title: Capillary electrophoretic analysis of genomic DNA methylation levels.
abstract: 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 micro g) 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.
authors.affiliation: Division of Molecular Toxicology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Stach
authors.forename: Oliver J
authors.initials: OJ
authors.lastname: Schmitz
authors.forename: Stephan
authors.initials: S
authors.lastname: Stilgenbauer
authors.forename: Axel
authors.initials: A
authors.lastname: Benner
authors.forename: Hartmut
authors.initials: H
authors.lastname: Döhner
authors.forename: Manfred
authors.initials: M
authors.lastname: Wiessler
authors.forename: Frank
authors.initials: F
authors.lastname: Lyko
mesh_terms: D003596:Cytosine / Q000378:metabolism; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics* / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D004273:DNA, Neoplasm; D003596:Cytosine