a) identification of differentially methylated regions:
Genomic DNA from tissue samples or cell cultures is treated with a frequent cutter whose recognition site occurs rarely within GC-rich and therefore potentially methylated regions (e.g. MseI which recognize TTAA).
The cleaved ends of the GC-rich fragments are ligated to linkers.
Repetetive sequences are removed by Cot-1 subtractive hybridization.
Half of the subtracted DNA is cut with a methylation sensitive endonuclease whose recognition sequence occurs frequently within CpG rich sites (e.g. BstUI which recognizes CGCG).
Both DNA-samples (one with methylation insensitive enzyme treatment and the one with methylation insensitive+sensitive treatment) are use as templates for linker PCR. Unmethylated genomic fragments have been cut and can not serve as template, whereas the fragments are amplified in the untreated control sample.
Both PCR fragment pools are used to screen libraries by hybridization. Library clones that show differences in the hybridization signal are candidates for differentially methylated regions.
b) estimation of degree of methylation
Probes for Southern Blotting are PCR generated from the library clones that showed positive signals in step (a). Digest with a methylation sensitive encyme and estimation of the degree of methylation from the fragment density patter delivers an estimation of the methylaton degree from 1 (complete methylation) to 0 (no methylation) in DHM units.