岩陀DNA提取方法研究
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摘要
以岩陀植物嫩叶为材料,用不同方法对岩陀叶片DNA进行提取,比较提取的质量差异,寻找适合岩陀叶片总DNA的提取方法,以满足多基原民族药岩陀DNA条形码识别系统构建的研究需要。采用十六烷基三甲基溴化铵(CTAB)法、改良的CTAB法、十二烷基硫酸钠(SDS)法及碱裂解法提取岩陀叶片总DNA,利用琼脂糖凝胶电泳、紫外分光光度法及PCR扩增效果检查DNA提取的质量。结果显示四种方法均能从岩陀植物叶片中提取到基因组DNA,改良CTAB法提取的DNA纯度和完整性明显好于其他方法,且采用此法进行PCR扩增获得的目标条带明亮单一,扩增效率达到100%。改良的CTAB法是岩陀叶片总DNA的提取中最为合适的提取方法,能够满足后续DNA条形码的研究需要。
Fresh leaves of Rodgersia pinnata were used as experimental materials, the differences of the DNA extraction effects among different methods were compared to find proper DNA extraction methods for meeting the study demands on constructing DNA barcode recognition system of R. pinnata with different origins. CTAB method, improved CTAB method, SDS method and alkaline lysis method were used to extract total DNA, the quality of DNA extraction were evaluated by agarose gel electrophoresis, UV spectrophotometry and PCR amplification effect. The results showed that all four methods could extract genomic DNA from the leaves of R.pinnata, DNA purity and integrity in improved CTAB method is significantly higher than that in other methods,in which the PCR amplification efficiency reaches 100%, and the amplified bands are bright and clear. The improved CTAB method can be used as a suitable method for extracting total DNA from the leaves of R. pinnata and could meet the study demand of DNA barcode.
Fresh leaves of Rodgersia pinnata were used as experimental materials, the differences of the DNA extraction effects among different methods were compared to find proper DNA extraction methods for meeting the study demands on constructing DNA barcode recognition system of R. pinnata with different origins. CTAB method, improved CTAB method, SDS method and alkaline lysis method were used to extract total DNA, the quality of DNA extraction were evaluated by agarose gel electrophoresis, UV spectrophotometry and PCR amplification effect. The results showed that all four methods could extract genomic DNA from the leaves of R.pinnata, DNA purity and integrity in improved CTAB method is significantly higher than that in other methods,in which the PCR amplification efficiency reaches 100%, and the amplified bands are bright and clear. The improved CTAB method can be used as a suitable method for extracting total DNA from the leaves of R. pinnata and could meet the study demand of DNA barcode.
引文
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[3]潘玉杰,蒋坤,夏文,等.岩陀中岩白菜素含量测定[J].医药导报,2016,35(10):1130-1133.
[4]黄梦玺,彭朝霞,杨朝莲,等.贵州地产岩陀的质量研究[J].医药与保健,2015,23(2):166-166.
[5]黄璐琦.分子生药学[M].2版,北京:北大医出版社,2006:38-40.
[6]何雪娇,郑涛,苏金强,等.改良CTAB法提取野牡丹科7种植物DNA[J].广东农业科学,2011,31(18):120-122.
[7]罗焜,马培,姚辉,等.中药DNA条形码鉴定中的DNA提取方法研究[J].世界科学技术-中医药现代化,2012,14(2):1433-1439.
[8]王茜,侯飞侠,王艺璇,等.碱裂解法快速提取虫草属真菌DNA研究[J].时珍国医国药,2016,27(4):995-997.
[9]陈士林.中药DNA条形码分子鉴定[M].北京:人民卫生出版社,2012.
[10]Fang G,Hammar S,et al.A quick and inexpensive method for removing polysaccharides from plant genomic DNA[J].BioTechniques,1992,13(1):52-56.
[11]肖智样,白冠章,张玲,等.改进的CTAB法提取胡杨(Populus euphratica)总DNA[J].塔里木大学学报,2009,21(2):29-32.
[12]王平康,骆延,王光志,等.磁珠法快速提取鉴定DNA的实验研究[J].生物学通报,2006,41(6):50-51.
[13]童巧珍,周日宝,刘湘,等.药用百合鳞叶中总DNA提取方法的研究[J].中国药房,2008,19(6):406-408.