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Accurate quantification of DNA methylation of DRD4 applying capillary gel electrophoresis with LIF detection
Author(s) -
Goedecke Simon,
Schlosser Sabrina,
Mühlisch Jörg,
Hempel Georg,
Frühwald Michael C.,
Wünsch Bernhard
Publication year - 2009
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800567
Subject(s) - capillary electrophoresis , chromatography , dna methylation , dna , gel electrophoresis , methylation , pulsed field gel electrophoresis , chemistry , microbiology and biotechnology , analytical chemistry (journal) , biology , gene , biochemistry , gene expression , genotype
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 ν ∼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.