DNA Methylation Products


A Central Source for DNA Methylation Products for Research and Development
Methylation, Ltd. Methylation Ltd., provides innovative reagents and kits for researchers working on DNA methylation and with DNA methyltransferases. Gene silencing through methylation is a well conserved and important feature of gene expression in virtually all eukaryotic and many prokaryotic organisms. In animals, the process is complex and essential in growth and development. There is additional evidence that inappropriate silencing can lead to serious genetic defects as well as cancer. Specifically, there is new evidence to suggest that the balance between growth promoting (oncogenes) and growth suppressing (tumor suppressor) genes is altered by inappropriate methylation. This can lead to aggressive cell growth associated with cancer. Inappropriate silencing can be partially reversed using hypomethylating agents such as the drug aza-deoxycytidine (aza-dC). Aza-dC (also aza-C) is in fact a useful tool to explore methylation events in vivo with endogenous methylation enzymes (DNA methyltransferases or DNMTs). The companys technology to examine methylation in vivo (the ICM Assay Kit) is based on use of aza-dC to actually track endogenous DNMTs on specific genes inside tumor cells. Kits are available to assay endogenous DNMTs (ICM Assay) and to recover sites of activity on the human genome. Also offered are in vitro assay kits for DNMT binding to DNA (SDSK+) as well as antibodies, DNA targets and drugs used in hypomethylation studies.

The ICM Kits are based on the finding that DNMT becomes irreversibly (covalently) bound to genomic DNA that is labeled with aza-deoxycytidine. This allows researchers to measure DNMT binding in vivo using antibodies (to specific isoforms of DNMT) to probe the DNA peak fractions of a CsCl gradient. Essentially, the ICM is a way to detect real time methylation activity on specific gene regions with specific DNMT isoforms. The method is based on the published paper by Liu et al, (2003) and is described in more detail in the PDF file: ICM KIT SUMMARY.

The key to the analysis is to separate DNA from free protein in a single step using CsCl density separation. One then uses Western slot or dot blots of the DNA peak to identify the presence of a given DNMT isoform. As shown above, cells labeled with the hypomethylating drug 5-aza-2-deoxycytidine, have DNMT-1 in the DNA fraction while control cells (no drug) have no detectable DNMT-1. Moreover, probing with a non-specific antibody (topoisomerase), fails to detect any signal in the DNA fractions either in the presence or absence of aza-deoxycytidine. Note also that the amount of signal is directly proportional to the amount of DNMT-1 active on the genome at the time of lysis by sarkosyl.

To demonstrate the quantitative aspects of the ICM Kit, the following experiment was done. HeLa cells were labeled for 1 hour with increasing amounts of aza-dC and DNA analyzed by ICM. The DNA fractions were pooled and its concentration accurately measured by fluorometry. Either 1,5 or 10 ug of DNA were slot blotted and probed with anti-DNMT1 antibody. The data show increasing amounts of DNMT-1 on the genome with increasing aza-dC treatment. If one is interested in a quantitative comparison of different isoforms, the ICM Kit should work extremely well using this quantitative adaptation

A Central Source for DNA Methylation Products for Research and Development