Chemical modification of DNA is one way that cells regulate which genes are expressed as proteins. Methylation of certain cytosines is a chemical modification that regulates gene expression in many normal biological contexts. In addition, inactivation of tumor suppressor genes by aberrant methylation of cytosines is frequently associated with cancer. Thus, mapping of methylation sites in DNA is important for understanding normal and pathological cellular processes. We have developed a method for determining the methylation states of many cytosines in a genomic DNA sample in parallel.

The method uses oligonucleotide arrays made with our Digital Optical Chemistry (DOC) system. Binding of a DNA sample to oligonucleotides on the array is used to determine the sequence of the DNA in the sample. Prior to sequence determination, the DNA is treated with sodium bisulfite, which converts unmethylated cytosines to deoxyruracils, but leaves methylated cytosines unchanged. Thus, the pattern of methylated cytosines determines the final sequence of the DNA, which is assessed by binding to the array of oligonucleotide probes. Thousands of cytosines widely dispersed through the genome can be analyzed in parallel this way. We are using this method to characterize tumor cells by their methylation states. We have analyzed the methylation of cytosines associated with 30 different genes in 13 different lung tumor cell lines and found that each cell line has a unique methylation profile. Interpretation of these profiles may provide new understanding about the process of tumorigenesis and may provide new tools for treatment and diagnosis of cancer.

Assay for CpG methylation by treatment with sodium bisulfite and sequence analysis with an oligonucleotide array. (a) Treatment with sodium bisulfite converts all unmethylated cytosines to deoxyuracils while methylated cytosines remain unconverted (b) Sequence analysis of a labeled representative of the bisulfite-treated DNA by hybridization to an array of oligonucleotides. The central base of each probe for a given position is varied to test for the identity of the base by hybridization. The probe with which the most label is associated identifies the base at the central position. A cytosine at the probed position indicates methylation that prevented conversion by sodium bisulfite.