| Project/Area Number |
10557250
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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 | Osaka University |
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Principal Investigator |
TATSUMI Ke-ita Osaka University Graduate School of Medicine, Lecturer, 医学研究科, 講師 (00222109)
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| Co-Investigator(Kenkyū-buntansha) |
KOMATSUBARA Hidesuke Toyobo Co. Ltd., Tsuruga Inst. of Biotechnology, Researcher, 敦賀バイオ研究所, 研究員
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 2000: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1999: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1998: ¥3,600,000 (Direct Cost: ¥3,600,000)
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| Keywords | genetic analysis / RT-PCR / mRNA / cDNA |
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| Research Abstract |
In genetic analysis, mRNA s, most beneficial when they can be obtained from tissues that are not difficult to obtain, as mRNAs contains both the nucleotide sequence information of the gene as well as the expression levels. But for genes that are only expressed in specific tissues that are difficult to obtain., and genomic DNAs are commonly used only when the genomic sturcture has been elucidated To analyze nucleotide sequence of genes that are only expressed in specific tissues that are difficult to obtain and whose genomic sturcture has not been elucidated, we attempted to develop a rapid genetic analysis method using nonspecific mRNA from blood samples so that neither the genomic DNA structure is not needed. After isolating RNA from peripheral blood samples, cDNAs for beta-actin or sodium/iodide symporter(NIS) could be amplified satisfactory using reverse transcription and polymerase chain reaction (RT-PCR), However, when blood Sample of a patient With combined heterozygous mutation of V59E/T354P in the NIS gene was used, V59E mutant sequence and T354P mutant sequence were not amplified efficiently compared to the wild type sequences. As amplification of these alleles were similar when 0.1 microgram genomic DNA was used, tremendous amplification from small amount of cDNA could have made prominent the biased amplification due to point mutation alone or with combination with change in the amplified sequence lacking intron sequence. In conclusion rapid genetic analysis method using nonspecific mRNA from blood samples is workable, but has a bottleneck of biased amplification.
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