Quantification of the common deletion in human testicular mitochondrial DNA by competitive PCR assay using a chimaeric competitor

Abstract

The "common" 4977 bp deletion in mitochondrial DNA (Delta4977) is commonly used as an indicator of tissue deterioration in ageing and bioenergetic diseases. Deletion levels are normally measured by a serial dilution polymerase chain reaction (PCR) approach, where test reactions are compared with dilutions of control amplifications of DNA from a similar sized stable region of the mitochondrial genome. The end-point of this assay is the dilution that can just detect any PCR product; however, this is an inherently unstable measure. We constructed a chimaeric DNA construct that binds to both control and deletion primers with similar annealing properties. This was used in a competitive PCR assay to quantify Delta4977 in human testicular tissues that had been well-characterized using the serial dilution approach. We found the competitive assay to be highly replicable as it compares the PCR product of the construct with that of test DNA samples during the linear growth phase of the PCR reaction. Moreover, the serial dilution assay was shown to significantly overestimate the amounts of deleted mitochondrial DNA present. The assay promises to throw new light on the role of mitochondrial DNA deletions in tissue dysfunction and ageing, as such deletions can now be determined with high accuracy and repeatability and is much cheaper to apply than real-time fluorescent quantitative PCR.

Mitochondrial DNA deletions and differential mitochondrial DNA content in Rhesus monkeys: Implications for aging

“…Our initial findings agree with other researchers’ reports of mtDNA deletions in oocytes [26] and skeletal muscle tissues [22,31,32] from rhesus monkeys. Our findings of 5.7 kb deletions in blood and brain samples from rhesus monkeys – together with these earlier studies [26,31–33] – suggest that the 5.7 kb deletion is present in different tissues in the rhesus monkey.…”

Comparison of Analytic Methods for Quantitative Real-Time Polymerase Chain Reaction Data

“…Quantification of an infectious microorganism assists the medical practitioner in determining when antimicrobial therapy should be used and the level of efficacy being achieved by an ongoing antimicrobial therapy (Mackay et al, 2007). However, common methods such as using reference genes or serial dilutions may not be sufficiently accurate for most clinical purposes due to the use of external standard reference curves, or limiting dilutions of samples (Mehmet et al, 2001). Minor differences in estimation, especially estimations of the initial DNA amount, may affect the therapeutic plan based on objective data.…”

Overview of Real-Time PCR Principles

“…This combination of allelespecific amplification with real-time PCR has been shown to reveal detection limits of down to 0.01% for SNPs (Maas, Schaap et al 2003). Real-time PCR is increasingly used in forensic analyses (Andreasson, Gyllensten et al 2002;von Wurmb-Schwark, Higuchi et al 2002;Ye, Parra et al 2002), but also to monitor disease-or age-related accumulation of deletions in the mitochondrial genome (Mehmet, Ahmed et al 2001;He, Chinnery et al 2002).…”