Analysis of DNA metabolic reactions based on single molecule visualization method.
| Project/Area Number |
24770164
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Multi-year Fund |
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| Research Field |
Molecular biology
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| Research Institution | Gunma University |
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Principal Investigator |
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| Project Period (FY) |
2012-04-01 – 2015-03-31
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| Project Status |
Completed (Fiscal Year 2014)
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| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2014: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2013: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2012: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
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| Keywords | １分子観察 / 計測技術 / 可視化技術 / ＤＮＡ代謝反応 / ＤＮＡ複製 / ＤＮＡポリメラーゼ / ヌクレアーゼ / ＤＮＡ結合タンパク質 / 分子計測 / DNA / DNAポリメラーゼ / １分子解析 / １本鎖DNA / マイクロ流路 / 形態制御 / タンパク質 / モニタリング / 表面修飾 |
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| Outline of Final Research Achievements |
DNA synthesis and DNA digestion reactions were studied using direct single-molecule observations in microflow channels. In the channels, ssλDNA shape was controlled as relaxed and stretched form by adjusting buffer flow rate, and DNA synthesis reactions by Klenow Fragment (3’-5’ exo-) were observed. Analysis of the synthetic reaction rates demonstrated that the DNA synthesis reaction rate of stretched ssλDNA was approximately 75% higher than that of random coiled ssλDNA . Also, DNA digestion reactions by T7 Exonuclease (T7 Exo) were directly observed both under pulse-chase conditions and under continuous buffer flow conditions with T7 Exo. Under pulse-chase conditions, the double-stranded regions of λDNA monotonously shortened by a DNA digestion reaction with a single T7 Exo molecule. Under continuous buffer flow conditions with T7 Exo, some pauses were observed during a DNA digestion reaction and double-stranded regions shortened linearly except during these pauses.
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Report
(4 results)
Research Products
(12 results)
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[Journal Article] Direct Single-Molecule Observations of Local Denaturation of a DNA Double Helix under a Negative Supercoil State.2015
Author(s)
Takahashi S, Motooka S, Usui T, Kawasaki S, Miyata H, Kurita H, Mizuno T, Matsuura S, Mizuno A, Oshige M, Katsura S.
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Journal Title
Analytical Chemistry
Volume: 87
Issue: 6
Pages: 3490-3497
DOI
Related Report
Peer Reviewed / Acknowledgement Compliant
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[Presentation] Direct observation of fluorescently labeled single-stranded λDNA molecules in a micro-flow channel.
Author(s)
Takahashi S, Kawasaki S, Yamaguchi K, Miyata H, Kurita H, Mizuno T, Matsu-ura SI, Mizuno A, Oshige M, Katsura S
Organizer
5th International Conference on Advanced Micro-Device Engineering (AMDE2013)
Place of Presentation
Kiryu, Gunma
Related Report
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