| Budget Amount *help |
¥19,630,000 (Direct Cost: ¥15,100,000、Indirect Cost: ¥4,530,000)
Fiscal Year 2010: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2009: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
Fiscal Year 2008: ¥8,190,000 (Direct Cost: ¥6,300,000、Indirect Cost: ¥1,890,000)
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| Research Abstract |
We developed direct electrochemical detection of DNA methylation using nanocarbon film formed by electron cyclotron resonance(ECR) sputtering. The developed method is based on oxidation potential difference between cytosine and methylated cytosine without any labeling. By optimizing electrode surface using electro- chemical pretreatment, we used this film to quantitatively detect single cytosine methylation regardless of the methylation position in the sequence including retino- blastoma gene fragment (24 mer). The nanocarbon film electrode also allowed us to realize the detection of DNA methylation ratios by measuring methylated 5'-cytosine- phosphoguanosine (CpG) repetition oligonucleotides (60 mer) with different methylation ratio. To improve the sensitivity for detecting the bases in the CpG oligonucleotides, we successfully used an exonuclease P1 to digest the target CpG oligonucleotide to an identical mononucleotide 5'-NMP. To develop the DNA methylation detection device, the microdisk array electrode which further improves sensitivity was fabricated using nanocarbon film which suppress biomolecules adsorption.
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