Genetic analysis of extinct and endangered species using formalin fixed specimens

使用福尔马林固定标本对灭绝和濒危物种进行遗传分析

• 批准号: 09839032
• 负责人: IDA Hitoshi
• 金额: $ 0.83万
• 依托单位: Kitasato University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09839032/
• 关键词: extinct species; endangered species; DNA extraction; mtDNA; PCR; formalin; Proteinase-K; Hydroxy appatite; 系統; シトクロームb

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.

研究灭绝或濒临灭绝的物种的系统发育。对福尔马林固定标本的DNA提取和纯化方法进行了研究。对以下的DNA提取方法进行了修改。结果表明：(1)物理干扰最小，避免了共用DNA；(2)改进了Tris-Glyine缓冲液的操作方法，提高了工作效率；(3)在DTT中加入4M高温培养的尿素缓冲液，连续加入Proteina Se-K是最优的；(4)与常规方法相比，羟基贴壁法在提取DNA的量和纯化方面都是最好的。用硅胶基质直接或提取纯化后的DNA不能扩增出足够数量的DNA。对20年前固定的鲑鱼标本提取的DNA，mtDNA中500bp的细胞色素b区的PCR扩增是成功的。但是，对从极其古老的标本中提取的DNA进行相同区域的扩增并不成功。POR的可能扩增长度似乎受到降解DNA的限制。微卫星DNA被认为比mtDNA更适合于这项研究，因为分析使用的是较短的片段。