Denaturing gradient gel method for mapping single base changes in human mitochondrial DNA.
Denaturing gradient gel method for mapping single base changes in human mitochondrial DNA.
复制标题
用于绘制人类线粒体 DNA 中单碱基变化的变性梯度凝胶法。
DOI：
10.1016/0003-2697(91)90489-g
复制
发表时间：
1991
影响因子：
2.9
通讯作者：
Aprille,JR
中科院分区：
文献类型：
--
作者：
Yoon,KL;Modica-Napolitano,JS;Ernst,SG;Aprille,JR
描述了一种变性梯度凝胶电泳（DGGE）方法，其检测线粒体DNA（mtDNA）中甚至单个碱基对的变化。在该方法中，线粒体DNA的限制性片段在60°C的尿素/甲酰胺梯度凝胶中进行电泳。每个mtDNA片段在凝胶中的迁移距离取决于反映碱基组成的熔解行为。用特异性mtDNA探针通过Southern印迹法定位片段。只需四种精心选择的限制性内切酶和50-100 ng的mtDNA，该方法几乎覆盖了整个人类线粒体基因组。为了证明该方法，分析了人类mtDNA。在6名正常人中，DGGE显示了限制性片段长度多态性（RFLP）分析在琼脂糖凝胶中未检测到的mtDNA片段的熔解行为多态性（MBPs）。另一个人，表现出在细胞色素B编码区的熔解行为多态性，进行了详细的研究。通过定位，推断该突变位于nt 14905和15370之间。PCR扩增并测序。在凝胶结果预测的区域中鉴定了特定的碱基变化。这种方法将是特别有用的线粒体疾病的诊断工具，快速定位的mtDNA突变的基因组的特定区域，但DGGE也可以补充RFLP分析作为一个更敏感的方法，在人类和动物种群的母系在各种研究领域。
A denaturing gradient gel electrophoresis (DGGE) method is described that detects even single base pair changes in mitochondrial DNA (mtDNA). In this method, restriction fragments of mtDNA are electrophoresed in a urea/formamide gradient gel at 60°C. Migration distance of each mtDNA fragment in the gel depends on melting behavior which reflects base composition. Fragments are located by Southern blotting with specific mtDNA probes. With just four carefully chosen restriction enzymes and as little as 50–100 ng of mtDNA, the method covers almost the entire human mitochondrial genome. To demonstrate the method, human mtDNA was analyzed. In six normal individuals, DGGE revealed melting behavior polymorphisms (MBPs) in mtDNA fragments that were not detected by restriction fragment length polymorphism (RFLP) analysis in agarose gels. Another individual, shown to have a melting behavior polymorphism in the cytochrome b coding region, was studied in detail. By mapping, the mutation was deduced to lie between nt 14905 and 15370. The affected fragment was amplified by PCR and sequenced. Specific base changes were identified in the region predicted by the gel result. This method will be especially useful as a diagnostic tool in mitochondrial disease for rapid localization of mtDNA mutations to specific regions of the genome, but DGGE also could complement RFLP analysis as a more sensitive method to follow maternal lineage in human and animal populations in a variety of research fields.