Abstract
BackgroundDNA methylation based techniques are important tools in both clinical diagnostics and therapeutics. But most of these methods only analyze a few CpG sites in a target region. Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing.ResultsWe have developed a novel approach for quantitative analysis of CpG methylation density on the basis of microarray-based hybridization and incorporation of Cy5-dCTP into the Cy3 labeled target DNA by using Taq DNA Polymerase on microarray. The quantification is achieved by measuring Cy5/Cy3 signal ratio which is proportional to methylation density. This methylation-sensitive technique, termed RMEAM (regional methylation elongation assay on microarray), provides several advantages over existing methods used for methylation analysis. It can determine an exact methylation density of the given region, and has potential of high throughput. We demonstrate a use of this method in determining the methylation density of the promoter region of the tumor-related gene MLH1, TERT and MGMT in colorectal carcinoma patients.ConclusionThis technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample. The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.
Highlights
DNA methylation based techniques are important tools in both clinical diagnostics and therapeutics
The results show that this technique allows for the quantitative analysis of regional methylation density with simplicity, rapidness, specificity and high-throughput
The unmethylated allele of a given DNA sequence is expected to have the unmethylated cytosine of the test CpG sites converted to thymine, whereas these CpG sequences remain unchanged in the methylated allele
Summary
DNA methylation based techniques are important tools in both clinical diagnostics and therapeutics Most of these methods only analyze a few CpG sites in a target region. GC-rich DNA sequences are found frequently within the promoter and first exon of ~50% of all genes [1] These sequences, known as CpG islands, can be targets of DNA methylation. An epigenetic phenomenon is known to be associated with genomic imprinting and X-chromosome inactivation, and essential for normal mammalian development [2]. Both global hypomethylation and regional hypermethylation have been described in human tumor cell lines and a wide spectrum of cancers [3]. Methylation profiles of multiple genes for each cancer type might have important prognostic implications for clinical monitoring, risk assessment, and even therapeutic considerations [6,7,8]
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