DNA methylation analysis by bisulfite conversion, cloning, and sequencing of individual clones

DNA methylation is an essential epigenetic modification in the human genome. For the investigation of DNA methylation patterns, bisulfite conversion and DNA sequencing is a method of choice, because it provides detailed information on the methylation pattern of individual DNA molecules at single CG site resolution. The method is based on the deamination of cytosine residues to uracils in the presence of NaOH and sodium bisulfite. Since methylcytosine is not converted under these conditions, the original methylation state of the DNA can be analyzed by sequencing of the converted DNA. After the conversion reaction, the DNA sequence under investigation is amplified by polymerase chain reaction (PCR) with primers specific for one strand of the bisulfite-converted DNA. The PCR product is cloned and individual clones are sequenced. Here, we describe an advanced protocol for bisulfite conversion, protocols for cloning, and tools for primer design (Methprimer, Bisearch). In addition, we present tools for the web display of primary data and data analysis (BiQ Analyzer, BDPC) and describe the setup of a sequencing and analysis pipeline for medium to high throughput.