A routine method for protein-free spreading of double- and single-stranded nucleic acid molecules.
A protein-free nucleic acid preparation method for electron microscopy is described. The basic procedure is very similar to the classical protein monolayer spreading techniques. The carrier protein (usually cytochrome c) is replaced by benzyldimethylalkylammonium chloride. Both the hypophase method and the microdiffusion or droplet method can be applied with this compound. Unlike cytochrome c, benzyldimethylalkylammonium chloride does not lead to any apparent thickening of the nucleic acid strands. Partially denatured DNA spread with this reagent shows a loosened structure with a foamy appearance in the regions previously considered to be "unmelted," which open up locally into melted loops of different size. Specifically bound proteins, such as RNA polymerase on bacteriophage T7 DNA, can be detected unambiguously.
D003090:Coliphages; D003574:Cytochrome c Group; D003584:Cytological Techniques; D004267:DNA Viruses; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D008854:Microscopy, Electron / Q000379:methods*; D009691:Nucleic Acid Denaturation; D009696:Nucleic Acids; D009698:Nucleoproteins; D011485:Protein Binding
D003574:Cytochrome c Group; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009696:Nucleic Acids; D009698:Nucleoproteins