ICON probes: synthesis and DNA methylation typing.

DNA methylation and demethylation significantly affect the deactivation and activation processes of gene expression, respectively. The determination of the location and frequency of DNA methylation is important for the elucidation of the mechanisms of cell differentiation and carcinogenesis and may be a useful and effective index for cancer diagnosis. We have developed an artificial DNA probe that induces a methylation detection reaction of a target cytosine in a long DNA sequence (ICON probe). This artificial DNA allows the rapid detection of a methyl group attached at the C5 position of the target cytosine. In addition, there is no nonspecific cleavage of genomic DNA in this reaction. The ICON probe also facilitates the quantification of methylation at the target cytosine using a small amount of genomic DNA sample. This unit provides a procedure for synthesizing bipyridine-modified adenosine phosphoramidite and preparation of ICON probes. Additionally, the protocol for the methylation quantification experiments by quantitative PCR utilizing ICON probes is also presented.