2002 Fiscal Year Final Research Report Summary
Development of preparative method of chromosomal DNA for AFM observation and application for genome analysis
| Project/Area Number |
12450165
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Measurement engineering
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| Research Institution | Toyohastii University of Technology |
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Principal Investigator |
MIZIMO Akira Toyohastii University of Technology, Faculty of Engineering, Professor, 工学部, 教授 (20144199)
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| 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)
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| Project Period (FY) |
2000 – 2002
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| Keywords | DNA stretching / mapping / SPM / globule / micro manipulation |
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| 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
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[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
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[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
