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-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.
Subject(s)
5-Methylcytosine/analysis , DNA Methylation , Oligonucleotide Array Sequence Analysis/methods , Antibody Specificity , Feasibility Studies , HL-60 Cells , Humans , Models, Biological , Nucleic Acid Hybridization , Polymerase Chain Reaction/methods , Sensitivity and SpecificityABSTRACT
Surfaces carrying a dense layer of poly(ethylene glycol) (PEG) were prepared, characterized, and tested as substrates for DNA oligonucleotide microarrays. PEG bis(amine) with a molecular weight of 2000 was grafted onto silanized glass slides bearing aldehyde groups. After grafting, the terminal amino groups of the PEG layer were derivatized with the heterobifunctional cross-linker succinimidyl 4-[p-maleimidophenyl]butyrate to permit the immobilization of thiol-modified DNA oligonucleotides. The stepwise chemical modification was validated with X-ray photoelectron spectroscopy. Goniometry indicated that the PEG grafting procedure reduced surface inhomogeneities present after the silanization step, while atomic force microscopy and ellipsometry confirmed that the PEG layer was dense and monomolecular. Hybridization assays using DNA oligonucleotides and fluorescence imaging showed that PEG grafting improved the yield in hybridization 4-fold compared to non-PEGylated maleimide-derivatized surfaces. In addition, the PEG layer reduced the nonspecific adsorption of DNA by a factor of up to 13, demonstrating that surfaces with a dense PEG layer represent suitable substrates for DNA oligonucleotide microarrays.
