Ultra-sensitive immunodetection of 5'methyl cytosine for DNA methylation analysis on oligonucleotide microarrays.
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-microm 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.
Pröll
Johannes
J
Elisabethinen Hospital, 1st Department of Internal Medicine Fadingerstrasse 1, A-4010 Linz, Austria. johannes.proell@uar.at
Födermayr
Mathilde
M
Wechselberger
Christian
C
Pammer
Patrick
P
Sonnleitner
Max
M
Zach
Otto
O
Lutz
Dieter
D
D044503:5-Methylcytosine / Q000032:analysis*; D000918:Antibody Specificity; D019175:DNA Methylation; D005240:Feasibility Studies; D018922:HL-60 Cells; D006801:Humans; D008954:Models, Biological; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D044503:5-Methylcytosine