适用于线粒体基因组测序的高纯度线粒体DNA提取和鉴定方法优化  

Optimization of extraction and identification methods of high-purity mitochondrial DNA suitable for mitochondrial genome sequencing

作　　者：李君 朱嘉晋 邓雨萍 吴文鹤[2] LI Jun;ZHU Jiajin;DENG Yuping;WU Wenhe(Medical Laboratory Center,Wenzhou People's Hospital in Zhejiang Provicne,Wenzhou325000,China;School of Laboratory Medicine(School of Life Sciences),Wenzhou Medical University,Wenzhou325035,China)

机构地区：[1]浙江省温州市人民医院医学检验中心,浙江温州325000 [2]温州医科大学检验医学院(生命科学学院),浙江温州325035

出　　处：《中国现代医生》2022年第4期40-43,共4页China Modern Doctor

基　　金：国家自然科学基金项目(81670712);浙江省温州市公益性科技计划项目(Y20170249)。

摘　　要：目的优化线粒体DNA(mtDNA)提取和纯度鉴定方法以满足线粒体基因组测序的要求。方法利用简化的蔗糖密度梯度离心法、DNA酶Ⅰ并结合质粒提取试剂盒提取纯化mtDNA、尽量降低核DNA(nDNA)污染的可能。获得的mtDNA经NanoDrop检测、琼脂糖凝胶电泳和荧光定量PCR绝对定量法测定nDNA/mtDNA拷贝数比值等鉴定其纯度。结果从小鼠肝脏和大脑组织中提取纯化得到mtDNA产率分别为311.7μg/g和59.1μg/g,其A_(260/280)均介于1.8~2.0,A_(260/230)浓度均大于2.0,荧光定量PCR绝对定量法测定nDNA/mtDNA拷贝数比值均低于0.005%。结论本研究优化了mtDNA提取和鉴定方法,得到的高纯度mtDNA适用于后续线粒体基因组测序等分子生物学研究。Objective To optimize mitochondrial DNA(mtDNA)extraction and purity identification methods to meet the requirements of mitochondrial genome sequencing.Methods Simplified sucrose density gradient centrifugation,DNase I,and plasmid extraction kit were used to extract and purify mtDNA and minimize the possibility of nuclear DNA(nDNA)contamination.The purity of the obtained mtDNA was determined by NanoDrop detection,agarose gel electrophoresis,and determination of nDNA/mtDNA copy number ratio by fluorescence quantitative PCR absolute quantitative method.Results The yields of mtDNA extracted and purified from mouse liver and brain tissues were 311.7μg/g and 59.1μg/g,respectively.The A_(260/280) ranged from 1.8 to 2.0,and the A_(260/230) concentration was greater than that of 2.0.The ratio of nDNA/mtDNA copy number determined by fluorescence quantitative PCR absolute quantitative method was lower than 0.005%.Conclusion This study optimizes the mtDNA extraction and identification methods,and the high purity mtDNA obtained is suitable for subsequent mitochondrial genome sequencing and other molecular biology research.

关 键 词：线粒体DNA 提取 纯化 核DNA 

分 类 号：R394.3[医药卫生—医学遗传学]