| Project/Area Number |
07557179
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 試験 |
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| Research Field |
Laboratory medicine
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| Research Institution | Tokai University School of Medicine |
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Principal Investigator |
INOKO Hidetoshi Tokai University School of Medicine,, 医学部, 教授 (10101932)
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| Co-Investigator(Kenkyū-buntansha) |
URYU Noboru Nichirei Genetic Research Laboratory, Researcher, 遺伝子ラボ, 研究員
ANDO Asako Tokai University School of Medicine, Lecturer, 医学部, 講師 (40101935)
TSUJI Kimiyoshi Tokai University School of Medicine, Professor, 医学部, 教授 (30055834)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥7,800,000 (Direct Cost: ¥7,800,000)
Fiscal Year 1996: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 1995: ¥4,100,000 (Direct Cost: ¥4,100,000)
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| Keywords | HLA / DNA typing / PCR-RFLP method / fluorescently labeling / dye-amidide / automated DNA sequencer / polymorphism / DQA1 gene / PCR-RFLP / 自動化 / 蛍光標識プライマー / 制限酵素 / GENESCAN / ジェノタイパ- |
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| Research Abstract |
We have previously developed the PCR-RFLP method which is sensitive and reliable in HLA class II genotyping. In this study, to develop an automated typing machine, we analyzed for fluorescent labeling of primers and optimized the condition for scanning of gel patterns of PCR products using an automated DNA sequencer. Dye-amidide was more reliable and stable fluorescent than aminolink-2 for labeling of the 5'-end of DQA1 and DQB1 primers. The PCR-RFLP method was performed using DQA1 primers that was fluorescently labeled with dye-amidide, and informative restriction enzymes. Using Genescan^<TM>672 and Genotyper^<TM>software kits, all DQA1 genotypes of four HLA homozygous typing cells (HTC) and fourteen heterologous healthy individuals were easily defined from the band patterns and automatical fragment size assignment. These typing results were completely identical to those by the conventional PCR-RFLP method. Each fragment size can be determined precisely even if the quantity of PCR products is only 1 ng. Therefore, this new PCR-RFLP method is highly sensitive and reliable in HLA class II genotyping. Discrimination between specific and non-specific fragments in the PCR products was relatively easy for the precision of size and quantitative amount assignment. Electrophoretic data can be also displayd as a multi color digitized gel pattern using several dyes labeling. Therefore, this system is highly efficient for application to a lot of samples.
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