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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.
Electrophoresis. 2005 Jun; 26(13):2591-8.E

Abstract

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.

Authors+Show Affiliations

Cornelius M
Division of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany.
Wörth CG
No affiliation info available
Kliem HC
No affiliation info available
Wiessler M
No affiliation info available
Schmeiser HH
No affiliation info available

MeSH

Boron CompoundsDNADeoxyadenine NucleotidesDeoxycytidine MonophosphateDeoxyguanine NucleotidesDeoxyribonucleotidesElectrophoresis, CapillaryEthylenediaminesFluorescent DyesLasersReproducibility of ResultsSensitivity and SpecificitySpectrometry, FluorescenceSulfitesThymidine Monophosphate

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15934055

Citation

Cornelius, Michael, et al. "Detection and Separation of Nucleoside-5'-monophosphates of DNA By Conjugation With the Fluorescent Dye BODIPY and Capillary Electrophoresis With Laser-induced Fluorescence Detection." Electrophoresis, vol. 26, no. 13, 2005, pp. 2591-8.
Cornelius M, Wörth CG, Kliem HC, et al. Detection and separation of nucleoside-5'-monophosphates of DNA by conjugation with the fluorescent dye BODIPY and capillary electrophoresis with laser-induced fluorescence detection. Electrophoresis. 2005;26(13):2591-8.
Cornelius, M., Wörth, C. G., Kliem, H. C., Wiessler, M., & Schmeiser, H. H. (2005). Detection and separation of nucleoside-5'-monophosphates of DNA by conjugation with the fluorescent dye BODIPY and capillary electrophoresis with laser-induced fluorescence detection. Electrophoresis, 26(13), 2591-8.
Cornelius M, et al. Detection and Separation of Nucleoside-5'-monophosphates of DNA By Conjugation With the Fluorescent Dye BODIPY and Capillary Electrophoresis With Laser-induced Fluorescence Detection. Electrophoresis. 2005;26(13):2591-8. PubMed PMID: 15934055.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Detection and separation of nucleoside-5'-monophosphates of DNA by conjugation with the fluorescent dye BODIPY and capillary electrophoresis with laser-induced fluorescence detection. AU - Cornelius,Michael, AU - Wörth,Christian G C T, AU - Kliem,Hans-Christian, AU - Wiessler,Manfred, AU - Schmeiser,Heinz H, PY - 2005/6/4/pubmed PY - 2005/9/20/medline PY - 2005/6/4/entrez SP - 2591 EP - 8 JF - Electrophoresis JO - Electrophoresis VL - 26 IS - 13 N2 - 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. SN - 0173-0835 UR - https://www.unboundmedicine.com/medline/citation/15934055/Detection_and_separation_of_nucleoside_5'_monophosphates_of_DNA_by_conjugation_with_the_fluorescent_dye_BODIPY_and_capillary_electrophoresis_with_laser_induced_fluorescence_detection_ DB - PRIME DP - Unbound Medicine ER -