Accurate quantification of DNA methylation of DRD4 applying capillary gel electrophoresis with LIF detection.
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.
Goedecke
Simon
S
Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität, Münster, Germany.
Schlosser
Sabrina
S
Mühlisch
Jörg
J
Hempel
Georg
G
Frühwald
Michael C
MC
Wünsch
Bernhard
B
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
C493599:DRD4 protein, human; D013447:Sulfites; D050638:Receptors, Dopamine D4; C042345:hydrogen sulfite