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Quantitative PCR for detection of DNA damage in mitochondrial DNA of the fission yeast Schizosaccharomyces pombe

2016; Elsevier BV; Volume: 127; Linguagem: Inglês

10.1016/j.mimet.2016.05.023

1872-8359

Takanori Senoo, Mayumi Yamanaka, Atori Nakamura, Tomoki Terashita, Shinji Kawano, Shogo Ikeda,

Fungal and yeast genetics research

Quantitative polymerase chain reaction (QPCR) has been employed to detect DNA damage and repair in mitochondrial DNA (mtDNA) of human and several model organisms. The assay also permits the quantitation of relative mtDNA copy number in cells. Here, we developed the QPCR assay primers and reaction conditions for the fission yeast Schizosaccharomyces pombe, an important model of eukaryote biology, not previously described. Under these conditions, long targets (approximately 10 kb) in mtDNA were quantitatively amplified using 0.1 ng of crude DNA templates without isolation of mitochondria and mtDNA. Quantitative detection of oxidative DNA damage in mtDNA was illustrated by using a DNA template irradiated with UVA in the presence of riboflavin. The damage to mtDNA in S. pombe cells treated with hydrogen peroxide and paraquat was also quantitatively measured. Finally, we found that mtDNA copy number in S. pombe cells increased after transition into a stationary phase and that the damage to mtDNA due to endogenous cellular processes accumulated during chronological aging.