi)系数S(Phi)=1-KPhi从最短碎片的1178减小到最长碎片的882。对于未带电的刚性棒，这些值比预测的要大得多，这表明存在与增强的纵横比和排除的体积相关的关联。从指数沉降-扩散平衡分布获得的分子量与根据碱基对数量计算的重量的精确匹配表明，任何缔合都是可逆的，并且在足够低的DNA浓度下消失。(C)2005 Elsevier Inc.保留所有权利。
We report sedimentation velocity and equilibrium measurements performed with an analytical ultracentrifuge to elucidate the effects of limited flexibility on the transport properties of semiflexible, monodisperse, double-stranded, blunt-ended DNA restriction fragments. We study a homologous series of fragments with 400, 800, and 1600 base pairs (3 to 11 persistence lengths), which are specifically designed and synthesized for this purpose (Part 1). The molecular weights following from the sedimentation measurements agree well with the values expected on the basis of the number of base pairs. The sedimentation coefficients at infinite dilution are in good agreement with theoretical predictions for wormlike cylinders. The first order in volume fraction (phi) coefficient K of the phi-dependent sedimentation coefficient s(phi) = 1 - K phi decreases from 1178 for the shortest fragment to 882 for the longest fragment. These values are much larger than predicted for uncharged rigid rods, indicating the presence of associates with an enhanced aspect ratio and excluded volume. The precise match of the molecular weights obtained from exponential sedimentation-diffusion equilibrium distributions with weights calculated from the number of base pairs shows that any association is reversible and disappears at sufficiently low DNA concentration. (c) 2005 Elsevier Inc. All rights reserved.