瘤胃微生物总DNA提取方法的比较

目前如何获得完整的瘤胃微生物总DNA是对瘤胃微生物在分子水平研究中关键的一步。瘤胃微生物总DNA分子片段很大，大概在15kb~20kb，因此需要选择一种合适的提取方法来获得完整的总DNA片段。笔者研究采用物理方法如玻璃珠研磨震荡法、反复冻融法、超声波破碎法、液氮研磨法等，化学方法如表面活性剂十二烷基磺酸钠（sodium dodecyl sulfate，SDS）、溴化十六烷三甲基铵（Cetyltrimethyl Ammonium Bromide，CTAB等），酶解法（溶菌酶、蛋白酶K等）相结合，并对其进行比较，选择最佳的提取方法，使其符合以后PCR扩增要求以及其它后续研究工作。试验结果表明用玻璃珠研磨震荡加CTAB提取液的方法、反复冻融加SDS提取液的方法提取的瘤胃微生物总DNA的效果高于其它方法，并且通过电泳以及PCR检测，所提取的DNA的量适于进一步的分子生物学研究。

Comparison Methods of DNA Extraction for Rumen Microbiology

How to get the total DNA of rumen microbes was the key factor on molecular research. The molecular size of the DNA of rumen microbes was very great, maximum size is 15 to 20kb. Therefore, an optimized method to get total DNA should be chosen for the future research. In this paper, some physical disruption methods were chosen, such as glass bead mill homogenization method, freeze-thaw treatment, ultrasonic lysis treatment, liquid N2 mill treatment etc, and some chemical method, such as sodium dodecyl sulfate solution, Cetyltrimethyl Ammonium Bromide solution and lysozyme pretreatment (lysozyme, proteinase K). And they were compared to choose the most optimized one to fit next PCR and other research. The results showed the total DNA quality of rumen microbes with the glass bead mill homogenization plus CTAB extraction solution method and the freeze-thaw treatment plus SDS extraction solution method were better than that with other methods, moreover, the sample of the total DNA can be used in the molecular experiment through electrophoresis and PCR detection.