Ultra-Sensitive Immunodetection of 5'Methyl Cytosine for DNA Methylation Analysis on Oligonucleotide Microarrays

J. Proll, M. Fodermayr, C. Wechselberger, P. Pammer, M. Sonnleitner, O. Zach, D. Lutz
2006 DNA Research  
For the determination of methylation levels in genomic regulatory DNA sequences a high-sensitive assay for detecting 5 0 methyl-cytosines (5 0 mC) in non-bisulfite-treated DNA has been established. The system is designed for the application of immunofluorescence using a monoclonal antibody that specifically recognizes 5 0 mC in single-stranded DNA hybridized to oligonucleotide microarrays. For assay readout an ultra-sensitive fluorescence scanner with submicrometer resolution was used. To
more » ... was used. To minimize autofluorescence 150-mm thin glass slides with an aldehyde-functionalized surface were developed. These methodological improvements allowed the detection of 5 0 mC in synthetic oligonucleotides hybridized to microarrays with atto molar analytical sensitivity. Using enzymatic fragmented genomic DNA from myeloid leukemia tumor cell lines differences in the methylation status of gene regulatory sequences for E-cadherin, p15/CDKN2b and p16/ CDKN2a were demonstrated. Thus, this novel technique can potentially be used for DNA methylation analysis in various scientific fields.
doi:10.1093/dnares/dsi024 pmid:16766511 fatcat:hzkvnglmb5gklhaozweciw4u3q