Methylation profiling by direct sequencing

Abstract

Introduction: DNA methylation takes part in the regulation of gene expression, although the exact mechanisms are incompletely understood. Therefore an exact determination of the degree and distribution of methylation is of great importance. Thus far methods used to examine methylation are mostly restricted to single or several CpG sites. Only sequencing methods provide information on the degree and distribution of methylation. Methods: Direct sequencing of bisulfite PCR products is used to determine the distribution and pattern of methylation. The benefit and restrictions of this method is evaluated using calibration series of cloned templates and representative samples. Results: Although quantification of methylation at some CpGs or for several sequences is impaired by sequencing artefacts it allows quantification of methylation in suitable sequences. Sequencing is highly reproducible (SD <0.03), but quantification is impaired by PCR-bias, when sequencing different PCR products (SD <0.2). Discussion: Due to the increased availability of capillary sequencers, direct sequencing is an easy to perform method, providing reproducible results. Artefacts impairing the results affect the whole sequence due to priming artefacts or single CpG, leaving a high number for evaluation. In contrast to methods restricted to single or multiple CpG, this methods allows the determination of methylation distribution in whole CpG-islands including gene regulatory binding sites. These observations may be correlated to expression and other epigenetic marks providing insight into the regulation of the respective gene. Supported by the C.D.-, the Sorensen-Haugstrup, the Cora-Lobscheid and the Sonja-Wasowicz Stiftung im Stifterverband fur die Deutsche Wissenschaft