Determination of the DNA methylation level in tumor cells by capillary electrophoresis and laser-induced fluorescence detection |
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Authors: | Wirtz Michaela Stach Dirk Kliem Hans-Christian Wiessler Manfred Schmitz Oliver J |
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Institution: | Department of Analytical Chemistry, University of Wuppertal, Gauss-Strasse 20, D-42119 Wuppertal, Germany. |
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Abstract: | An analytical method to determine the genome-wide DNA methylation in only 100 ng DNA is presented. The analysis is based on DNA isolation and hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with a fluorescence dye (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride, Bodipy FL EDA). The separation of the derivatives was carried out by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. To calculate the methylation level, the derivatization factor and the quantum yields of the Bodipy conjugates of 2'-desoxycytidine-3'-monophosphate (dCMP) and 2'-desoxy-5-methylcytidine-3'-monophosphate (5m-dCMP) were determined by measurement of methylated Lambda DNA. The assignment was made by cochromatography with the synthesized and characterized standard compound 5m-dCMP. After optimization of the method it was possible to determine the methylation level in 100-ng DNA samples with a standard deviation of less than 5%. |
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Keywords: | Cancer Capillary electrophoresis – laser‐induced fluorescence DNA adducts DNA methylation 5‐Methylcytosine |
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