Abstract:
Research was conducted to evaluate the boiling water bath, alkaline lysis, and acid precipitation plasmid isolation techniques on clinical samples of Bordetella pertussis. Variations on these techniques included plasmid purification procedures, experimental temperature differences, cell lysis methods and centrifugation times. The protocols that were examined all require less than two hours preparation time from pelleted, fresh growth of the test organisms to purified plasmid lysate. None of the methods require laboratory equipment more complicated than a microcentrifuge. Agarose gel electrophoresis was used to detect the presence of plasmid DNA in the lysates. A distinctive 21 kb DNA band was produced by each isolation method. The alkaline lysis procedure produced the most intense DNA bands. The boiling water bath and acid precipitation methods were
second and third in intensity. Digestion of the isolated plasmid lysates was attempted with the following restriction endonucleases: Bgl I, EcoRl, Hae II, Hind III and Hinc II. None of the restriction enzymes produced a distinctive plasmid profile, however, Bgl I, Hae II, and Hinc II showed promise as possible tools for the "fingerprinting" (profiling) of Bordetella plasmids.
