Development of a methylated DNA sensing system for circulating tumor cells detection

2019 Fiscal Year Final Research Report

• Project/Area Number: 17K06933
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Biofunction/Bioprocess
• Research Institution: Tokyo University of Technology
• Principal Investigator: YOSHIDA Wataru 東京工科大学, 応用生物学部, 講師 (10599806)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Keywords: メチル化CpG / メチル化アデニン / グアニン四重鎖 / 定量PCR / 熱安定性

Outline of Final Research Achievements

We have previously reported that PCR amplification efficiency decreased when the template DNA containing VEGF G-quadruplex (G4) was CpG methylated. In this project, thermal stability of the CpG methylated VEGF G4 structure was analyzed. The results revealed that the G4 structure was stabilized by CpG methylation in the presence of 20 mM NaCl and 2 mM MgCl2. Thermal analysis also demonstrated that c-kit1 G4 structure was destabilized by N6-methyladenosine modifications. Moreover, 9,651 G4 clusters were identified in human genomic DNA by high-throughput sequencing of whole genome amplified products with a G4 ligand.

Free Research Field

核酸工学

Academic Significance and Societal Importance of the Research Achievements

本研究により、CpGのメチル化とアデニンのメチル化がG4構造の熱安定性に影響を与えることが示された。さらに、本メチル化レベル測定法の標的となるG4構造形成配列が含まれるヒトゲノムDNA領域を網羅的に同定することに成功した。つまり、今後これらの知見を基に、定量PCRを行うだけで簡便に標的遺伝子のCpGメチル化レベルやアデニンメチル化レベルを測定できる方法が開発され、種々の疾病の診断法へ利用されることが期待される。また、メチル化レベル測定法の開発だけでなく、メチル化G4構造の生体内での機能解明にも貢献することが期待される。