The scope of dansyl vs fluorescein label in fluorescence postlabeling assay for DNA damage.
The 5'-phosphate groups of the normal nucleotides of DNA (10 nmol) were labeled with two different fluorescent reagents, namely, dansyl chloride and fluorescein-isothiocyanate, via phosphoramidate derivatives of ethylene and hexamethylenediamines. Using a conventional detector, HPLC analysis showed a linear relationship of fluorescence signal as an integrated peak area and the amounts of nucleotide injected from 2 pmol to 200 fmol (r = 0.996, n = 3) for dansyl-labeled dAmp and from 2 pmol to 90 fmol (r = 0.997) for fluorescein-labeled dAmp, respectively. Although fluorescein-labeled nucleotides are more sensitive than dansylated nucleotides (detection limit < 2 times, S/N = 3), the excess labeling reagent from the fluorescein labeling reaction interferes with HPLC resolution of the labeled nucleotides. The phosphoramidate derivatives of the nucleotides require purification prior to labeling with fluorescein isothiocyanate. As a result, the overall assay procedure becomes complicated. The potential of dansylated nucleotides to assay DNA modifications has been demonstrated by HPLC resolution of the labeled nucleotides of both normal and modified bases without further manipulation of the reaction intermediates.