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

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.

Dynamic Patterns of Histone Methylation Are Associated with Ontogenic Expression of the Cyp3a Genes during Mouse Liver Maturation

“…Although this method cannot resolve DNA methylation at base pair resolution as shotgun bisulphate sequencing does (Cokus et al, 2008), it has been used for ultrasensitive detection and genome-wide high-resolution mapping of DNA methylation (Pröll et al, 2006;Zhang et al, 2006). Two active regions of DNA methylation were identified in the Rnf6 and Cdk8 genes, but the contribution of DNA methylation on regulation of their gene expression needs to be determined.…”

“…Genomic DNA from the liver tissues was isolated by a ChargeSwitch gDNA Mini Tissue Kit (Invitrogen, Carlsbad, CA) and sonicated to an average length of 300 to 500 bp. An antibody against 5-methyl-cytosine (ab51552; Abcam Inc. Cambridge, MA) was used for immunoprecipitation of methylated DNA fragments as described previously (Pröll et al, 2006;Zhang et al, 2006). The purified ChIP-enriched fragments were amplified by using Sigma GenomePlex Kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's protocol (O'Geen et al, 2006).…”

A novel method to assess the full genome methylation profile using monoclonal antibody combined with the high throughput based microarray approach

“…The presence of DNA cytosine methylation in the hybridized DNA is detected by employing Cy3 labelled anti 5mC (5-methyl cytidine) antibody (monoclonal). Such an approach has been demonstrated by Proll et al 14 as a viable method for detecting methylation in hybridized DNA. They used a set of three genes using a microscope to detect florescence.…”

Single Molecule Bioanalysis

“…Besides RNA expression profiling, the technology paves the way for numerous further applications [90,91]. In particular, single molecule DNA sequencing comes into reach [18].…”