Detection method for methylation density on microarray using methyl-CpG-binding domain protein

A method for determining methylation density of target CpG islands has been established. In the method, DNA microarray was prepared by spotting a set of PCR products amplified from bisulfite-converted sample DNAs. The PCR products on the microarray were treated by SssI methyltransferase and labeled with TAMRA fluorescence. A recombinant, antibody-like methyl-CpG-binding protein labeled with Cy5 fluorescence was used to identify symmetrical methyl-CpG dinucleotide of the PCR products on the microarray. By use of a standard curve with control mixtures, the ratio of two fluorescence signals can be converted into percentage values to assess methylation density of targeted fragments. We obtained the methylation density of six CpG islands on the two tumor suppressor genes of CDK2A and CDK2B from seven cancer cell line samples and two normal blood samples. The validity of this method was tested by bisulfite sequencing. This method not only allows the quantitative analysis of regional methylation density of a set of given genes but also could provide information of methylation density for a large amount of clinical samples.