Microtechnique for electron microscopy of DNA.

In recent studies on kinetoplast DNA, the mitochondrial DNA of trypanosomatids (1), we had insufficient quantitites for standard electron microscopy (EM) procedures. We therefore developed a new microtechnique for sample preparation. EM of DNA is commonly done by the Kleinschmidt method (2), in which the nucleic acid is first adsorbed to a positively charged protein monolayer formed at an air-water interface. The spread DNA is then picked up on a film (e.g. nitrocellulose) supported on an EM grid. Using a common modification of the Kleinschmidt method, by Davis et al. (3), this technique requires about 25 ng of plasmid DNA. Since kinetoplast DNA is in the form of a large network of several thousand minicircles, the requirements are closer to 50—100 ng. Our new method is a variation of the Davis technique using much smaller amounts of DNA for each spreading. Furthermore, this technique allows preparation of grids for multiple samples in considerably less time than required by traditional methods while maintaining the quality of the micrographs. Therefore, we now use mis method even if we have abundant supplies of DNA. The method should be applicable for any DNA, especially when it is available only in small quantitites (e.g. from microorganisms that are difficult to culture or from in vitro enzyme reactions). Spreadings were conducted in a Teflon block (12.5 cmx 8.5 cmx 1.3 cm) drilled with 50 cylindrical holes, each 6.3 mm in diameter and 9.3 mm in depth. The holes were spaced in the block in 5 rows with 10 holes per row. Commercially available