| Project/Area Number |
13660339
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied molecular and cellular biology
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| Research Institution | Tokyo University of Agriculture & Technology |
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Principal Investigator |
IKEBUKURO Kazunori Tokyo University of Agriculture & Technology, Faculty of Technology, Associate Professor, 工学部, 助教授 (70251494)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2001: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | Zinc linger proiein / Detection of pathogenic bacteria / PCR product / Phage display / Detection of double stranded DNA / Fluorescence polarization / Zif 268 / 有機微生物検出 / PCR / 分子進化工学 / 遺伝的アルゴリズム |
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| Research Abstract |
In 2002, we succeeded in preparing the zif268 displayed phage with which we can directly detect the specific base sequence of the double stranded DNA. Zif 268 is one of the zinc finger protein and it is a translation factor of mouse. It specifically recognizes the 9 sequential base pairs, GCGTGGGCG and binds the double stranded DNA bearing this sequence with nano molar level dissociation constant. We tried to design and express the new zinc finger protein recognizing the specific sequential 9 base pairs in the invA gene specific to Salmonella which is one of the most popular pathogenic bacteria causing food poisoning in Japan by mutating the base sequence of the finger motif which recognize the base sequence, but the one showing strong and specific binding was not obtained.
Then we tried to develop the rapid and simple detection method of the double stranded DNA for the PCR product using fluorescence polarization method and zif268 displayed phage. We were able to detect the fluorescein
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labeled double stranded DNA bearing the target sequence of zif268, GCGTGGGCG by using zif268 displayed phage at once. We can observe the binding between two target molecules with fluorescence polarization method in solution simultaneously without the separation of bound and free molecules so that this method is one of the most simple and rapid method lo detect binding. The detection of Salmonella requires high sensitivity since it can cause food poisoning even a few bacteria, so that the PCR amplification of the DNA is required to obtain high sensitivity. However PCR product is double stranded and denaturing was required for hybridization of the DNA probe bearing complementary sequence to the target gene. By using this method developed in this research project, PCR product can be directly detected without pretreatment, therefore we can say that this is one of the most easy and rapid method to detect pathogenic bacteria. The wide application of this method to different bacteria would be expected. Less
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