Abstract
Mitochondrial dysfunction is associated with several diseases such as neuronal degeneration and diabetes, and has also been linked to ageing. Mitochondrial DNA (mtDNA) damages by reactive oxygen species appear to be a major contributor to the dysfunction. To determine mtDNA damages, this study used the combination of long polymerase chain reaction (PCR) and fluorescence correlation spectroscopy (FCS) which is suitable for mass examination. FCS permits the sensitive estimation of the number and the length of PCR products without the electrophoresis. The long PCR assay is based on the assumption that DNA lesions such as strand breaks and base modifications will block the progression of the polymerase. The band of 16 kbp-PCR products on the gel was detected with total DNA isolated from 293 cells. The fragment lengths of the product digested with Eco 47I were same as those predicted from human mitochondrial genome on the database. When the product was stained with YOYO-1, a large fluorescence enhancement was observed due to the intercalation. To monitor the degradation of the labeled DNA with Hae III, FCS measurements were carried out. The number of DNA molecules in volume element increased from 5 to 66, corresponding to fragmentation of the PCR product. The diffusion time derived from Brownian motion of the DNA molecules was also shortened from 19.5 ms to 10.1 ms. These data demonstrate that the mtDNA PCR product is detected by FCS and suggest the feasibility to measure mtDNA damage by oxidative stress. [Jpn J Physiol 54 Suppl:S80 (2004)]
