摘要
气候变化和人类活动胁迫致使许多海洋鱼类种群结构发生了显著变化。为了摸清鱼类种群结构对气候变化和人类胁迫活动的响应机理,利用福尔马林固定标本研究较早年代海洋鱼类DNA是一种有效的途径。为了解决福尔马林固定鱼类样本DNA提取质量差的难题,以福尔马林固定近50年的金线鱼(Nemipterus virgatus)标本为例,研究DNA提取过程中多个因素对DNA提取产量的影响。以取样部位、除甲醛处理、消化时间、抽提方法为考察因素,以DNA提取浓度为评价指标,通过SPSS软件设计L8(4×24)正交实验。结果,得到的最优处理组为:肌肉组织样品经GTE缓冲液浸泡与酒精梯度处理后,90℃水浴30 min,真空干燥处理,加入裂解液与蛋白酶K,55℃水浴消化3 h,试剂盒法提取DNA。除甲醛处理是提高DNA产量的关键,处理步骤越多效果越好。90℃水浴后再消化能够显著提高DNA提取效率。通过对比标本DNA与新鲜样品DNA,PCR扩增的结果,证明使用优化方法提取的DNA能够满足后续研究的需求。
In recent decades,the population structure of many marine fish has dramatically changed under the influ-ence of anthropogenic activities and climate change.To better understand the change mechanism,DNA extracted from formalin-fixed marine fish specimens is required.However,obtaining high quality DNA from formalin-fixed specimens is difficult.To obtain knowledge for improving the quantity of DNA extracted from marine fish specimens,ten Nemipterus bathybius specimens that were fixed in 1962 were used as samples,and an orthogonal experiment was designed following the L8（4×24） table.The DNA concentration was taken as the evaluation index.The tissue types,anti-formaldehyde treatments,digestive times and extraction methods were taken as selection factors.The best group was determined to be as follows： muscle samples wash for an extended time in buffer GTE and an alcohol gradient,90℃ annealing for 30 min and drying in a vacuum centrifuge,3 h of digestion with Proteinase K within a waterbath of 55℃,and DNA extraction by kit.Anti-formaldehyde treatments significantly improved the production of DNA and the use of various anti-formaldehyde methods obtained the best effect.Digestion after a 90℃ waterbath was efficient.The quality of specimen DNA was assessed by PCR amplification and sequencing.The sequences be-tween specimens and fresh tissue were compared,which validated that the methods were reliable.
出处
《中国水产科学》
CAS
CSCD
北大核心
2012年第6期1068-1073,共6页
Journal of Fishery Sciences of China
基金
广东省重大科技计划项目(2009B030600002)
中央级公益性科研院所基本科研业务费专项资金项目(2010ZD01,2011YD03)
广东省科技计划项目(2011B031100001)
广东省自然科学基金资助项目(9451030002002475)
关键词
福尔马林
金线鱼
DNA提取
正交设计
Formalin
Nemipterus virgatus
DNA extraction
orthogonal design
