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Development of preparative method of chromosomal DNA for AFM observation and application for genome analysis

Research Project

Project/Area Number 12450165
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Measurement engineering
Research InstitutionToyohastii University of Technology

Principal Investigator

MIZIMO Akira  Toyohastii University of Technology, Faculty of Engineering, Professor, 工学部, 教授 (20144199)

Co-Investigator(Kenkyū-buntansha) TAKASHIMA Kazunori  Toyohastii University of Technology, Faculty of Engineering, Research Associate, 工学部, 助手 (60303707)
MATSUZAWA Yukiko  Toyohastii University of Technology, Faculty of Engineering, Research Associate, 工学部, 助手 (10273335)
KATSURA Shinji  Toyohastii University of Technology, Faculty of Engineering, Associate Professor, 工学部, 助教授 (10260598)
Project Period (FY) 2000 – 2002
Project Status Completed (Fiscal Year 2002)
Budget Amount *help
¥12,300,000 (Direct Cost: ¥12,300,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥10,400,000 (Direct Cost: ¥10,400,000)
KeywordsDNA stretching / mapping / SPM / globule / micro manipulation / AFM / DNA伸張 / 染色体 / DNA / 伸長固定 / 遺伝子
Research Abstract

We have been studying micromanipulation technique of single DNA molecule for rapid genome analysis. Because human genome project has been just finished, function of protein and protein-DNA complex (nucleosome), and genetic differences between individual persons such as single nucleotide polymorphisms (SNPs) must be studied in the next stage. To reveal a function of protein and nucleosome gives us many information of our body. And to detect SNPs or genetic errors is helpful for human health.
In this study, at first, we have analyzed a structure of RecA-DNA complex. RecA is known as one of proteins that play on important role on DNA recombination and DNA repair. RecA protein forms helical complex with DNA molecules. Analysis of electron microgram found that the pitch of helix was10nm, however, no one has ever seen the 10nm pitch of RecA-DNA complex in three-dimensional structure directly. we tried to directly observe the form of RecA-DNA complex by atomic force microscope (AFM) with a car … More bon nanotube (CNT) attached cantilever. AFM is a powerful tool for analysis of biological molecules. It has an atomic resolution and can be used in atmosphere, solution, and high vacuum. A CNT has very small diameter (several nm 〜 some ten nm) and high aspect ratio, and is flexible and stiffness. So a CNT attached cantilever can realize higher resolution than a normal fabricated silicon cantilever. We observed the 10nm pitch of RecA-DNA complex by AFM with a CNT cantilever.
Second, we have developed a method to detect specific sites of DNA such as mismatched base pairs by AFM. We can determine the mismatch position precisely and rapidly if we can observe a protein that specifically binds to mismatched nucleotides on a stretched DNA molecule. In this study, we tried to observe restriction enzyme-DNA complexes as a model of protein bound on the mismatch point on stretched DNA molecules. DNA molecules can be stretched and immobilized by meniscus or flow force by freezing DNA solution. We prepared restriction enzyme-DNA complexes without magnesium ions in solution and clearly observed the restriction enzyme attached to a specific site of DNA molecule on an amino-silanized mica surface. Less

Report

(4 results)
  • 2002 Annual Research Report   Final Research Report Summary
  • 2001 Annual Research Report
  • 2000 Annual Research Report
  • Research Products

    (16 results)

All Other

All Publications (16 results)

  • [Publications] Michihiko Nakano, Jun Komatsu, Shun-ichi Matsuura, Kazunori Takashima, Shinji Katsura, Akira Mizuno: "Single molecule PCR using water-in-oil emulsion"Journal of Biotechnology, vol. 203, 117-124, 2003. 203. 117-124 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] S.Matsuura, H.Kurita, M.Nakano, J.Komatsu, K.Takashima, S.Katsura, A.Mizuno: "One-End Immobilization of Individual DNA Molecules on a Functional Hydrophobic Glass Surface"J. of Biomolecular Structure & Dynamics. 20. 429-435 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] A.Mizuno, S.Katsura: "Manipulation of a Large DNA Molecule using the Phase Transition"J. of Biological Physics. 28. 587-603 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Y.Matsuzawa, K.Hirano, A.Mizuno, M.Ichikawa, K.Yoshikawa: "Geometric manipulation of DNA molecules with a laser"Applied Physics Letter. 81. 3494-3496 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] K.Hirano, Y.Baba, Y.Matsuzawa, A.Mizuno: "Manipulation of single DNA molecules by laser clustering of microparticles"Applied Physics Letter. 80. 515-517 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] K.Umemura, J.Komatsu, T.Uchihasi, N.Choi, S.Ikawa, T.Hishinaka, T.Shibata, Y.Nakayama, S.Katsura, A.Mizuno, et al.: "Atomic Force Microscopy of RecA-DNA Complexes Using a Carbon Nanotube Tip"Biochemical and Biophysical Research Communications. 281. 390-395 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Michihiko Nakano, Jun Komatsu, Shun-ichi Matsuura, Kazunori Takashima, Shinji Katsura and Akira Mizuno: "Single molecule PCR using water-in-oil emulsion"Journal of Biotechnology. vol.203. 117-124 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] S.Matsuura, H.Kurita, M.Nakano, J.Komatsu, K.Takashima, S.Katsura and A.Mizuno: "One-End Immobilizaiton of Individual DNA Molecules on a Functional Hydrophobic Glass Surface"J.of Biomolecular Structure & Dynamics. Vol.20, No.3. 429-435 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] A.Mizuno and S.Katsura: "Manipulation of a Large DNA Molecule using the Phase Transition"J.of Biological Physics. vol.28. 587-603 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Y.Matsuzawa, K.Hirano, A.Mizuno, M.Ichikawa and K..Yoshikawa: "Geometric manipulation of DNA molecules with a laser"Applied Physics Letter. Vol.81, No.18. 3494-3496 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] K.Hirano, Y.Baba, Y.Matsuzawa and A.Mizuno: "Manipulation of single DNA molecules by laser clustering of microparticles"Applied Physics Letters. vol.80, No.3. 515-517 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] K.Umemura, J.Komatsu, T.Uchihasi, N.Choi, S.Ikawa, T.Hishinaka, T.Shibata, Y.Nakayama, S.Katsura, A.Mizuno, H.Tokumoto, M.Ishikawa, and R.Kuroda: "Atomic Force Microscopy of RecA-DNA Complexes Using a Carbon Nanotube Tip"Biochemical and Biophysical Research Communications. Vol.281, No.2. 390-395 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Jun Komatsu, Shunichi Matsuura, Michihiko Nakano, Kazunori Takashima, Shinji Katsura, Akira Mizuno: "Detection of restriction enzyme on the stretched DNA molecules by atomic force microscope"Proceedings ESA-IEJ Joint Meeting 2002. 26-31 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 小松 旬, 松浦 俊一, 佐藤 知也, 高島和則, 松澤 有希子, 桂 進司, 水野 彰: "針電極とグロビュール相転位を用いたDNA伸張固定技術の開発"静電気学会講演論文集'01. 131-132 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] 小松 旬, 高島和則, 桂 進司, 水野 彰: "DNA結合タンパク質-DNA複合体の原子間力顕微鏡による解析"2002年度静電気学会春期講演会論文集. 45-46 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] 佐藤知也,松浦俊一,平野研,松澤有希子,桂進司,水野彰: "微小針電極を用いたグロビュールDNAの伸長・固定技術の開発"静電気学会講演論文集'00. 105-106 (2000)

    • Related Report
      2000 Annual Research Report

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Published: 2000-04-01   Modified: 2016-04-21  

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