47] The analysis of nucleic acids in gels using glyoxal and acridine orange

Abstract

Publisher Summary This chapter discusses the analysis of nucleic acids in gels using glyoxal and acridine orange. To determine the molecular weights of nucleic acids using gel electrophoresis techniques, the molecules must have equivalent conformations. This is achieved by removing native secondary and tertiary structure with a variety of chemical denaturants, thus reducing the electrophoretic mobility to a simple function of polynucleotide molecular weight, gel composition, and voltage gradient applied. The chapter describes the method for denaturing RNA or DNA molecules with glyoxal, followed by electrophoresis through either polyacrylamide- or agarose-containing slab gels in a low ionic strength buffer. Using this method reliable molecular weight estimates for RNA and DNA molecules of varying sizes and G + C contents were obtained; glyoxalated DNA and RNA molecules were shown to lie on the same log molecular weight versus mobility curve, or on very similar curves. The chapter describes the use of the metachromatic stain, acridine orange, for the visualization of nucleic acids in gels. When intercalated between the stacked bases of double-helical nucleic acids, acridine orange manifests a green fluorescence. Its metachromasy visually confirms glyoxal denaturation and makes it a powerful reagent for rapid determination of gross nucleic acid structure after gel electrophoresis.