Molecular Human Reproduction, Vol. 7, No. 3, 301-306,
March 2001
© 2001 European Society of Human Reproduction and Embryology
Reproductive genetics |
Quantification of the common deletion in human testicular mitochondrial DNA by competitive PCR assay using a chimaeric competitor
1 Fertility West, Suite 47, Mount Medical Centre, 146 Mounts Bay Road, Perth, Western Australia, 2 Department of Biochemistry, The University of Western Australia, Nedlands, Western Australia 6009, 3 Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Western Australia 6150, and 4 Genetics Division, Princess Margaret Hospital, Thomas Street, Subiaco, Western Australia 6008, Australia
Abstract
The `common' 4977 bp deletion in mitochondrial DNA (
4977) 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 4977 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.
common deletion/competitive PCR/mitochondrial DNA/polymerase chain reaction
Notes
5 To whom correspondence should be addressed. E mail: cummins{at}central.murdoch.edu.au
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