使用碱裂解法快速提取药材DNA方法的研究

目的: 建立药材DNA快速提取方法。 方法: 使用碱裂解缓冲液提取药材DNA,考察提取液配方组成和中和剂种类对药材DNA纯度、浓度的影响。利用优化提取液提取了144个中药材DNA,动物药使用COⅠ、植物药使用psbA-trnH通用引物进行PCR扩增。 结果: 0.5 mol·L^-1氢氧化钠、1% PVP 和1% Triton×100具有最佳的DNA提取效果。对144个中药材进行PCR扩增,利用琼脂糖凝胶电泳可检测到123个中药材DNA的PCR产物。碱裂解法可用于快速提取蛇类药材DNA,并成功用于乌梢蛇、金钱白花蛇的分子鉴定。 结论: 优化的碱裂解法可用于提取中药材基因组DNA,其提取时间可控制在10 min左右,且避免了酚、三氯甲烷等对人体有毒试剂的使用,对药材快速分子鉴定体系的建立将发挥重要作用。

Objective: To develop a rapid method to extract DNA from Chinese medicinal materials. Methods: The alkaline lysis buffer was used for rapid extraction of DNA from traditional Chinese medicines(TCM) and the effects of the extracting solution's formula composition and types of neutralizing reagents on the purity and concentration of Chinese medicines DNA were investigated.DNA of 144 Chinese medicinal materials were extracted using the optimized DNA extraction buffer,and psbA-trnH primers were used to amplify PCR products for plant materials and COⅠ primers for animal materials. Results: The results showed that the buffer of 0.5 mol·L^-1 sodium hydroxide,1% PVP and 1% Triton×100 had the optimum effect on DNA extraction of TCM.After PCR amplification for 144 Chinese medicinal materials,the PCR products from 123 Chinese medicinal materials DNAs were obtained by agarose gel electrophoresis.The alkaline lysis method could be used for rapid extraction of snake medicine DNA,and was successfully used in molecular identification of medicinal snakes Zaocys and Bungarus Parvus. Conclusion: The optimized alkaline lysis method is suitable to extract DNA from Chinese medicinal materials.The whole process of this method could be finished in 10 minutes and the new method can also avoid the use of toxic reagents such as chloroform or phenol,which will play an important role in establishing the rapid identification system of medicine material molecules.