Electron Microscopy of Nucleic Acids
New techniques of molecular cloning and subcloning have stimulated renewed interest in the structural analysis of nucleic acid molecules by electron microscopy (EM). The specialized procedures for visualizing both naturally occurring nucleic acid molecules from cells or viruses and laboratory-constructed hetero- duplexes and R-loop molecules could, and perhaps should, be a part of the technical capability of every fully equipped EM laboratory. While requirements for specialized equipment are minimal, this technique does require a separate set of interpretive skills including an appreciation of the fact that individual molecules are assayed without the benefit of the selective pressure present in other techniques, as well as a clear understanding of the relationship between preparative procedure and image quality. It is this latter relationship that can be most troublesome and one that I hope to deal with effectively in this chapter. My aim, then, rather than providing a comprehensive review of all of the modifications and permutations of the basic technique that have appeared in the literature over the years, is to describe a broad range of procedures that have, in my laboratory, most often provided useful and consistent structural data on nucleic acids. Since sample requirements for successful structural analysis of nucleic acid molecules often exceed those of other techniques, it will also be my aim to provide detailed purification, handling, and storage procedures that will provide samples of sufficient quantity and quality for ultrastructural analysis. Finally, I intend to “troubleshoot” the procedures as I go on to define those factors that are potential sources of problems and emphasize those procedures that most often provide crisp and unambiguous results.
KeywordsContour Length Nucleic Acid Molecule Strand Separation Stainless Steel Screen Denaturation Profile
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