| Project/Area Number |
09839032
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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 |
自然史科学
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| Research Institution | Kitasato University |
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Principal Investigator |
IDA Hitoshi Kitasato University School of Fisheries Science, Professor, 水産学部, 教授 (90050533)
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| Co-Investigator(Kenkyū-buntansha) |
ASAHIDA Takashi Kitasato University School of Fisheries Science, Lecturer, 水産学部, 講師 (00296427)
HAYASHIZAKI Ken-ichi Kitasato University School of Fisheries Science, Lecturer, 水産学部, 講師 (80208636)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1998: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1997: ¥800,000 (Direct Cost: ¥800,000)
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| Keywords | extinct species / endangered species / DNA extraction / mtDNA / PCR / formalin / Proteinase-K / Hydroxy appatite / 系統 / シトクロームb |
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| Research Abstract |
To examine phylogeny of extinct or endangered species. DNA extraction and purification methods from formalin-fixed specimens were studied. For DNA extraction following protocols were modified. (1) disruption of tissue to ease protein digestion (2) predigestion process to quench formaldehyde activity (3) optimization of proteinase-K digestion (4)retrieval of DNA from digestions.
As results (1) physical disturbance should be minimal to avoid sharing DNA.(2) protocol was modified to work Tris-Glycine buffer more efficiently, (3) serial addition of Proteina se-K in DTT added 4M Urea buffer incubated at high temperature was optimum, (4) hydroxy appatite method performed best in both amount of retrieved DNA and purification comparing ordinary methods. Direct or post retrieval purification from digestins using silica-matrix could not yield enough amount of DNA for amplification.
PCR amplification of 500 bp of Cytochrome b region in mtDNA was successful for DNA extracted from chum salmon specimens fixed 20 years ago. But amplifications of the same region for DNA extracted from extremely old specimens were not successful. The possible amplification length using POR seemed to be limited by degraded DNA.Microsatellite DNA thought to be more appropriate for this study than mtDNA because shorter fragments were used for the analysis.
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