提取线粒体DNA方法的比较及较纯线粒体基因获取方法的确立

Analysis of Extracting mtDNA Methods and Establishment of a Pure Mitochondrial Gene Acquisition Method

目的:对2种常用的线粒体DNA提取方法进行比较,分析提取物中核DNA的存在情况,同时建立新检测方法降低线粒体假基因干扰。方法:以仅在核DNA中存在的基因β-actin作为核DNA存在的标定基因,通过PCR方法扩增线粒体DNA上的一段基因MTND5-2,并以β-actin做参比,比较常用的2种提取线粒体DNA方法的优劣,即碱法Ⅰ(先提取完整线粒体后从中获得线粒体DNA)和碱法Ⅱ(根据线粒体DNA与核DNA的结构差异,从中获得双链环状的线粒体DNA)。结果:2种线粒体DNA提取方法并不能获得仅含线粒体DNA的纯提取物,碱法Ⅰ获得的线粒体DNA纯度相对较高;以碱法Ⅰ提取物为模板进行PCR,可获得更多较纯的线粒体目的基因。结论:碱法Ⅰ较碱法Ⅱ可获得更纯的线粒体来源的目的基因;新建方法可获得较纯的线粒体基因,且是一种简单、方便、经济的方法。

Objective： To compare two usual methods of extracting mitochondrialDNA（mtDNA） and analyze nu-clearDNA（nDNA） content.Also,to develope a new method to reduce the effect of nuclear mitochondrial pseudo-genes.Methods： β-actin was used as a marker gene,which only existed in nDNA,to roughly quantify nDNA con-tent.Two usual methods of extracting mtDNA（alkaline lysis methodⅠ and alkaline lysis method Ⅱ） were com-pared through PCR to amplify MTND5-2,a gene in mtDNA,and marker gene β-actin.Results： The two methodsof extracting mtDNA cannot obtain pure mtDNA,and the extracts of alkaline lysis method Ⅰ contained lessernDNA.Moreover,we established a new method to obtain pure mitochondrial gene.Conclusion： Alkaline lysismethod Ⅰ can obtain purer mitochondrial gene than alkaline lysis method Ⅱ.The new method is a simple,rapidand inexpensive testing,through which pure mitochondrial gene can been acquired.