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DNA Research Advance Access published online on February 22, 2006
DNA Research, doi:10.1093/dnares/dsi024
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© The Author 2006. Kazusa DNA Research Institute.
Received October 20, 2005
Revised December 12, 2005

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

Johannes Pröll 1 *, Mathilde Födermayr 1, Christian Wechselberger 2, Patrick Pammer 2, Max Sonnleitner 2, Otto Zach 3, and Dieter Lutz 3

1 Elisabethinen Hospital, 1st Department of Internal Medicine, Fadingerstrasse 1, A-4010 Linz, Austria; Upper Austrian Research GmbH, Center for Biomedical Nanotechnology, Scharitzer Strasse 6, A-4020 Linz, Austria
2 Upper Austrian Research GmbH, Center for Biomedical Nanotechnology, Scharitzer Strasse 6, A-4020 Linz, Austria
3 Elisabethinen Hospital, 1st Department of Internal Medicine, Fadingerstrasse 1, A-4010 Linz, Austria

* To whom correspondence should be addressed.
Johannes Pröll, E-mail: johannes.proell{at}uar.at


  Abstract

For the determination of methylation levels in genomic regulatory DNA sequences a high-sensitive assay for detecting 5'methyl-cytosines (5'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'mC in single-stranded DNA hybridized to oligonucleotide microarrays. For assay readout an ultra-sensitive fluorescence scanner with submicrometer resolution was used. To minimize autofluorescence 150-µm thin glass slides with an aldehyde-functionalized surface were developed. These methodological improvements allowed the detection of 5'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.

Keywords: epigenetics; DNA methylation; oligonucleotide microarray; immunofluorescence.
Communicated by Michio Oishi
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