Quantitative aspects of mercurial-agarose gel electrophoresis

Abstract

Single-stranded nucleic acids are capable of extensive intramolecular base pairing as well as intermolecular aggregation. Consequently, electrophoretic studies of single-stranded nucleic acids are most effective when conducted under denaturing conditions. A number of techniques are available for nucleic acid denaturing gel electrophoresis (1–3). In this paper we describe certain quantitative features of one of these techniques, mercurial-agarose gel electrophoresis (4–7). Specifically, we address the questions of resolution and base composition dependence and we introduce a new mercurial for agarose gel electrophoresis, p-chloromercuriphenyl-sulfonic acid. In a previous publication we demonstrated that methylmercury was an effective denaturant in an agarose gel (4). The mechanism of denaturation is presumably the disruption of hydrogen bonding by the reversible binding of methylmercury to uridine and guanosine imino nitrogens. At saturating mercurial concentrations accurate molecular weights can be determined, free of conformation effects. The presence of methylmercury has no observable effect on the mechanical properties of the gel. Hence the denaturing power of the gel can be readily varied. The strength and rigidity of agarose gels make them considerably easier to handle than acrylamide gels, and the large pore size ensures a system compatible with high molecular weight RNA.