| Project/Area Number |
09557217
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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 | 展開研究 |
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| Research Field |
Laboratory medicine
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
UEDA Kunihiro KYOTO UNIVERSITY, INSTITUTE FOR CHEMICAL RESEARCH, PROFESSOR, 科学研究所, 教授 (00027070)
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| Co-Investigator(Kenkyū-buntansha) |
KITAGAWA Kohichiro DAKO JAPAN CO., LTD., RESEARCH AND DEVELOPMENT DEPARTMENT, MANAGER, 研究開発部, 部長
KIDO Takahiro KYOTO UNIVERSITY, COLLEGE OF MEDICAL TECHNOLOGY, ASSISTANT PROFESSOR, 医療技術短期大学部, 助手 (60234308)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥12,400,000 (Direct Cost: ¥12,400,000)
Fiscal Year 1999: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1998: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1997: ¥6,800,000 (Direct Cost: ¥6,800,000)
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| Keywords | PNA probe / MRSA / CSA / PNA-CSA method / IM-PCR method / in situ PCR / direct PCR / Ampdirect / ペプチド核酸 / 遺伝子診断 / mecA / in situ PCR / 点変異 / K-ras / 結核菌 / mec A / 16S rRNA / PNA / 黄色プドウ球菌 |
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| Research Abstract |
We developed a novel diagnostic method using PNA (Peptide Nucleic Acid). We first examined physicochemical and biological properties of PNA probe. We confirmed its characters superiority over DNA probe, such as the ability to freely penetrate through cell membranes, a high affinity to DNA or RNA target, the precise sequence specificity, and resistances to nuclease and protease, but the sensitivity proved to be insufficient for practical use. We then tried to combine PNA with the CSA (Catalyzed Signal Amplification) method, which utilizes ABC (avidin-biotin complex) formation with tyramine radicals. We applied this combination to detection of mecA gene in MRSA (methicillin-resistant Staphylococcus aureus), and obtained a remarkable improvement of the sensitivity. In addition to this PNA-CSA method, we investigated three other methods for efficient gene diagnosis. The first is IM (Intercalation-Monitering)-PCR, which enabled a real-time measurement of amplified DNA with a fluorescent probe to be intercalated into double-strand DNA. The second is in situ PCR, a technique to amplify DNA in pathological specimen. Our method enabled detection of point mutation of K-ras gene in lung cancer tissues. The third is direct PCR, that is amplification without DNA isolation from various clinical samples. We showed that AmpdirectィイD1TMィエD1, a commercial reagent cocktail to overcome inhibition of Taq polymerase by endogenous inhibitory substances, such as hemoglobin and bile acids, is useful for direct PCR from as well as feces.
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