The development of a reaction for detecting the presence/absence of one methyl group in a long DNA strand is a chemically and biologically challenging research subject. A newly designed chemical assay on a chip for the typing of DNA methylation has been developed. A methylation-detection probe fixed at the bottom of microwells was crosslinked with methylated DNA mediated by osmium complexation and contributes to selective amplification of methylated DNA. 1. Introduction Gene expression is well regulated by the epigenetic modification of DNA and histone tails independent of their primary sequences. In particular, cytosine methylation, in which the C5 position of the cytosine base is methylated enzymatically, plays a crucial role in the regulation of chromatin stability, gene regulation, parental imprinting, and X-chromosome inactivation in females [1–4]. Therefore, detection of cytosine methylation is very important, and much effort has gone into developing a simple reaction for 5-methylcytosine ( ) detection. For the evaluation of the methylation status of genes, several conventional methods have so far been used, such as a cleavage assay with methylation-insensitive restriction enzymes [5–7], hydrolysis and sequencing with a bisulfite salt [8–10], and immunofluorescence with anti-5-methylcytosine antibody [11, 12]. Although the conventional methods have many merits, there are many disadvantages, and methylation detection assays must be further improved through another approach. The existence of a more rapid and selective chemical reaction capable of distinguishing between methylcytosine and unmethylated cytosine on a chip has promise as a good method for efficiently analyzing the status of cytosine methylation at a specific site in a gene. The sequence-selective DNA methylation-detection probe, ICON (interstrand complexes formed by osmium and nucleic acids), may be effective for the development of an on-chip analysis of DNA methylation [13–15]. In the presence of osmium oxidants and a bipyridine ligand, 5-methylcytosine forms a stable osmium-centered complex, in contrast to unmethylated cytosine (Figure 1) [16–19]. ICON probes form a crosslink with a specific 5-methylcytosine in the probe-hybridizing DNA mediated by osmium-centered complex formation. This function will be effective for the capture of methylated DNA on a chip for sequence-selective methylation analysis. Figure 1: Structures of the “B” nucleotide of the ICON probe for 5-methylcytosine selective crosslink formation and the “D 514” nucleotide of the Exciton primer for real-time PCR
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