Method combining BAC film and positive staining for the characterization of DNA intermediates by dark-field electron microscopy
Résumé
DNA intermediate structures are formed in all major pathways of DNA metabolism. Transmission electron microscopy
(TEM) is a tool of choice to study their choreography and has led to major advances in the understanding of these mechanisms,
particularly those of homologous recombination (HR) and replication. In this article, we describe specific TEM procedures
dedicated to the structural characterization of DNA intermediates formed during these processes. These particular
DNA species contain single-stranded DNA regions and/or branched structures, which require controlling both the DNA molecules
spreading and their staining for subsequent visualization using dark-field imaging mode. Combining BAC (benzyl dimethyl
alkyl ammoniumchloride) film hyperphase with positive staining and dark-field TEM allows characterizing synthetic
DNA substrates, joint molecules formed during not only in vitro assays mimicking HR, but also in vivo DNA
intermediates.
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