Determination of the DNA methylation level in tumor cells by capillary electrophoresis and laser-induced fluorescence detection.
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%.
Department of Analytical Chemistry, University of Wuppertal, Gauss-Strasse 20, D-42119 Wuppertal, Germany.
Michaela
M
Wirtz
Dirk
D
Stach
Hans-Christian
HC
Kliem
Manfred
M
Wiessler
Oliver J
OJ
Schmitz
D010582:Bacteriophage lambda / Q000737:chemistry / Q000235:genetics; D001896:Boron Compounds / Q000737:chemistry; D001943:Breast Neoplasms / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000032:analysis*; D003843:Deoxycytidine Monophosphate / Q000031:analogs & derivatives* / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007834:Lasers; D009369:Neoplasms / Q000737:chemistry / Q000175:diagnosis* / Q000235:genetics
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D003843:Deoxycytidine Monophosphate; C007261:deoxy-5-methylcytidylic acid