Quantitative analysis of DNA methylation in the promoter region of the methylguanine-O(6) -DNA-methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis.
Along with histone modifications, RNA interference and delayed replication timing, DNA methylation belongs to the key processes in epigenetic regulation of gene expression. Therefore, reliable information about the methylation level of particular DNA fragments is of major interest. Herein the methylation level at two positions of the promoter region of the gene methylguanine-O(6) -DNA-Methyltransferase (MGMT) was investigated. Previously, it was demonstrated that the epigenetic status of this DNA region correlates with response to alkylating anticancer agents. An automated CGE method with LIF detection was established to separate the six DNA fragments resulting from combined bisulfite restriction analysis of the methylated and non-methylated MGMT promoter. In COBRA, the DNA was treated with bisulfite converting cytosine into uracil. During PCR uracil pairs with adenine, which changes the original recognition site of the restriction enzyme Taql. Artificial probes generated by mixing appropriate amounts of DNA after bisulfite treatment and PCR amplification were used for validation of the method. The methylation levels of these samples could be determined with high accuracy and precision. DNA samples prepared by mixing the corresponding clones first and then performing PCR amplification led to non-linear correlation between the corrected peak areas and the methylation levels. This effect is explained by slightly different PCR amplification of DNA with different sequences present in the mixture. The superiority of CGE over PAGE was clearly demonstrated. Finally, the established method was used to analyze the methylation levels of human brain tumor tissue samples.
Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität, Münster, Germany.
Simon
S
Goedecke
Klinik und Poliklinik für Kinder- und Jugendmedizin - Pädiatrische Hämatologie und Onkologie, Münster, Germany.
Jörg
J
Mühlisch
Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität, Münster, Germany.
Georg
G
Hempel
Swabian Children's Cancer Center, Children's Hospital, Augsburg, Germany.
Michael C
MC
Frühwald
Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität, Münster, Germany.|Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), Westfälische Wilhelms-Universität Münster, MüNster, Germany.
Bernhard
B
Wünsch
D001932:Brain Neoplasms / Q000235:genetics*; D002138:Calibration; D004247:DNA / Q000737:chemistry; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics* / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000235:genetics* / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D013447:Sulfites / Q000737:chemistry; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
D013447:Sulfites; D025521:Tumor Suppressor Proteins; D004247:DNA; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes; C042345:hydrogen sulfite
Capillary gel electrophoresis; Combined bisulfite restriction analysis (COBRA); DNA methylation; Epigenetics; Laser induced fluorescence detection