Detection and quantitation by competitive PCR of an age-associated increase in a 4.8-kb deletion in rat mitochondrial DNA

Abstract

Recent studies on human tissues have shown that the guantity of partially deleted mitochondrial DNA (mtDNA) increases with age. In this study, mtDNAs from the livers of young adult and old Wistar rats were analyzed by PCR. Evidence for partially deleted mtDNAs was found, with a 4834-bp deletion present in all animals and most easily detected in samples from senescent rats. The deletion breakpoint occurs at a 16-bp direct repeat present in the cytochrome oxidase I and ATPase 6 genes. This deletion in rats is similar in size and location to the 5.0-kb deletion observed in human mtDNA. The proportion of rat mtDNA with this 4.8-kb deletion was quantitated by a competitive PCR assay. The ratio of partially deleted mtDNA/ total mtDNA in liver mtDNA from individual 6 month old rats ranged from 5 × 10⁻⁶ to 3 × 10⁻⁵, while the ratio in 24 month old rats ranged from 8 × 10⁻⁴ 5 × 10⁻³, with a mean 100-fold increase with age. These increases are in the range observed for human mtDNA during aging. Thus senescent rats can be used as a model to study this type of mitochondrial DNA damage in aging. The method and reagents described should prove useful of the mechanism(s) underlying deletions, their significance to the aging process, and testing of various compounds or interventions for their ability to slow the process.