title: Accurate quantification of DNA methylation of DRD4 applying capillary gel electrophoresis with LIF detection.
abstract: Aberrant DNA methylation of gene promoters may be investigated by an array of different technologies. Besides DNA sequencing techniques following bisulfite treatment and determination of overall methylation by quantification of 5-methylcytosine/cytosine ratio following DNA hydrolysis, most approaches rely on PCR amplification of a defined template and subsequent analysis by conventional gel electrophoresis. As an additional analytical tool, a capillary gel electrophoresis method has been developed to quantify the methylation in combined bisulfite restriction analysis products of the gene dopamine receptor D4 (DRD4). Analyses were carried out in a bare fused-silica capillary dynamically coated with a 1% w/w solution of PVA (M(r)=72,000). A buffer (pH 7.3) containing 3% w/w 2-hydroxyethylcellulose (M(nu) approximately 90,000 g/mol) was used as sieving matrix. With 1/x weighted regression the accuracy (bias) of the method is within +/-10% and the precision (expressed as RSD) also meets the common acceptance criteria of 15% (20% near lower LOQ). It overcomes the limitations of standard gel electrophoresis, which allows only one single run per analysis and requires large amounts of DNA. Therefore, the method represents a valuable tool for routine quantitative analysis of the methylation status of DRD4 and other target genes.
authors.affiliation: Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität, Münster, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Goedecke
authors.forename: Sabrina
authors.initials: S
authors.lastname: Schlosser
authors.forename: Jörg
authors.initials: J
authors.lastname: Mühlisch
authors.forename: Georg
authors.initials: G
authors.lastname: Hempel
authors.forename: Michael C
authors.initials: MC
authors.lastname: Frühwald
authors.forename: Bernhard
authors.initials: B
authors.lastname: Wünsch
mesh_terms: D002138:Calibration; D002531:Cerebellum / Q000737:chemistry; D019175:DNA Methylation; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D007223:Infant; D016014:Linear Models; D008297:Male; D050638:Receptors, Dopamine D4 / Q000737:chemistry*; D015203:Reproducibility of Results; D013447:Sulfites / Q000737:chemistry
chemical_list: C493599:DRD4 protein, human; D013447:Sulfites; D050638:Receptors, Dopamine D4; C042345:hydrogen sulfite