Analysis of bromodeoxyuridine incorporation into DNA: Comparison of gas chromatographic/mass spectrometric, CsCl gradient sedimentation, and specific radioactivity methods

A sensitive new method for the quantitation of 5-bromodeoxyuridine (BrdUrd) incorporated into DNA by GC/MS analysis of enzymatically released Thy and bromouracil (BrUra) is presented. The hydrolysis procedure was characterized and found to give uniform results when sample size was 1-10 [mu]g DNA and incubation time for DNA digestion was between 40 min and 16 h. Samples of DNA containing 3H-labeled BrdUrd were analyzed in parallel by the GC/MS technique and by specific radioactivity and buoyant density measurements, in order to compare the three methods. The GC/MS procedure gave values for percentage replacement of Thy by BrUra which were higher than those obtained by specific activity and lower than those obtained by buoyant density. This GC/MS method can detect 1% replacement in a 1-[mu]g DNA sample, equivalent to approximately 105 cells or 0.1 mg tissue, and will permit sensitive and quantitative analysis of the presence of this chemotherapeutic agent/radiosensitizer in cellular DNA from biopsy samples of normal or tumor tissue.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26801/1/0000357.pd