Determination of DNA methylation by COBRA: a comparative study of CGE with LIF detection and conventional gel electrophoresis.
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.
Goedecke
Simon
S
Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität, Münster, Germany.
Schlosser
Sabrina
S
Klinik und Poliklinik für Kinder- und Jugendmedizin - Pädiatrische Hämatologie und Onkologie, Münster, Germany.
Mühlisch
Jörg
J
Klinik und Poliklinik für Kinder- und Jugendmedizin - Pädiatrische Hämatologie und Onkologie, Münster, Germany.
Hempel
Georg
G
Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität, Münster, Germany.
Frühwald
Michael C
MC
Klinik und Poliklinik für Kinder- und Jugendmedizin - Pädiatrische Hämatologie und Onkologie, Münster, Germany.
Wünsch
Bernhard
B
Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität, Münster, Germany.
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
D005456:Fluorescent Dyes; D004247:DNA
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