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Dynamics of Gestalt Polymer Chains in Gel and Separation Mechanism of DNA under Gel Electrophoresis

凝胶中格式塔聚合物链的动力学及凝胶电泳下DNA的分离机制

基本信息

批准号：
01470107
负责人：
KOTAKA Tadao
金额：
$ 4.54万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

The object of this study was to clarify dynamics of gestalt polymeric chains in gels, and to clarify the mechanism of molecular weight dependent separation in gel electrophersis of DNA. We also attempted to establish the optimum electropheric condition that enabled us to separate enormously large DNA more than 10^6 basepairs (bp). First, we examined dielectric relaxation of gest polyisoprene, PI, that exhibits the dielectric normal mode prucess, dissolved in much longer PI matrix, in polybutadiene (PB) matrix, and also in crosslinked PB matrix with varying molecular weight M. Then, we found that when gest chains were too short to entangle with the gel matrix, their relaxation time was proportinal to M^2, and this could be explained by Rouse-Zimm theory. On the other hand, when the gest chains was so long as to entangle with the gel, the relaxation time was proporinal to M^3, and this well suited with Doi-Edowards theory that is invalid in monodisperse systems.Then, we proposed a new electric field that has a sinusoidal field with amplitude E_s and frequency f superposed on a steady biase field E_b ; Biased Sinusolidal Field. We found that in a condition of E_b < E_s, DNAs longer than 20 x 10^3 bp had minimum in mobilityat a particular M-dependent frequency, which was called pin-down frequency, f_p. We also confirmed the relation of f_p alpha M^<-1>C_<gel>^<-1>E_S^0E_b^1 from experiments with various agarose gel concentration C_<gel>. Biased reptation model explained molecular weight dependence of mobility in steady field electrophoresis qualitatively, but not quantitatively. More realistic model describing dynamics of large DNA in gel electrophoresis is required for more detailed discussion.

本研究的目的是阐明凝胶中完形聚合物链的动力学，并阐明凝胶电泳中分子量依赖分离的机理。我们还试图建立最佳的电泳条件，使我们能够分离超过10^6碱基对（bp）的巨大的DNA。首先，我们研究了溶解在更长的PI矩阵、聚丁二烯（PB）矩阵以及不同分子量M的交联PB矩阵中表现出介电正模过程的聚异戊二烯（PI）的介电弛豫。然后，我们发现当聚异戊二烯链太短而不能与凝胶基体纠缠时，它们的弛豫时间与M^2成正比，这可以用rose - zimm理论来解释。另一方面，当最大的链长到与凝胶纠缠时，弛豫时间与M^3成正比，这很适合在单分散体系中无效的doi - edward理论。然后，我们提出了一个新的电场，其振幅E_s和频率f的正弦电场叠加在稳定偏置场E_b上；偏置正弦场。我们发现，在E_b < E_s的条件下，长度大于20 × 10^3 bp的dna在一个特定的m依赖频率（称为pin-down频率，f_p）上的迁移率最小。通过不同琼脂糖凝胶浓度C_<gel>的实验，证实了f_p α M^<-1>C_<gel>^<-1>E_S^0E_b^1的关系。偏倚重复模型定性地解释了稳定场电泳中迁移率对分子量的依赖，但不能定量地解释。更现实的模型描述动力学大DNA在凝胶电泳需要更详细的讨论。