Analysis of the methylated CpG-binding protein-mediated inhibition mechanism of isothermal DNA amplification and its application

2023 Fiscal Year Final Research Report

• Project/Area Number: 21H02457
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 43050:Genome biology-related
• Research Institution: Hirosaki University
• Principal Investigator: Fujita Toshitsugu 弘前大学, 医学研究科, 准教授 (10550030)
• Co-Investigator(Kenkyū-buntansha): 元岡 大祐 大阪大学, 微生物病研究所, 講師 (10636830)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Keywords: メチル化DNA / RPA / 等温増幅反応 / エピジェネティクス

Outline of Final Research Achievements

CpG methylation in specific genomic regions is involved in cell differentiation and proliferation, as well as in the onset of intractable diseases such as cancer. In this study, we combined the Recombinase Polymerase Amplification (RPA) method, which is an isothermal DNA amplification reaction, with methylated CpG-binding proteins to establish a bisulfite treatment-free, rapid, and simple method for evaluation of the CpG methylation status in template DNA. We also examined in detail the inhibition mode of the RPA reaction with methylated CpG-binding proteins, as well as the inhibitory effect on genome-wide scale. We also succeeded in establishing a rapid and accurate real-time CpG methylation evaluation technology.

Free Research Field

分子生物学

Academic Significance and Societal Importance of the Research Achievements

本研究では、バイサルファイト処理を必要とせずに、超高速・高精度にCpGメチル化を判定できる技術の開発に成功しており、分子生物学・医学分野でのCpGメチル化解析が飛躍的に進むとともに、CpGメチル化を指標とした癌細胞の検出技術として、医療分野への貢献も期待される。本研究成果は、多種類のエピジェネティック修飾を解析する技術に発展する可能性を秘めており、エピジェネティクス分野での波及効果が非常に高い。