Detection of oxidative stress-induced mitochondrial DNA damage using fluorescence correlation spectroscopy.
Using fluorescence correlation spectroscopy (FCS), we tested the feasibility of rapid detection of oxidative damage of mitochondrial DNA (mtDNA) in a small volume. The complete mtDNA genome was amplified by long polymerase chain reaction (LPCR), and the product was fluorescently labeled with an intercalating dye, YOYO-1. The fluorescence autocorrelation function was analyzed using a simple two-component model with the diffusion time of 0.21 ms for the LPCR primer and 18 ms for the mtDNA LPCR product. When human embryonic kidney 293 (HEK-293) cells were exposed to 0.4 mM H2O2, the fraction of the mtDNA LPCR product decreased significantly. In contrast, the fraction of the nuclear-encoded beta-globin LPCR product remained unchanged. The analysis time of FCS measurement was very short (5 min) compared with that of gel electrophoresis (3 h). Thus, FCS allowed the rapid detection of the vulnerability of mtDNA to oxidative stress within a small volume element at the subfemtoliter level in solution. These results suggest that the LPCR-FCS method can be used for epidemiological studies of diseases caused by mtDNA damage.
Nomura
Yasutomo
Y
Department of Environmental Life Science, Graduate School of Medical Science, Yamagata University, Yonezawa, Yamagata 992-8510, Japan. ynomura@yz.yamagata-u.ac.jp
Fuchigami
Hirobumi
H
Kii
Hiroaki
H
Feng
Zhonggang
Z
Nakamura
Takao
T
Kinjo
Masataka
M
D001583:Benzoxazoles; D002478:Cells, Cultured; D004249:DNA Damage / Q000187:drug effects; D004272:DNA, Mitochondrial / Q000737:chemistry*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018384:Oxidative Stress; D016133:Polymerase Chain Reaction / Q000379:methods; D011806:Quinolinium Compounds; D013050:Spectrometry, Fluorescence / Q000379:methods*
D001583:Benzoxazoles; D004272:DNA, Mitochondrial; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D006861:Hydrogen Peroxide