RNA MOLECULAR-WEIGHT DETERMINATIONS BY GEL-ELECTROPHORESIS UNDER DENATURING CONDITIONS, A CRITICAL RE-EXAMINATION
RNA MOLECULAR-WEIGHT DETERMINATIONS BY GEL-ELECTROPHORESIS UNDER DENATURING CONDITIONS, A CRITICAL RE-EXAMINATION
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DOI：
10.1021/bi00640a033
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发表时间：
1977-01-01
期刊：
影响因子：
2.9
通讯作者：
BOEDTKER, H
中科院分区：
文献类型：
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作者：
LEHRACH, H;DIAMOND, D;BOEDTKER, H
RNA [小鼠、鸡、大肠杆菌辛德毕斯病毒和烟草花叶病毒rRNA] MW测量通过凝胶电泳在4种不同的变性条件下进行，包括99%甲酰胺、10 mM甲基汞、2.2 M甲醛和6 M尿素，pH 3.8。在一系列的凝胶浓度和至少2个不同的电压梯度的电泳导致在一些RNA物种表现出明显的MW随凝胶浓度和电压梯度。三个不同的偏离流体力学等效构象的要求进行了观察：非常大的RNA的无规卷曲结构的变形在适度高的凝胶浓度和电压梯度，导致在极端情况下，在MW独立的迁移的RNA分子;不完全变性的RNA分子与非常GC丰富的螺旋区域;和不同的电荷/质量比，由于在pH 3.8的差异质子化。可靠地测量高达4.0x的RNA分子的MW 106含有GC丰富的螺旋区域，只能在与2.2 M甲醛或10 mM氢氧化甲基汞反应后在稀释的（0.5-1.0%）琼脂糖凝胶上制备。使用的经验日志MW流动性关系的理论依据。一系列同源无规卷曲的凝胶电泳行为可以近似为一系列半径与无规卷曲回转半径的平方根成比例的球体。如果将MW的平方根与对数迁移率作图，则变性RNA的MW测定，尤其是通过外推法获得的MW测定更可靠。
RNA [mouse, chicken, Escherichia coli Sindbis virus and Tobacco Mosaic virus rRNA] MW measurements were carried out by gel electrophoresis under 4 different denaturing conditions including 99% formamide, 10 mM methyl mercury, 2.2 M formaldehyde and 6 M urea at pH 3.8. Electrophoresis at a series of gel concentrations and at least 2 different voltage gradients resulted in some RNA species exhibiting apparent MW that vary with both gel concentration and voltage gradient. Three different deviations from the requirement for hydrodynamically equivalent conformations were observed: deformation of the random coil structure of very large RNA at moderately high gel concentrations and voltage gradients resulting, in extreme cases, in a MW independent migration of RNA molecules; incomplete denaturation of RNA molecules with very GC rich helical regions; and varying charge/mass ratio due to differential protonation at pH 3.8. Reliable MW measurements of RNA molecules as large as 4.0 .times. 106 containing GC rich helical regions could only be made on dilute (0.5-1.0%) agarose gels after reaction with either 2.2 M formaldehyde or 10 mM methyl mercury hydroxide. A theoretical justification for the use of the empirical log MW-mobility relation is presented. The gel electrophoretic behavior of a homologous series of random coils can be approximated by a series of spheres with radii proportional to the square root of radius of gyration of a random coil. MW determinations of denatured RNA, especially those obtained by extrapolation, are more reliable if the square root of the MW is plotted vs. log mobility.