| Project/Area Number |
10557074
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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 |
Pediatrics
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| Research Institution | TOHOKU UNIVERSITY |
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Principal Investigator |
NARISAWA Kuniaki MEDICAL GENETICS, TOHOKU UNIV., PROFESSOR, 大学院・医学系研究科, 教授 (90004647)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Yoichi MEDICAL GENETICS, TOHOKU UNIV., ASSOCIATE RESEARCHER, 大学院・医学系研究科, 助手 (80216457)
KURE Shigeo MEDICAL GENETICS, TOHOKU UNIV., ASSOCIATE RESEARCHER, 大学院・医学系研究科, 助手 (10205221)
MATSUBARA Yoichi MEDICAL GENETICS, TOHOKU UNIV., ASSOCIATE PROFESSOR, 大学院・医学系研究科, 助教授 (00209602)
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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 |
¥9,000,000 (Direct Cost: ¥9,000,000)
Fiscal Year 1999: ¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 1998: ¥5,100,000 (Direct Cost: ¥5,100,000)
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| Keywords | KNOWN MUTATION / TAQ MAN-ASA / SYBR-GREEN-ASA / ALLELE SPECIFIC PRIMER / PHENYLKETONURIA / GLYCOGEN STORAGE DISEASE TYPE 1A / MEDIUM CHAIN ACYL-COA DEHYDROGENASE / Multiplex primer extension法 / Extention プライマー / 糖原病1型 / ホロカルボキシラーゼ合成酵素欠損症 / Tay-Sachs病 / 軟骨無形成症 / 自動既知変異検出システム |
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| Research Abstract |
We have developed two novel methods for the detection of point mutations, TaqMan-ASA and SYBR Green-ASA. APCR amplicon using two sets of allele-specific primers in the presence of a TaqMan probe or SYBR Green dye was monitored in real time with a fluorescence detector (PRISM 7700 Sequence Detection System). The difference in amplification efficiency between the two PCR reactions was determined by "threshold" cycles to differentiate mutant and normal alleles. These methods eliminate the requirement for subsequent gel electrophoresis or additional hybridization steps by directly detecting positive reactions. We applied these methods to detect R111X, IVS-4, Y204C, R241C, R243Q, R245V, R252W, R278W, IVS-9, Y356X and R413P mutations in patients with phenylketonuria, a prevalent 727g>t mutation in Japanese patients with glycogen storage disease type la, and a common K329E mutation in Caucasian patients with medium-chain acyl-CoA dehydrogenase deficiency. These techniques can be automated and are useful for the genotype analysis of a variety of single nucleotide polymorphisms and mutations.
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