STRUCTURAL-ANALYSIS OF NUCLEIC-ACIDS BY PRECISE DENATURING GRADIENT GEL-ELECTROPHORESIS .1. METHODOLOGY
STRUCTURAL-ANALYSIS OF NUCLEIC-ACIDS BY PRECISE DENATURING GRADIENT GEL-ELECTROPHORESIS .1. METHODOLOGY
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DOI：
10.1093/oxfordjournals.jbchem.a123728
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发表时间：
1992-02-01
影响因子：
2.7
通讯作者：
HUSIMI, Y
中科院分区：
文献类型：
--
作者：
NISHIGAKI, K;TSUBOTA, M;HUSIMI, Y
开发了一种将传统变性梯度凝胶电泳转化为高度重复性实验系统的方法。它基于以下实验结果； (i) 染料是小分子，不会表现出由于其构象变化而导致的迁移率变化，而核酸则会表现出迁移率变化； (ii) 大部分由实验波动引起的迁移率变化可以通过标准化样品相对于相应染料的迁移率来消除。该方法涉及内参染料与样品（核酸）的共迁移以及计算机辅助数据处理，使我们能够获得整个变性梯度中核酸相对于染料的相对迁移率。由此获得的相对迁移率的总体模式，称为归一化迁移率谱（NMP），与变性效应引起的大分子构象变化很好地对应。该方法为我们提供了客观的数据，无需使用内部大分子参考，既保证了精度，又扩展了变性梯度法的应用范围。
A method to convert the conventional denaturing gradient gel electrophoresis into a highly reproducible experimental system was developed. It was based on the following experimental findings; (i) dyes, which are small molecules, do not exhibit mobility changes attributed to their conformational change while nucleic acids do; and (ii) most of the mobility shifts caused by experimental fluctuations could be cancelled by normalizing the mobility of a sample with respect to the corresponding one of a dye. The method involves co-migration of internal reference dyes with samples(nucleic acids), and computer-aided data processing, allowing us to obtain the relative mobility of nucleic acids with respect to a dye throughout the denaturing gradient. The overall pattern of the relative mobilities thus obtained, named the normalized mobility profile (NMP), corresponded well to conformational changes of a macromolecule induced by denaturing effects. This method provides us with objective data without using internal macromolecular references, which not only guarantees the precision but also extends the range of application of the denaturing gradient method.