title: Determination of DNA methylation by COBRA: a comparative study of CGE with LIF detection and conventional gel electrophoresis.
abstract: DNA methylation as an epigenetic modification of the human genome is under emphatic investigation. Several studies have demonstrated a role of DNA methylation in oncogenesis. In conjunction with histone modifications, DNA methylation may cause the formation of heterochromatin and thus mediate the inactivation of gene transcription. It is important to develop methods that allow for an accurate quantification of the amount of DNA methylation in particular DNA regions, to gain information concerning the threshold of methylation levels necessary for gene inactivation. In this article, a CGE method with on-column LIF detection using SYBR Green is compared with a conventional slab-gel electrophoresis. We thus investigate the validity to analyze DNA methylation in the samples of a combined bisulfite restriction analysis. It is demonstrated that CGE is superior to gel electrophoresis in means of linearity, precision, accuracy, automatization (high throughput), and sample consumption. However, gel electrophoresis is easier to perform (simple devices, no PC usage), and the running costs are comparatively low. A further advantage of CGE is the sparse use of toxic compounds (MeOH and SYBR Green), whereas gel electrophoresis is performed in polyacrylamide gels with ethidium bromide staining.
authors.affiliation: Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität, Münster, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Goedecke
authors.affiliation: Klinik und Poliklinik für Kinder- und Jugendmedizin - Pädiatrische Hämatologie und Onkologie, Münster, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Schlosser
authors.affiliation: Klinik und Poliklinik für Kinder- und Jugendmedizin - Pädiatrische Hämatologie und Onkologie, Münster, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Mühlisch
authors.affiliation: Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität, Münster, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Hempel
authors.affiliation: Klinik und Poliklinik für Kinder- und Jugendmedizin - Pädiatrische Hämatologie und Onkologie, Münster, Germany.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Frühwald
authors.affiliation: Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität, Münster, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Wünsch
mesh_terms: D002138:Calibration; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D019075:Electrophoresis, Capillary / Q000191:economics / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000191:economics / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D016014:Linear Models; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D005456:Fluorescent Dyes; D004247:DNA
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