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单条线虫DNA提取方法优化-给专家电子版-审回稿修改

单条线虫DNA提取方法

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 宁波 315012 2 嘉兴出入境检验检疫局 嘉兴 314000)

Method of extract DNA from a single nematode. Wang Jiangling1, Zhang Jiancheng2, Gu Jianfeng1*(1Ningbo Entry-exit Inspection and Quarantine Bureau, Ningbo 315012; 2Jiaxing Entry-exit Inspection and Quarantine Bureau, Jiaxing 314000)

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 suddenly. 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 system 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 disruption 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 effective 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 molecular experiments chances. Key words nematode, optimized method, DNA, proteinase K, temperature

摘 要: 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PCR扩增有干扰,从而影响下一步的研究。本研究用液氮快速冷冻单条线虫,继以85℃快速变温,用温度的剧烈变化使线虫裂解,再以蛋白酶K降解蛋白质,提高了获得的DNA的纯度和数量。通过多种线虫的验证试验表明,该方法高效、快速、稳定。该试验方法有如下优点:(1)使用Taq酶附带的PCR Buffer代替WLB裂解液和SDS裂解液,减少了配置的不确定性和外来离子的影响。(2)整个提取过程在一个PCR管中完成,并可直接用于PCR扩增,没有溶液的转移和过多的溶质添加,减少污染和对后续PCR的影响。(3)使用变温处理代替其他手工破碎方法,减少了外界污染物的掺入,不需要手工操作技巧。与另外两种提取线虫DNA的蛋白酶K方法相比,本研究提取单条线虫DNA的时间短,效率高,PCR扩增结收稿日期 :

基金项目 :国家质量监督检验检疫总局科技项目(2010IK269);宁波市农业科研攻关合作项目 (2008C10014);宁波市自然科学基金项目(2010A610018)

作者简介 :王江岭(1982—),男,河南开封人,助理农艺师,硕士,主要从事检疫性植物病原线虫的检测鉴定. E-mail : jiangling0916@http://www.wenkuxiazai.com 通讯作者 :顾建锋(1972-),男,浙江余姚人,高级农艺师,大学本科,从事检疫性植物病原线虫及真菌的检测鉴定. Tel:0574-87022839;E-mail : gujf@http://www.wenkuxiazai.com.

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