2000 Fiscal Year Final Research Report Summary
Detection of 1 mutant cell in normal 10^5〜10^6 cells : Genetic diagnosis using enriched PCR
| Project/Area Number |
10470267
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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 | 一般 |
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| Research Field |
Thoracic surgery
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| Research Institution | Tohoku University |
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Principal Investigator |
SAGAWA Motoyasu Tohoku University Hospital, Research Associa, 医学部・附属病院, 助手 (70292274)
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| Co-Investigator(Kenkyū-buntansha) |
SATO Masami Tohoku University Hospital, Research Associa, 医学部・附属病院, 助手 (30250830)
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| Project Period (FY) |
1998 – 2000
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| Keywords | enriched PCR / PCR / point mutation |
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| Research Abstract |
Although sputum cytology and brushing cytology under the bronchoscope are useful for the detection of early lung cancer, it is impossible to detect cancer cells which have mutant DNA, but is not accompanied by the morphological change. On the other hand, it is important to detect very small ratio of mutant cells which exists in many normal cells in the clinical sample. In such case, detection method which has higher sensitivity is required. Enriched polymerase chain reaction method is the technology developed on the basis of the PCR-RFLP method, which can detect 1 mutant cell in normal 105〜106 cells. The principle of this method is as follows. After the samples are amplified (1st PCR), using the primer which introduced restriction enzyme cleavage site, normal DNA (the PCR product) was cut by the restriction enzyme. And then, only abnormal DNA was amplified by the second PCR.The problem of this method is misreading of the Taq DNA polymerase. In our study, DNA polymerase other than Taq was used, because some DNA polymerase has proofreading exonuclease activity. We have investigated various condition for enriched PCR with cultured cell, for the detection of point mutation of K-ras. Then, the investigation was proceeded to the establishment of the condition for the enriched PCR with pathological and cytological specimens.
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