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Nucleic Acids Research Advance Access first published online on February 5, 2007
This version published online on March 13, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm010
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

DNA supercoiling suppresses real-time PCR: a new approach to the quantification of mitochondrial DNA damage and repair

Jinsong Chen1, Fred F. Kadlubar2 and Junjian Z. Chen1,*

1Department of Surgery, Division of Urology, McGill University Health Centre and Research Institute, Montreal, Quebec H3G 1A4, Canada and 2Department of Epidemiology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

*To whom correspondence should be addressed. Tel: +1 514 934 1934 x 44601(o); Fax: +1 514 934 8261; Email: junjian.chen{at}mcgill.ca

Received September 15, 2006. Revised November 26, 2006. Accepted December 27, 2006.

As a gold standard for quantification of starting amounts of nucleic acids, real-time PCR is increasingly used in quantitative analysis of mtDNA copy number in medical research. Using supercoiled plasmid DNA and mtDNA modified both in vitro and in cancer cells, we demonstrated that conformational changes in supercoiled DNA have profound influence on real-time PCR quantification. We showed that real-time PCR signal is a positive function of the relaxed forms (open circular and/or linear) rather than the supercoiled form of DNA, and that the conformation transitions mediated by DNA strand breaks are the main basis for sensitive detection of the relaxed DNA. This new finding was then used for sensitive detection of structure-mediated mtDNA damage and repair in stressed cancer cells, and for accurate quantification of total mtDNA copy number when all supercoiled DNA is converted into the relaxed forms using a prior heat-denaturation step. The new approach revealed a dynamic mtDNA response to oxidative stress in prostate cancer cells, which involves not only early structural damage and repair but also sustained copy number reduction induced by hydrogen peroxide. Finally, the supercoiling effect should raise caution in any DNA quantification using real-time PCR.


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