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Detection and separation of nucleoside‐5'‐monophosphates of DNA by conjugation with the fluorescent dye BODIPY and capillary electrophoresis with laser‐induced fluorescence detection
Author(s) -
Cornelius Michael,
Wörth Christian G. C. T.,
Kliem HansChristian,
Wiessler Manfred,
Schmeiser Heinz H.
Publication year - 2005
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200410405
Subject(s) - bodipy , chemistry , capillary electrophoresis , dna , deoxyadenosine , fluorescence , chromatography , micrococcal nuclease , oligonucleotide , biochemistry , microbiology and biotechnology , biology , chromatin , physics , quantum mechanics , nucleosome
We investigated the separation and detection of the 5'‐monophosphates of 2'‐deoxynucleosides selectively conjugated with 4,4‐difluoro‐5,7‐dimethyl‐4‐bora‐3a,4a‐diaza‐ s ‐indacene‐3‐propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'‐phosphate group using capillary electrophoresis with laser‐induced fluorescence detection (CE‐LIF). BODIPY conjugates of the four common deoxynucleoside‐5'‐monophosphates (2'‐deoxyguanosine‐5'‐monophosphate, 2'‐deoxyadenosine‐5'‐monophosphate, 2'‐deoxycytidine‐5'‐monophosphate, and thymidine‐5'‐monophosphate) were prepared and subjected to CE‐LIF to serve as standard compounds for peak assignment and to develop separation conditions for the analysis of DNA. BODIPY conjugates were detected and resolved by CE‐LIF after digestion of DNA or an oligonucleotide to 5'‐monophosphates by nuclease P1 (NP 1) and fluorescence labeling without further purification step. Comparative analyses of calf‐thymus DNA digested either with micrococcal nuclease/spleen phosphodiesterase to 3'‐monophosphates or with NP 1 to 5'‐monophosphates showed that both versions of the fluorescence postlabeling assay were equally efficient and sensitive. Moreover, using the same assay, 2'‐deoxyuridine and 2'‐deoxy‐5methylcytidine were identified in bisulfite treated DNA after NP 1 digestion indicating that fluorescence postlabeling of 2'‐deoxyribonucleoside‐5'‐monophosphates with BODIPY FL EDA and detection by CE‐LIF has the potential to determine DNA damage and genomic DNA methylation.