线粒体在themitochondriaperformfourcentral-第三军医大学学报.docVIP

三种线粒体DNA提取方法的比较 杨玉有1，扎拉嘎白乙拉1，云利兵2， 叶懿3，侯一平1 ( 610041 成都，四川大学，华西基础医学与法医学院：法医遗传学教研室1，法医病理学教研室2，法医毒物分析教研室3） [摘要] 目的 将高盐沉淀法、碱变性法和Triton法三种线粒体DNA(mitochondrial DNA, mtDNA)提取方法加以比较，以得到产量高、核DNA(nuclear DNA, nDNA)污染少的mtDNA提取方法。方法 分离外周血白细胞，用高盐沉淀法、碱变性法和Triton法三种方法提取mtDNA，每种方法提取10例样本。紫外分光光度法检测提取产物中DNA含量，琼脂糖凝胶电泳检测mtDNA纯度。用线粒体ND1基因PCR扩增产物鉴定mtDNA，用12号染色体上GAPDH基因PCR扩增产物鉴定nDNA。结果 高盐沉淀法产物以nDNA为主，碱变性法提取mtDNA产量高且nDNA污染少，Triton法提取DNA产量极低。结论 碱变性法提取mtDNA产量高、nDNA污染少、操作简单、重复性好，优于另外两种方法，适宜于mtDNA的提取。 [关键词] DNA ；线粒体 ；抽提纯化 [中图法分类号] Q523;Q731;R-331 Comparison of three methods for isolation of mitochondrial DNA Yang Yuyou1, Zha Lagabaiyila1,Yun Libing2, Ye Yi3, Hou Yiping1 (1Department of Forensic Genetics, 2Department of Forensic Pathology, 3Department of Forensic Analytical Toxicology，West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, Sichuan 610041, China ) [Abstract] Objective To find out an isolation method which yields more mitochondrial DNA (mtDNA) and less nuclear DNA(nDNA), an evaluation of three mtDNA isolation methods was carried out. Methods White blood cells (WBC) were prepared from peripheral blood. MtDNA were extracted by using three mtDNA isolation methods, namely the salting out, the alkaline lysis with sodium dodecyl sulfonate (SDS) and the lysis with Triton solution methods, 10 WBC samples for each method. Extracted DNA were detected by agarose gel electrophoresis and quantified with spectrophotometer. The presence of mtDNA and nDNA in the extracted DNA samples was examined using polymerase chain reaction (PCR) amplification of a fragment of the mtDNA NADH dehydrogenase subunit 1 (ND1) gene or the glyceraldehyde-3-phosphate dehydrogenase(GAPDH) gene located in chromosome 12. Results The extracts of the salting out method were mostly nDNA rather than mtDNA. The triton based method showed poor yield and reproducibility. In contrast, the alkaline lysis with SDS method yielded more mtDNA and less nDNA. Conclusion The alkaline lysis with SDS meth