摘要
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。
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.
出处
《水生生物学报》
CAS
CSCD
北大核心
2007年第4期485-491,共7页
Acta Hydrobiologica Sinica
基金
国家基础科学人才培养基金(NSFC-J0030092)
中国科学院院长基金(A2901046)资助
