Chan Sam W, Chen Junjian Z
Department of Surgery, Division of Urology, McGill University Health Centre and Research Institute, Montreal, QC, Canada.
Methods Mol Biol. 2009;554:183-97. doi: 10.1007/978-1-59745-521-3_12.
Compromised mitochondrial DNA structural integrity can have functional consequences for mitochondrial gene expression and replication leading to metabolic and degenerative diseases, aging, and cancer. Gel electrophoresis coupled with Southern blot and probe hybridization and long PCR are established methods for detecting mtDNA damage. But each has its respective shortcomings: gel electrophoresis is at best semi-quantitative and long PCR does not offer information on the structure. To overcome these limitations, we developed a new method with real-time PCR to accurately quantify the mtDNA structural damage/repair and copy number change. We previously showed that the different mtDNA structures (supercoiled, relaxed circular, and linear) have profound influences on the outcome of the real-time PCR amplification. The supercoiled structure is inhibitory to the PCR amplification, while relaxed structures are readily amplified. We will illustrate the use of this new method by quantifying the kinetics of mtDNA damage and repair in LNCaP prostate cancer cells induced by exogenous H2O2 treatments. The use of this new method on clinical samples for spontaneous mtDNA damage level will also be highlighted.
线粒体DNA结构完整性受损会对线粒体基因表达和复制产生功能性影响，进而导致代谢性疾病、退行性疾病、衰老和癌症。凝胶电泳结合Southern印迹和探针杂交以及长链PCR是检测线粒体DNA损伤的常用方法。但每种方法都有其各自的缺点：凝胶电泳充其量只是半定量的，而长链PCR无法提供有关结构的信息。为了克服这些局限性，我们开发了一种利用实时PCR的新方法，以准确量化线粒体DNA的结构损伤/修复以及拷贝数变化。我们之前表明，不同的线粒体DNA结构（超螺旋、松弛环状和线性）对实时PCR扩增结果有深远影响。超螺旋结构会抑制PCR扩增，而松弛结构则易于扩增。我们将通过量化外源性过氧化氢处理诱导的LNCaP前列腺癌细胞中线粒体DNA损伤和修复的动力学来阐述这种新方法的应用。还将强调这种新方法在临床样本中用于检测自发线粒体DNA损伤水平的应用。
