哈蟆油药材基因组DNA提取方法的比较  

Comparison of Genomic DNA Extraction Methods for Ranae Oviductus

作　　者：刘建辉[1] 石林春 唐先明[1] 王洋[1] 王姝 

机构地区：[1]哈尔滨市食品药品检验检测中心,哈尔滨150525 [2]中国医学科学院·北京协和医学院药用植物研究所,北京100193

出　　处：《中国实验方剂学杂志》2018年第4期27-31,共5页Chinese Journal of Experimental Traditional Medical Formulae

摘　　要：目的：建立一种适合于哈蟆油药材基因组DNA的提取方法,为该药材的后续分子鉴定奠定基础。方法：采用十二烷基硫酸钠（SDS）法,改良SDS法,Chelex-100法,异硫酸氰胍（Gu SCN）法,试剂盒法及改良试剂盒法提取哈蟆油基因组DNA,利用琼脂糖凝胶电泳、紫外分光光度法及细胞色素C氧化酶Ⅰ（COⅠ）序列的引物聚合酶链式反应（PCR）扩增对DNA产量及质量进行检测。结果：Chelex-100法,改良SDS法,Gu SCN法及改良试剂盒法均能从哈蟆油药材中提取得到基因组DNA,SDS法和试剂盒法提取不到。其中改良SDS法得到的DNA质量良好,得率高,但操作繁琐;改良试剂盒方法得到的DNA质量较好,但得率低,成本高;Chelex-100法操作简单快速、得率高,但得到的DNA质量较差;Gu SCN法提取的DNA含有较多杂质,会抑制PCR反应。结论：Chelex-100法、改良SDS法及改良试剂盒法均可得到满足PCR扩增要求的DNA,实践中可根据条件选择适宜方法来提取哈蟆油基因组DNA。Objective： To explore a extracting method of genomic DNA from Ranae Oviductus and lie a foundation for further molecular experiments of this medicinal herb.Method： Sodium dodecyl sulfate（SDS）method,improved SDS method,Chelex-100 method,guanidine thiocyanate（Gu SCN） method,kit method and improved kit method were used to extract genomic DNA from Ranae Oviductus.The yield and quality of genomic DNA were evaluated by agarose gel electrophoresis,ultraviolet spectrophotometry and polymerase chain reaction（PCR） amplification of primer with cytochrome C oxidase Ⅰ（COⅠ） sequence.Result： Chelex-100 method,improved SDS method,Gu SCN method and improved kit method could extract genomic DNA from Ranae Oviductus,while its yields of SDS method and kit method were little.The quality and yield of genomic DNA extracted by improved SDS method were high with complex operation.The improved kit method cost too much and the yield of DNA was low.The Chelex-100 method was easy to operate and cost less time,but the quality of DNA was poor.DNA extracted by Gu SCN method contained many PCR inhibitors.Conclusion： The genomic DNA extacted by Chelex-100 method,improved SDS method and improved kit method all can meet the demands of PCR amplification,suitable method can be selected to extract genomic DNA from Ranae Oviductus according to the actual conditions of the laboratory.

关 键 词：哈蟆油 基因组DNA 提取方法 改良十二烷基硫酸钠法 异硫酸氰胍 Chelex-100 聚合酶链式反应 

分 类 号：R283.6[医药卫生—中药学] Q78[医药卫生—中医学]