| Project/Area Number |
10557252
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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 | Tokai University |
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Principal Investigator |
INOKO Hidetoshi Tokai University School of Medicine, Professor, 医学部, 教授 (10101932)
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| Co-Investigator(Kenkyū-buntansha) |
NISHIMURA Naoyuki Shimazu Factory Inc., Researcher, 基盤技術研究所・医療グループ, 主任研究員
ANDO Asako Tokai University School of Medicine, Lecturer, 医学部, 講師 (40101935)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥12,200,000 (Direct Cost: ¥12,200,000)
Fiscal Year 1999: ¥5,800,000 (Direct Cost: ¥5,800,000)
Fiscal Year 1998: ¥6,400,000 (Direct Cost: ¥6,400,000)
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| Keywords | HLA typing / mass spectrometry / automated analysis / allele / high-performance liquid chromatography / 自動判定 |
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| Research Abstract |
In order to develop an automated HLA typing machine, we have estimated the molecular weight of PCR products after DNA amplification of the HLA DRB1 gene using electrospray ionization mass spectrometry (MS) and optimized the condition for purification of PCR products. The application to MS of the PCR-RFLP method was hindered due to impurities in PCR products after restriction enzyme digestion and purification. In contrast, PCR products amplified with sequence specific primer sets for the PCR-SSP method allowed measurement of their reliable molecular mass. Further, the typing software was revised for the numerous sequence specific primer sets. However, in several heterozygous samples, it is difficult to assign the alleles precisely even if we use the revised software. To validate the combination of the MS and PCR-SSP methods, we have optimized the size of PCR products and minimized the time for measurement by slide chips of mass spectrometer. The molecular mass of PCR products with a 90 bp-length can be determined precisely by MS after their simplified purification. This combination of the MS and PCR-SSP methods is a simple and sensitive HLA typing system, and so may be suited for application to identify one base pair mismatch in the typing of heterozygous samples using relatively short length PCR products, especially their molecular weight of less than 50 kDa.
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