| Project/Area Number |
09672365
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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 |
Laboratory medicine
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| Research Institution | Showa University |
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Principal Investigator |
ARAKAWA Hidetoshi Showa University, School of Pharmaceutical sciences, Associate Professor, 薬学部, 助教授 (70129807)
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| Co-Investigator(Kenkyū-buntansha) |
KOKADO Amane Showa University, School of Pharmaceutical sciences, Assistant Professor, 薬学部, 助手 (20266159)
MAEDA Masako Showa University, School of Pharmaceutical sciences, Professor, 薬学部, 教授 (00053869)
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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 |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1999: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1998: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1997: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Osteoporosis / Vitamin D receptor / ELISA / Bioluminescence / SSCP / Capillary electrophoresis / RFLP / DNA polymorphism |
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| Research Abstract |
Osteoporosis is a major public health problem which affects quality of life and raises costs for health care providers. Although an osteoporotic fracture is caused by multiple factors, the risk of osteoporotic fracture in later life is determined by the peak bone density achieved in early adulthood in relation to age and menopause-related bone loss. It is reported that common allelic variation in the vitamin D receptor locus (VDR) can be used to predict bone turnover and bone mineral density. Japanese premenopausal women, homozygous subjects in whom the ApaI in intron 8 and TaqI site in exon 9 is present (AA,tt) were found to have lower BMD than those with the (aa, TT) genotype respectively. Analysis of VDR gene polymorphism is generally done by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and single strand DNA confirmational polymorphism. These traditional techniques are not suitable for routine clinical analysis because of problems of rapidity, reproducibility and use of electrophoresis gel separation (time consuming). Recently, capillary electrophoresis (CE) is used for analysis of RFLP and single strand DNA conformation polymorphism technique (SSCP). Further, enzyme linked immunosorbent assays (ELISA) combined with RFLP and hybridization using a DNA probe are used for analysis of PCR products. These methods have enabled the fast detection and quantification of PCR products. At present, a more sensitive and simpler method for DNA diagnosis is required. In the research reported here, we developed a sensitive and rapid PCR-RFLP ELISA using acetate kinase (AK) and firefly luciferase as a detection system and SSCP analysis by CE with laser-induced fluorescence detection for VDR plymorphism analysis. DNA polymorphism types of VDR could be clearly determined by these methods. These system is suitable for determining simultaneously DNA polymorphisms of a large number of samples.
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