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首页> 美国卫生研究院文献>Nucleic Acids Research >Analysis of differential DNA damage in the mitochondrial genome employing a semi-long run real-time PCR approach
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Analysis of differential DNA damage in the mitochondrial genome employing a semi-long run real-time PCR approach

机译：使用半实时实时PCR方法分析线粒体基因组中的差异DNA损伤

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摘要

The maintenance of the mitochondrial genomic integrity is a prerequisite for proper mitochondrial function. Due to the high concentration of reactive oxygen species (ROS) generated by the oxidative phosphorylation pathway, the mitochondrial genome is highly exposed to oxidative stress leading to mitochondrial DNA injury. Accordingly, mitochondrial DNA damage was shown to be associated with ageing as well as with numerous human diseases including neurodegenerative disorders and cancer. To date, several methods have been described to detect damaged mitochondrial DNA, but those techniques are semi-quantitative and often require high amounts of genomic input DNA. We developed a rapid and quantitative method to evaluate the relative levels of damage in mitochondrial DNA by using the real time-PCR amplification of mitochondrial DNA fragments of different lengths. We investigated mitochondrial DNA damage in SH-SY5Y human neuroblastoma cells exposed to hydrogen peroxide or stressed by over-expression of the tyrosinase gene. In the past, there has been speculation about a variable vulnerability to oxidative stress along the mitochondrial genome. Our results indicate the existence of at least one mitochondrial DNA hot spot, namely the D-Loop, being more prone to ROS-derived damage.
机译：维持线粒体基因组完整性是适当的线粒体功能的前提。由于由氧化磷酸化途径产生的高浓度的活性氧（ROS），线粒体基因组高度暴露于氧化应激，导致线粒体DNA损伤。因此，线粒体DNA损伤被证明与衰老以及许多人类疾病有关，包括神经退行性疾病和癌症。迄今为止，已经描述了几种检测受损的线粒体DNA的方法，但是这些技术是半定量的并且经常需要大量的基因组输入DNA。我们开发了一种快速定量的方法，通过使用实时荧光定量PCR扩增不同长度的线粒体DNA片段来评估线粒体DNA中的相对损伤水平。我们调查了接触过氧化氢或酪氨酸酶基因过表达引起的SH-SY5Y人成神经细胞瘤细胞中线粒体DNA的损伤。过去，一直有人猜测线粒体基因组中的氧化应激易受伤害。我们的结果表明至少存在一个线粒体DNA热点，即D环，更容易受到ROS衍生的损害。

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