Sequencing of sodium-bisulfite modified genomic DNA originally introduced by M. Frommer (Frommer et al., 1992) is a widely used “gold standard” method for DNA-methylation analysis. Since this method relies on a harsh chemical treatment of DNA it causes a lot of DNA damage and hence a dramatic loss of high quality DNA for PCR amplification and further analysis. In the meantime several commercial kits are available for this procedure which work reasonably well when starting with large amounts of DNA.
Here we describe a protocol for small numbers of cells and little DNA which requires some specific handling. The protocol is based on a strategy originally introduced by our lab (Hajkova et al., 2002) using agarose embedded DNA. This physical trapping helps to avoid DNA loss during the various incubation steps while maintaining a good bisulphite conversion rate. We will introduce two alternative procedures to perform bisulphite treatment of agarose embedded small DNA aliquots or cells and guide through some generally critical points in the bisulphite reaction and primer design. We also include tips for the process of data processing after sequencing which is facilitated by a new and very useful software tool (BiQ Analyzer). This tool allows rapid and reproducible processing and evaluation of bisulphite sequencing data. It generates standardized table output formats allowing direct database integration.
Saarland University, FR 8.3 Biosciences
Dept. of Genetics/Epigenetics
Campus Saarbrücken - 66123 Saarbrücken, Germany