单条线虫DNA提取方法.docVIP

2011 年第 2 期  植物检疫 PLANT QUARANTINE 单条线虫 DNA 提取方法  Vol． 25 No． 2  王江岭  1  张建成  2  顾建锋  1* （ 1． 宁波出入境检验检疫局技术中心 浙江宁波 315012； 2． 嘉兴出入境检验检疫局）  Method of extract DNA from a single nematode． Wang Jiangling1 ，Zhang Jiancheng2 ，Gu Jianfeng1* （ 1． Ning- bo Entry － exit Inspection and Quarantine Bureau，Ningbo 315012，China； 2． Jiaxing Entry － exit Inspection and Quarantine Bureau） Abstract There are many problems in extracting DNA from large number of nematodes． The nematodes could be a mixed specie． The extracted DNA were decreasing in experiment process． Amplification might be cumbered by adding more solutes，and even to effect on the next experiments． In this method a single nematode was freezed by liquid nitrogen and heated at 85 to disrupte the structure of nematode cells by changing the temperature sudden- ly． Then the proteins were dissolved easily by proteinase K and more DNA were extracted． This method was proved effective，rapid，and stabile by kinds of nematodes． The advantages of this method are： （ 1） PCR Buffer attached to Taq enzyme take the place of WLB and SDS lysis liquids，which reduces the instability of reaction sys- tem and the effect of added solutes． （ 2） All the process is finished in one PCR tube，no liquid transported and less solutes added． So the pollution and bad effect to the later PCR process are greatly reduced． （ 3） Manual dis- ruption of nematode is replaced by poikilothermic treatment； pollution is avoided and no experiment technique is required． Compared with two other proteinase K methods of nematode DNA extraction，this method was proved ef- fective and stabile，and could be used in rapid test； nematode proteins were dissolved completely and more DNA released，the PCR result is good even when the extracted DNA were diluted for 32 time，which procides more mo- lecular experiments chances． Key words 摘要 nematode； optimized method； DNA extraction； proteinase K； temperature 大量线虫 DNA 的提取可能因线虫不纯而受到污染，溶液多次转移造成 DNA 损失，并且在提取 过程中加入过多的溶质离子也可能对