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The dangers of extended PCR in the clinic

Nature Medicine volume 5, pages 965–966 (1999)Cite this article

To the editor—Rearranged mitochondrial (mt) DNA molecules are associated with an increasing spectrum of human diseases1. To be pathologically important, such molecules must constitute a major fraction of total mtDNA, at least within affected tissues. Although this is easily demonstrated by Southern blot analysis, it is a relatively slow and cumbersome technique, requiring substantial amounts of intact, highly purified and high-molecular-weight DNA. PCR-based methods to detect mtDNA deletions in the clinical context seem preferable, as they are quick, inexpensive, require only very small amounts of biopsy material, are tolerant of poor sample quality and can be automated. However, PCR-based methods are notoriously prone to artefacts, and are at best semi-quantitative.

One particular technique, long-extension PCR (LX-PCR), is commonly used to detect the full range of deleted mitochondrial DNA species associated with muscle disease, such as dilated cardiomyopathy2, aging or other conditions. In contrast to conventional PCR, in which most deleted mtDNA molecules of clinical relevance cannot be detected by any given primer pair, LX-PCR uses two outwardly oriented, adjacent primers whose sequences are located within a region that is rarely, if ever, deleted. This approach leads to the amplification of full-length mtDNA (16.6 kb) plus any subgenomic, deleted molecules that may be present.

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Figure 1: Detection limit for deleted mtDNAs by LX-PCR.

References

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Acknowledgements

We thank M. Niittylahti and O.Lumme for technical assistance, and P. Rustin, I. Holt, S. Khogali and N.-G. Larsson for discussions. This work was supported by grants from the Finnish Academy, Muscular Dystrophy Group, Royal Society, Tampere University Hospital Medical Research Fund, Yrjö Jahnsson Foundation, Finnish Foundation of Alcohol Research and the Pirkanmaa Region Fund of the Finnish Cultural Foundation.

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Authors and Affiliations

  1. Institute of Medical Technology & Tampere University Hospital, University of Tampere, Finland

    Olli A. Kajander, Johannes N. Spelbrink, Anja Rovio & Howard T. Jacobs

  2. Department of Forensic Medicine & Tampere University Hospital, University of Tampere, Finland

    Olli A. Kajander & Pekka J. Karhunen

  3. Department of Paediatrics, University of Oxford, England

    Joanna Poulton

  4. Institute of Biomedical and Life Sciences, University of Glasgow, Scotland

    Howard T. Jacobs

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  1. Olli A. Kajander
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  2. Joanna Poulton
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  4. Anja Rovio
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  6. Howard T. Jacobs
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Correspondence to Howard T. Jacobs.

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Kajander, O., Poulton, J., Spelbrink, J. et al. The dangers of extended PCR in the clinic. Nat Med 5, 965–966 (1999). https://doi.org/10.1038/12379

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