Isolation and characterization of DNA from Tritrichomonas foetus and Trichomonas vaginalis |
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| Authors: | A L Wang C C Wang |
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| Institution: | Department of Pharmaceutical Chemistry School of Pharmacy, University of California San Francisco, CA 94143, U.S.A. |
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| Abstract: | High molecular weight DNA samples free of contaminating proteins or RNA were obtained from Tritrichomonas foetus or Trichomonas vaginalis by lysing the cells in 4 M guanidinium thiocyanate before centrifuging in CsCl density gradient and then purifying the DNA band by NACS-37 column chromatography. The bulk DNA from either organism acted as a single component in ion-exchange chromatography, agarose gel electrophoresis, CsCl density gradient centrifugation and thermal denaturation. T. foetus DNA showed a melting temperature (Tm) of 82 degrees C corresponding to a 31% GC content whereas T. vaginalis DNA melted at 84 degrees C to suggest 36% GC. Both DNA samples demonstrated 35 to 42% hyperchromicity when fully melted. Cot analysis revealed the presence of repetitive sequences in both DNAs: approximately 46.7% in T. foetus DNA and 53.3% in T. vaginalis DNA. The unique sequences of these two protozoan DNAs are of a similar size of about 2.5 X 10(7) base pairs. Agarose gel electrophoresis of restriction fragments of the two purified DNA samples gave distinct banding patterns that were characteristic of the two species of protozoan parasites. |
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| Keywords: | DNA Guanidinium thiocyanate Transition temperature Endonuclease digestion Double-stranded RNA PBS phosphate-buffered saline TE 10 mM Tris-HCl pH 7 8 and 1 mM EDTA TBE 89 mM Tris-borate pH 8 3 and 2 5 mM EDTA SSC 15 mM sodium citrate pH 7 0 and 0 15 M NaCl melting temperature kb kilobase SDS sodium dodecyl sulfate |
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