Epigenetics includes DNA methylation in which a genome is physiologically modified and posttranslational modification of chromatin which is a complex of DNA and proteins and a large number of proteins constituting chromatin, which integrally control the gene expression.
Regarding modification of histones in chromatin, structural alteration of chromatin by histone modification plays an important role in the induction of transcription. For example, acetylation by a histone acetylation enzyme triggers the induction of remodeling of chromatin, so that transcription is started by general transcription factors and a RNA polymerase. In addition, methylation or phosphorylation of histones controls the transcription and causes silencing, chromatin condensation, and the like.
In addition, in the genomes of many eukaryotes, 60 to 90% of CpG dinucleotides are methylated at the 5-carbon atom position of cytosine. The methylated CpG is found in heterochromatins and transposons containing many repeated sequences, and thought to suppress the activation of viruses and transposons. In addition, the CpG methylation and the histone modification coordinate with each other.
An exception is that CG-rich regions (CpG islands) present in promoter regions of many genes are not methylated. In addition, an exception to the exception is that CpG islands are methylated in genes to be imprinted and in the inactive X chromosome of females. In addition, CpG islands are methylated also in the promoter regions of tumor suppressor gene in cancer cells. Hence, methylation of cytosine can be used as a marker of development, recurrence, and metastasis of cancer. In this respect, a simple method for detecting whether or not cytosine in a gene is methylated has been sought.
As described above, 5-methylcytosine, which is a methylated cytosine (C), has been reported to be present in the DNA, and has been known to play an important role in the developmental genetics, so far. Recently, a new base (5-hydroxymethylcytosine) has been discovered in a study of Purkinje cells (Non Patent Literature 1). It is pointed out that 5-hydroxymethylcytosine is a key for elucidating the mechanism of DNA demethylation (reprogramming). Hence, specifying the presence and the position of 5-hydroxymethylcytosine in a DNA is important and essential as a core technology for investigating the reprogramming of a gene function in all the fields associated with epigenetics technologies, such as the fields of cancer, aging, and regenerative medicine.
Various methods have been proposed for chemically and easily detecting methylcytosine in a DNA. Patent Document 1 describes a method for distinguishing cytosine from methylcytosine. In addition, Patent Document 2 describes a method in which a target DNA is selectively cleaved at the position of 5-methylcytosine to obtain a 5′-fragment, an enzymatic reaction is carried out by using a FRET probe capable of hybridizing with the 5′-fragment and a flap endonuclease, and then the fluorescence is detected.
However, none of these methods can be applied directly to the detection of 5-hydroxymethylcytosine.
Known methods for detecting 5-hydroxymethylcytosine in a DNA include a method in which 5-hydroxymethylcytosine in a fragmented DNA sample is detected by applying the conventional immunoprecipitation method using an anti-5-hydroxymethylcytosine antibody (Non Patent Literatures 2 and 3), a method in which 5-hydroxymethylcytosine is modified with a sugar by using an enzyme capable of the sugar modification, and the modified 5-hydroxymethylcytosine is isolated (Non Patent Literature 4), and the like.
However, each of the method requires that a DNA sample be fragmented in advance, and has a problem that even when a fragment containing 5-hydroxymethylcytosine is isolated, the position of the 5-hydroxymethylcytosine in the fragment cannot be specified.
Hence, there is a demand for a method for chemically and easily detecting 5-hydroxymethylcytosine in a nucleic acid such as a DNA.