Genetic analysis of extinct and endangered species using formalin fixed specimens

研究已灭绝或濒临灭绝物种的繁殖史。研究了甲醛固定标本中DNA的提取和纯化方法。对于DNA提取，修改以下方案。(1)（2）预消化过程以淬灭甲醛活性（3）优化蛋白酶-K消化（4）从消化物中回收DNA。(2)（3）在DTT +4 M尿素缓冲液中连续加入ProteinaSe-K的方法最佳;（4）羟基磷灰石法在回收量和纯化效果上均优于常规方法。用硅胶基质直接或回收后纯化的方法不能获得足够的DNA用于扩增，用20年前固定的大麻哈鱼标本提取的DNA进行PCR扩增，成功地扩增了线粒体DNA中500 bp的细胞色素B区。但是从非常古老的标本中提取的DNA的相同区域的扩增并不成功。PCR扩增的长度可能受到降解DNA的限制，微卫星DNA比mtDNA更适合于本研究，因为它可以使用较短的片段进行分析。

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.