福尔马林保存鱼类标本中大片段DNA的提取

从福尔马林保存的鱼类标本中获得高质量DNA是比较困难的。我们对前人的方法进行了如下改进：1）在标本的前处理过程中,通过长时间的缓冲液浸泡、短暂的加温、真空干燥来消除福尔马林对样品的影响;2）在样品消化过程中,加入相对过量的蛋白酶K和还原剂;3）提取DNA后立即进行PCR反应,并增加反应的循环次数和提高退火温度。通过这些改进,我们成功地从福尔马林保存的鱼类标本中提取出了高质量DNA;通过对比不同方法（福尔马林、酒精及冰冻）处理过的标本的DNA测序结果,表明该方法是值得信赖的;标本从死亡到用福尔马林处理之间的时间延搁可能是影响所提取的DNA质量的重要因素。

Discussing on the feasibility of large fragments DNA extraction from formalin-fixed fish specimens

Formalin-fixed specimens were of profound importance to the study of evolution,systematic,phylogeny and genetic structure of populations.However,the PCR analysis of DNA isolated from formalin-fixed tissue could be difficult because of the inherently poor quality of template DNA available for amplification.As a consequence of fixation process,DNA was complex with proteins and is often nicked,giving relatively short fragments.Such samples were often of low DNA concentration and poor quality.Formalin-fixation had a profound effect on the molecular arrangement of nucleic acids in the tissue and resulted almost invariably in DNA degradation of various degrees.Extraction,purification and amplification of DNA from formalin-fixed tissues were influenced by a multitude of factors.In this report,we introduced a method of DNA extraction and PCR amplification of relatively large target fragments from formalin-fixed,fluid-preserved tissues.Main modifications include: long-time preservation in buffer,short-time heating,and drying in a vacuum centrifuge during pre-disposal;more proteinase K and sodium SDS during extraction;performing PCR immediately after digestion,prolonging annealing time,and increasing the number of cycles.The feasibility of this method was tested by PCR amplification.The nucleotide DNA of cloned products from formalin-fixed specimens were sequenced and the results were compared with the sequences obtained from fresh and ethanol-fixed tissue and published data.Comparison of the DNA sequence data from the formalin-fixed tissues with that from the frozen and ethanol-fixed tissues demonstrated this method is reliable.We also found that the pre-fixation time could be an important factor determining the quality of DNA.