Methylmercury as a reversible denaturing agent for agarose gel electrophoresis

Abstract

A method for agarose gel electrophoresis under denaturing conditions, with methylmercuric hydroxide as the denaturing agent, is described. Methylmercuric hydroxide is a strong and reversible denaturing agent. It appears that complete denaturation of any base-paired secondary structural feature of a nucleic aicd can be achieved at practical concentrations of CH 3 HgOH. The method has been tested by showing that singly nicked circular duplex PM2 DNA is dissociated into more rapidly migrating linear and circular single-strand forms by a CH 3 HgOH concentration within the 2.5–5.0 m m range. The mobilities of single-strand RNA molecules are decreased in the presence of sufficient CH 3 HgOH because their secondary structure is disrupted. For HeLa 28S rRNA, the melting transition occurs mainly in 2–3 m m range of CH 3 HgOH. For a series of RNA's, at 5 m m CH 3 HgOH, corresponding to complete denaturation, there is a linear dependence of log (molecular weight) on electrophoretic mobility and of log (mobility) on agarose concentration, just as in other gel electrophoresis systems.