| Project/Area Number |
16591347
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Digestive surgery
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| Research Institution | Iwate medical University School of Medicine |
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Principal Investigator |
OTSUKA Koki (2005) Iwate Med.Univ., School of Med., Dept.of Surg.1., Research Associate, 医学部, 助手 (50316387)
樋口 太郎 (2004) 岩手医科大学, 医学部, 講師 (40244909)
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| Co-Investigator(Kenkyū-buntansha) |
MAESAWA Chihaya Iwate Med. Univ. School of Med., Dept of Pathol., Lecturer, 医学部, 助教授 (10326647)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2004: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Keywords | k-ras / MGB / colorectal cancer / real-time PCR / p53 / APC / 大腸癌 |
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| Research Abstract |
Tumor-specific point mutations are stable biomarkers compared with tumor-specific mRNA expression, and are therfore useful to detect occult tumor cells. These mutations have never been used for real-time quantitative polymerase chain reaction (RQ-PCR) assays, because the ability of conventional probes to discriminate between wild-type and mutant alleles is poor. Recently, DNA probes with conjugated minor groove binder (MGB) have been developed. Because of their high melting temperature, these probes achieve high performance in detecting single nucleotide mismatches. Using the MGB technology, we developed a new RQ-PCR system for detecting occult tumor cells in patients with colorectal cancer (CRC), targeting K-ras point mutations. Sixteen MGB-conjugated DNA probes were designed for all previously reported K-ras mutations. The performance of these probes was examined with plasmid DNAs into which K-ras point mutations had been inserted, 32 cancer cell lines and 338 lymph nodes obtained from 15 CRC patients. Fifteen of the 16 MGB probes designed were useful for accurate quantitative assessment, and achieved high sensitivity (1/10^4-10^5 background cells) and high reproducibility (coefficients of variation < 10%). Performance in discriminating single nucleotide mismatches was superior for MGB probes compared with non-MGB probes. We detected a micrometastasis (5.85/10^4 cells equivalent) in one (0.9%) of 110 lymph nodes obtained from 6 patients with K-ras mutations. There was no true false-positive result in 209 lymph nodes obtained from 9 patients without K-ras mutations. The MGB RQ-PCR assay targeting K-ras mutations is an accurate quantitative method for detecting occult tumor cells in CRC.
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