An alteration in DNA methylation is one of the most prominent changes associated with early embryo development, cellular differentiation, and progression of many types of cancers, including breast and prostate cancers. In the past several years we have been developing techniques to detect alterations in DNA methylation, targeting potential clinical applications for cancer diagnosis. The DNA Microarray MS- AFLP method that we developed has been proven to be useful in detecting DNA methylation alterations at the Notl landmarks in the genome. However, the utility has been limited due to the absence of commercial interest in producing the DNA microarrays based on the technique. The dependence on Notl restriction enzymes to identify the methylated regions has also restricted the scope of interrogation of the genome. Keeping this in mind, we propose, in this Exploratory/Developmental R21 Research Grant application, to perform several pilot experiments to test the feasibility of a conceptually novel method for the detection of alterations in DNA methylation that can utilize the DNA microarrays that are currently available. As with any pilot study, this project is risky in nature because we have not experimentally tested the concept yet. However, if proven feasible, the new method may achieve a revolutionary role in analyzing the genome- wide detection of global changes in DNA methylation. In this application, we propose the following two specific aims. 1. To examine the utility of anti-5-methylcytosine (anti-5mC) antibodies toward the magnetic bead-based separation of methylated and unmethylated DNA fragment populations. We will establish the experimental conditions under which 2 different fractions rich in methylated and unmethylated DNA fragments may be separated, using anti-5mC. 2. To examine the utility of methylated and unmethylated DNA fragment populations separated above toward the array-based detection of differences in DNA methylation. We will fluorescently label DNA from those 2 fractions and perform DNA microarray hybridization experiments. We will evaluate the sensitivity, specificity, and reproducibility of the method, using BAG clone microarrays, DNA fragment microarrays, and oligonucleotide microarrays. ? ? ?
