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DNA Research Advance Access originally published online on February 22, 2006
DNA Research 2006 13(1):37-42; doi:10.1093/dnares/dsi024
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© The Author 2006. Kazusa DNA Research Institute
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

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

Johannes Pröll1,2,*, Mathilde Födermayr1,2, Christian Wechselberger2, Patrick Pammer2, Max Sonnleitner2, Otto Zach1 and Dieter Lutz1

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

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.

Key words: epigenetics; DNA methylation; oligonucleotide microarray; immunofluorescence

*To whom correspondence should be addressed. Tel. +43-732-6060-7929, Fax. +43-732-6060-7930, E-mail: johannes.proell{at}uar.at

Communicated by Michio Oishi


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