悬铃叶苎麻基因组DNA的六种提取方法比较

Comparison of Six Methods for the Extraction of Genomic DNA from Boehmeria tricuspis( Hance) Makino

悬铃叶苎麻(Boehmeria tricuspis(Hance)Makino)是一种多年生草本植物,含有较多的多酚、多糖等次生代谢物质,这些次生代谢物质能够与基因组DNA结合从而影响其DNA的提取。本文以悬铃叶苎麻幼嫩叶片为试材,采用6种方法提取基因组DNA,并比较其提取效果,以期寻找到正确的、快速的、能够得到高质量的基因组DNA的方法。琼脂糖凝胶电泳、紫外分光光度仪、SSR-PCR扩增和限制性内切酶酶切等方法检测不同方法提取的全基因组DNA的质量和纯度。结果显示:采用方法 1(Tian Gen试剂盒)提取的溶液中含有杂质,DNA含量较少;方法 2(常规CTAB法)提取的DNA含有较多杂质;方法 3(硅胶-CTAB法)和方法 4(酚抽提-CTAB法)没有提取到DNA;方法 5(高盐预先去杂-CTAB法)得到的基因组DNA溶液含有较多的蛋白质或盐;方法 6(葡萄糖预先去杂-CTAB法)是唯一能获得高质量的基因组DNA,能够满足PCR、限制性内切酶酶切等分子实验对DNA的要求。本研究找到了一种通用、简单易行,成本低且安全的去除植物DNA提取过程中多糖等杂质的方法,为悬铃叶苎麻后续的其他分子生物学的研究奠定了基础。

Boehmeria tricuspis （Hance） Makino is a perennial herbaceous plant which contains ex- ceptionally high amounts of po]yphenols, po]ysaccharides, and other secondary metabelltes that interfere with DNA iso]ation. Six methods were compared for the extraction of genomic DNA from young leaves of Boehmeria tricuspis, and we expected to find a correct method which can make us quickly obtained high quality genomie DNA. The total DNAs extracted by different methods were detected by agarose gel elec- trophoresis, the ultraviolet absorbency, the amplification of DNAs with SSR primer and restriction endo- nuclease reaction. The results indicate the solution of genomic DNA extracted by method 1 （Tian - C, en kit） contains impurities and little DNA, method 2 （ simp]e CTAB） produces a lot of impurities, method 3 （silica gel - CTAB） and method 4 （phenol extracting - CTAB） can＇ t extract genomic DNAs, and ]ots of proteins or salts in the solution of genomic DNA is obtained by method 5 （ high - salt predeimpurity - CTAB）, while the method 6 （glucose predeimpurity -CTAB ） is the best way to obtain high quality ge- nomic DNA and can meet the requirement for PCR, restriction endonuclease reaction and other DNA mo-lecular experiments. This study found a generally simple,low cost and safe way to remove impurities such as polysaccharides in plant DNA extracted process, which laid the foundation of follow -up study of ram- ie in other molecular biology.