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目的将提取线粒体 DNA的碱变性法、Triton法、改进高盐沉淀法加以比较 ,以得到最方便快速提取线粒体 DNA的方法。方法分离 Wistar大鼠小肠上皮细胞 ,用 3种方法提取线粒体 DNA,紫外分光光度法定量。用琼脂糖凝胶电泳和线粒体 ATPase 8亚基基因 PCR扩增产物鉴定所提取的线粒体 DNA。结果改进高盐沉淀法线粒体 DNA量最多 ,Triton法最少。 OD2 6 0 / OD2 80均在 1.78～ 1.85间。将改进高盐沉淀法提取线粒体 DNA用于PCR扩增 ,测定出了线粒体 DNA ATPase 8亚基基因序列。结论改进高盐沉淀法提取线粒体 DNA具有操作简单 ,产量多的优点 ,该法所提取 mt DNA可用于 mt DNA测序
OBJECTIVE To compare mitochondrial DNA alkaline denaturation method, Triton method and modified high-salt precipitation method to obtain the most convenient and rapid method for extracting mitochondrial DNA. Methods Wistar rat intestinal epithelial cells were isolated and the mitochondrial DNA was extracted by three methods and quantified by UV spectrophotometry. The extracted mitochondrial DNA was identified by agarose gel electrophoresis and PCR amplification of the mitochondrial ATPase 8 gene. Results The highest amount of mitochondrial DNA was found in high salt precipitation and the least was Triton method. OD2 60 / OD2 80 are between 1.78 ~ 1.85. Mitochondrial DNA was extracted by high salt precipitation method for PCR amplification. The mitochondrial DNA ATPase 8 subunit gene sequence was determined. Conclusion The improvement of high-salt precipitation extraction of mitochondrial DNA has the advantages of simple operation, the yield of more, the method extracted mt DNA can be used for mt DNA sequencing