Study of the Possibility of the Detection of Fluorescent Dyes by Cathodoluminescence in the Scanning Electron Microscope and Partial Denaturation Maps of Lambda DNA using Methylmercuric Hydroxide

Citation

Mohr, Douglas Crane (1973) Study of the Possibility of the Detection of Fluorescent Dyes by Cathodoluminescence in the Scanning Electron Microscope and Partial Denaturation Maps of Lambda DNA using Methylmercuric Hydroxide. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/1F4Q-0K50. https://resolver.caltech.edu/CaltechTHESIS:02052018-145823945

Abstract

Part I. The possibilities of using ethidium bromide, quinacrine hydrochloride, and dansyl chloride as cathodoluminescent stains for scanning electron microscopy have been investigated. Nucleic acid specimens stained with the dyes gave best results when they were embedded in frozen glycerol and examined at liquid nitrogen temperature. Under these conditions, the excitation cross section for ethidium bromide is about 0.5 A² and the destruction cross section 5 A². The other dyes were destroyed quite rapidly.

Even under the best conditions, the minimum amount of ethidium bromide detectable by cathodoluminescence is about the same as detectable by fluorescence microscopy.

Part II. Methylmercuric hydroxide preferentially reacts with thymine. This reaction disrupts the Watson-Crick base pairing, the mercurated regions appearing as loops in the electron microscope. Lambda DNA has been partially denatured by methylmercuric hydroxide and mounted for electron microscopy by the basic film technique.

The results indicate that there are four regions which readily denature, located on one side of the lambda genome. This result is in agreement with other thermal and alkaline partial denaturation data.

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