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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(GuSCN)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,GuSCN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;GuSCN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満足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 establish a suitable extraction method for genomic DNA of Rana oil medicine and lay a foundation for the subsequent molecular identification of this medicinal material. Methods: The genomic DNA of Toona sinensis was extracted by sodium dodecyl sulfate (SDS) method, modified SDS method, Chelex-100 method, GuSCN method, kit method and modified kit method. Gel electrophoresis, UV spectrophotometry and cytochrome C oxidase I (COI) sequence were used to detect the yield and quality of DNA. Results: Chelex-100 method, modified SDS method, GuSCN method and modified kit method were all able to extract genomic DNA from Oviducta Rugosae crude oil, which could not be extracted by SDS method and kit method. The quality of the DNA obtained by the modified SDS method is good, the yield is high, but the operation is cumbersome. The quality of the DNA obtained by the improved kit method is better, but the yield is low and the cost is high. The Chelex-100 method is simple and rapid in operation with high yield, The quality of the obtained DNA is poor; the DNA extracted by GuSCN method contains more impurities and inhibits the PCR reaction. Conclusion: Chelex-100 method, modified SDS method and modified kit method all can get the DNA that is enough for PCR amplification. In practice, the appropriate method can be used to extract the genomic DNA of Tana Ranae.