Electron microscopy of nucleic acid.
Electron microscopy (EM) of nucleic acids was first described by Kleinschmidt and Zahn 1959 (1). By complexing duplex DNA with basic proteins and spreading the complexed DNA onto the surface of an aquous phase it was possible to obtain well stretched DNA molecules that could be examined by EM. Addition of formamide to DNA spreading solution as well as to hypophase prevented single-stranded DNA and RNA from forming secondary structures thus providing conditions for analysis of these molecules. EM studies have provided information about molecular weight, molecular configuration and homogeneity/heterogeneity of the DNA preparations examined. Furthermore, intramolecular heterogeneity in base sequence could be analyzed by partial denaturation of duplex DNA. By partial denaturation mapping information of the orientation of DNA molecules was obtained. Sequence homology between two DNA molecules were studied by heteroduplex analyses where the two DNAs to be studied were denatured to full strand separation followed by reassociation. This method has been widely used to analyze DNA mutations, and to discover sequence homologies within a DNA molecule or between two DNA molecules. Heteroduplex analyses between DNA and RNA (R-loop formation) provided information about distribution of ribosomal RNA genes and gave the first information of eukaryotic post transcriptional processing of mRNA. Also the various modes for DNA replication and transcription were studied by EM. It is possible to study duplex DNA not complexed by basic proteins by EM. The DNA is then adsorbed to an activated carbon film and rotary shadowed with finely grained metals to enhance the contrast. Using this method it was possible to study binding of proteins to DNA. This method was also used in the study of chromatin structure where it provided information of the size and distribution of nucleosomes as well as packaging of DNA in chromatin fibres. The complicated nature of DNA strand exchange and repair reactions was studied by in-vitro systems where EM analysis was used in the description of the presynoptic filament and structure of the joined molecules. Furthermore, EM provided the first direct evidence for the existence of the paranemic joint, where the DNA strands are joined without Watson-Crick base-pairing.
Christiansen
G
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Institute of Medical Microbiology, University of Aarhus.
D000818:Animals; D002843:Chromatin / Q000648:ultrastructure; D004247:DNA / Q000648:ultrastructure*; D005057:Eukaryotic Cells / Q000648:ultrastructure; D008854:Microscopy, Electron / Q000379:methods; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis; D009710:Nucleotide Mapping; D011485:Protein Binding; D011995:Recombination, Genetic; D014158:Transcription, Genetic
D002843:Chromatin; D009692:Nucleic Acid Heteroduplexes; D004247:DNA