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Determination of the DNA methylation level in tumor cells by capillary electrophoresis and laser‐ induced fluorescence detection
Author(s) -
Wirtz Michaela,
Stach Dirk,
Kliem HansChristian,
Wiessler Manfred,
Schmitz Oliver J.
Publication year - 2004
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.200305761
Subject(s) - derivatization , capillary electrophoresis , chemistry , bodipy , chromatography , dna , fluorescence , micellar electrokinetic chromatography , laser induced fluorescence , gel electrophoresis , biochemistry , high performance liquid chromatography , physics , quantum mechanics
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%.