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Nucleic Acids Research, 2003, Vol. 31, No. 2 e2
© 2003 Oxford University Press

Capillary electrophoretic analysis of genomic DNA methylation levels

Dirk Stach, Oliver J. Schmitz1, Stephan Stilgenbauer2, Axel Benner3, Hartmut Döhner2, Manfred Wiessler and Frank Lyko*,4

Division of Molecular Toxicology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, 1 Department of Analytical Chemistry, University of Wuppertal, Gaußstrasse 20, 42119 Wuppertal, Germany, 2 Department of Internal Medicine III, University of Ulm, Robert-Koch-Strasse 8, 89081 Ulm, Germany, 3 Central Unit Biostatistics and 4 Research Group Epigenetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

*To whom correspondence should be addressed. Tel: +49 6221 423800; Fax: +49 6221 423802; Email: f.lyko{at}dkfz.de

Changes in DNA methylation have been found in the large majority of tumors. This phenomenon includes both genome-wide hypomethylation and gene- specific hypermethylation. However, the clinical relevance of either mechanism has remained contentious. In order to determine DNA methylation levels from a large number of clinical samples, we have established a method for accurate high-throughput quantification of 5-methylcytosine in genomic DNA. Our protocol requires a small amount (<1 µg) of DNA that is enzymatically hydrolyzed to single nucleotides. Single nucleotides are then derivatized with a fluorescent marker and separated by capillary electrophoresis. After calibration of the method, we have determined cytosine methylation levels from tumor samples of 81 patients that had been diagnosed with chronic lymphocytic leukemia (CLL). These patients showed a high variability in their methylation levels with a general trend towards hypomethylation. Because of its high accuracy and throughput our method will be useful in determining the role of genomic DNA methylation levels in tumorigenesis.


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