Synthesis of fluorescently labeled alkylated DNA adduct standards and separation by capillary electrophoresis.
DNA adducts are regarded as individual internal dosimeters for the exposure to chemical carcinogens. To date, the most sensitive method for DNA adduct analysis is the radioactive 32P-postlabeling method, which allows the detection of one adduct in 10(10) unmodified nucleotides in microg amounts of DNA. However, this technique suffers from disadvantages such as working with radioactive phosphorus and time-consuming chromatographic separation procedures. In addition, the simultaneous detection of adducts from different classes of carcinogens in a DNA sample is difficult. In order to overcome these drawbacks, we are developing a new detection method, comprising fluorescence labeling of DNA adducts, capillary electrophoretic (CE) separation, and on-line detection by monitoring laser-induced fluorescence (LIF). So far, we have evaluated the separation power and the detection limit of CE with fluorescently labeled standard compounds such as unmodified nucleotides or alkylated thymidines. For this purpose, we developed a universal method for labeling 5'-OH-mononucleosid-3'-dicyanoethyl-phosphates with fluorescent dyes based on the phosphoramidite technology for DNA synthesis. The separation of N3-methylated, N3-, O2- and O4-butylated thymidines from the unmodified nucleotide within a few minutes recommends CE-LIF as a powerful method for DNA adduct analysis.
Wörth
C C
CC
Molekulare Toxikologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany. c.woerth@dfkz-heidelberg.de
Schmitz
O J
OJ
Kliem
H C
HC
Wiessler
M
M
D000478:Alkylation; D002851:Chromatography, High Pressure Liquid; D018736:DNA Adducts / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods; D005456:Fluorescent Dyes; D015394:Molecular Structure; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013936:Thymidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000302:isolation & purification
D018736:DNA Adducts; D005456:Fluorescent Dyes; D013936:Thymidine