Structural analysis of nucleic acids by precise denaturing gradient gel electrophoresis: I. Methodology

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.