| Project/Area Number |
07557017
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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 | 展開研究 |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
YUASA Yasuhito Tokyo Medical and Dental University, Department of Hygiene and Oncology, Professor, 医学部, 教授 (80111558)
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| Co-Investigator(Kenkyū-buntansha) |
SUEHIRO Takeshi Nippon Bio-Rad Laboratories, Technical Support Planning Department Manager, 企画部, 室長
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥15,600,000 (Direct Cost: ¥15,600,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1996: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1995: ¥11,300,000 (Direct Cost: ¥11,300,000)
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| Keywords | capillary electrophoresis / gene diagnosis / PCR / mutation / microsatellite / colorectal cancer / SSCP |
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| Research Abstract |
We developed a method to analyze a polyadenine tract, the (A)_<10> repeat, within the cysteine-rich domain of the transforming growth factor-beta (TGF-beta) type II receptor gene using a non-gel-sieving capillary electrophoresis technique and applied it to the DNA diagnosis of colorectal cancers. This method consists of single-strand DNA amplification of the (A)_<10> repeat by an asymmetric PCR technique and capillary electrophoresis. A higher concentration of dATP in the PCR reaction mixture led to more specific amplification of the (A)_<10> repeat. Under the optimal electrophoretic conditions, one nucleotide difference could be determined in 8 to 32 nucleotides. One or two base deletions of the (A)_<10> repeat in colorectal cancers could be detected under these conditions within 30 min, and the results coincided with those obtained on DNA sequencing analyzes. According to a sensitivity study, we could detect the deleted sequence if it was present in 12.5% or more of the wild-type allele. The reproducibility of this technique was satisfactory because the intraassay imprecision (CV) (n=10) was 1.4%. These results indicate that capillary electrophoretic analysis of small repeated sequences results in easier handling and more feasible automation, com-pared with conventional gel electrophoretic analysis.
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