Reliable detection of DNA CpG methylation profiles by the isoschizomers MspI/HpaII using oligonucleotide stimulators.

The increasing interest in methylation patterns of mammalian DNA demands a simple and reliable method to clearly differentiate between cytosine (C) and 5-methylcytosine (5-MeC) within a specific DNA region. The simplest and most commonly used approach is the analysis with restriction endonucleases exhibiting different methylation sensitivities, like the MspI/HpaII pair of isoschizomeric enzymes (New England Biolabs, Beverly, MA, USA). Whereas MspI cleaves the recognition sequence 5′-CCGG independently of the methylation state of the internal C, cleavage by HpaII is blocked by the presence of 5-MeC at this site (3). The method is compromised by the fact that methylation can be detected only in those CpGs that are located within the tetranucleotide recognition sequence. Another problem is that not all restriction endonucleases cleave their respective unmethylated recognition sequences completely. An incomplete digestion of unmethylated DNA is typically observed when DNA substrates with a low frequency of recognition sites are incubated with restriction endonucleases requiring at least two copies of the recognition site for cleavage activity (2). HpaII seems to belong to this group of restriction endonucleases, because genomes possessing only one or very few recognition sites [simian virus 40 (SV40) (Reference 4 and unpublished) and hamster polyoma virus (unpublished)] exhibit an intrinsic resistance towards this enzyme. This means that unmethylated DNA molecules are not (or are not completely) digested by the enzyme, mimicking a