Doctoral Dissertations

Date of Award

12-1988

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary Sayler

Committee Members

Gary Stacey, Russell Lewis, David White, Stuart Riggsby

Abstract

A procedure for the extraction and purification of microbial DNA from sediments has been developed. This technique is based on the lysis of cells in the presence of the sediment and the subsequent extraction and purification of the DNA. The DNA recovered is of high quality as judged by ultraviolet spectrophotometry and is susceptible to clevage by common restriction endonucleases, which renders it amenable for use in advanced techniques for the study of environmental community structure. Susceptibility to attack by restriction endonucleases is not a characteristic of all DNA obtained by this method, however. This procedure has the advantage over existing methods in that it allows a more representative sample of DNA to be recovered.

DNA adsorption isotherms were analyzed for a broad range of soil and sediment types, which allowed the major factors controlling DNA adsorption to be identified. Minerology and pH were the primary factors, with phosphate ion concentration content being less important. This knowledge led to the development of a procedure for the quantitative extraction of free DNA from sediments, an important phase in the total purification procedure.

This procedure also allowed the discrimination between extracellular and intracellular DNA to be made, which resulted in the first isolation and molecular analysis of sediment extracellular DNA to be made. A method for the rapid and accurate measurement of extracellular DNA concentrations was developed which utilizes high performance liquid chromatography and a fluorescence detection system.

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