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.

Rapid screening of the heterogeneity of DNA methylation by single‐strand conformation polymorphism and CE‐LIF in the presence of electro‐osmotic flow

“…3A and B. The level of methylation increases with the decrease in fluorescence intensity of the PCR products . This result corresponds to the tendency shown in Fig.…”

“…Recently, Goedecke et al. reported a fast and sensitive method for quantitation of DNA methylation of the DRD4 gene by CE‐LIF . This method differentiates the rare methylation level from PCR products, indicating that the COBRA‐based techniques could successfully be combined with the CE‐LIF analysis to obtain more reliable experimental results.…”

Determination of DNA methylation by COBRA: A comparative study of CGE with LIF detection and conventional gel electrophoresis

“…The capillary was then filled with BGE from the flush vial (2 min, 30 psi) and the samples were injected hydrodynamically (20–50 s, 0.5 psi). The postconditioning procedure consisted of flushing with MeOH (1 min, 40 psi) to remove the gel buffer from the capillary 16.…”

“…Focusing on the last‐mentioned COBRA, it is necessary to develop alternative analytical strategies that can be performed in a time‐ and cost‐efficient way, lead to a better precision of the results, and moreover allow automatization. We have recently published a native CGE method 16 to investigate methylation levels in the 5′CpG island of the dopamine receptor D4 ( DRD4 ) gene promoter region, which meets all of the above‐mentioned requirements. Herein, we report on a comparison of this method with a commonly used slab‐gel electrophoresis method.…”

Comparative microRNA detection from precursor‐microRNA‐transfected hepatocellular carcinoma cells by capillary electrophoresis with dual‐color laser‐induced fluorescence

“…CE‐LIF is a sensitive and popular detection method for the –omics related research fields including genomics , epigenetics , metabolomics , and proteomics . CE‐LIF provided distinct advantages, such as good separation efficiency, high sensitivity, and smaller sample requirements; it can also be automated to facilitate high‐throughput screening.…”