烟草碱法提取基因组DNA的改进  

Improvement of Alkaline Lysis Method for DNA Extraction from Tobacco

作　　者：徐宗昌[1,2] 王萌[1,2] 孔英珍[1] 

机构地区：[1]中国农业科学院烟草研究所中国农业科学院烟草遗传改良与生物技术重点开放实验室,青岛266101 [2]中国农业科学院研究生院,北京100081

出　　处：《生物技术通报》2017年第2期59-65,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31470291;31670302);国家科技支撑计划项目(2015BAD15B03-05);中国农业科学院青年英才计划

摘　　要：为能够快速高效的对大量转基因烟草样品进行DNA提取,对碱法提取烟草组织DNA的方法进行了改进。采用0.01mol/L的Na OH溶液为提取液,破碎转基因烟草叶片组织后无需经过加热煮沸中和等步骤,上清液可直接用作PCR反应的模板。结果表明,0.01-0.1 mol/L之间的Na OH溶液制备的DNA模板均能成功扩增,该方法制备的DNA模板不依赖特殊的反应条件,多种品牌的PCR Mix均能成功进行扩增反应。与传统SDS提取法和试剂盒提取法获得的DNA相比,PCR结果没有明显差异。此提取方法在小麦、高粱、水稻、玉米等作物上也取得了很好的实验效果。通过本方法制备的DNA模板,其PCR扩增的产物可直接用来测序分析。该方法使用仪器少,样品消耗少,快速、简便、成本低,没有交叉污染,能够在短时间内处理大量样品,因此能够对大量的转基因烟草样品进行DNA的快速提取和鉴定。To expeditiously extract DNA templates from a large number of transgenic tobacco samples, the alkaline lysis method of extracting DNA from tobacco was improved in this study. Solution of 0.01 mol/L NaOH was used as the extraction buffer and the supernatant could be directly used as a template for PCR reaction, after crushing transgenic tobacco leaf tissues without heating/boiling and neutralizing procedures. The results showed that DNA template produced by NaOH solution of 0.01-0.1 mol/L was amplified successfully. The DNA template produced by the improved method did not rely on particular reaction condition, a variety of types of PCR Mix buffer all amplified successfully. Compared with the traditional SDS extraction method or commercial kits method, PCR results showed no significant differences. In addition, the !reproved method presented promising results while it was applied to other crops such as wheat, sorghum, rice and maize. The PCR products amplified from the DNA template by the improved method was able to be directly used for sequencing analysis. In conclusion, the improved method is in need of fewer instruments and less sample consumption, rapid, simple, low cost, no cross-contamination, and very efficient to handle a large number of samples, thus, it may rapidly extract and identify the DNA from a large number of transgenic tobacco samples.

关 键 词：烟草 DNA提取 碱裂解 PCR 转基因检测 

分 类 号：S572[农业科学—烟草工业] Q943.2[农业科学—作物学]