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Published online 29 March 2004

Nucleic Acids Research, 2004, Vol. 32, No. 6 e56
© 2004 Oxford University Press

Instant evaluation of the absolute initial number of cDNA copies from a single real-time PCR curve

Stéphane Swillens*, Jean-Christophe Goffard, Yoann Maréchal, Alban de Kerchove d’Exaerde1 and Hakim El Housni2

Institut de Recherche Interdisciplinaire, Faculté de Médecine, Université Libre de Bruxelles, CP 602, Belgium 1 Laboratoire de Neurophysiologie, Faculté de Médecine, Université Libre de Bruxelles, CP 601, Belgium and 2 Laboratoire de Génétique Médicale, Hopital Erasme, route de Lennik 808, B-1070 Brussels, Belgium

*To whom correspondence should be addressed. Tel: +32 2 555 4160; Fax: +32 2 555 4655; Email: swillens{at}ulb.ac.be
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

Received October 31, 2003; Revised February 26, 2004; Accepted March 15, 2004

Amplification of a cDNA product by quantitative PCR (qPCR) is monitored by a fluorescent signal proportional to the amount of produced amplicon. The qPCR amplification curve usually displays an exponential phase followed by a non-exponential phase, ending with a plateau. Contrary to prevalent interpretation, we demonstrate that under standard qPCR conditions, the plateau can be explained by depletion of the probe through Taq polymerase- catalysed hydrolysis. Knowing the probe concentration and the fluorescence measured at the plateau, a specific fluorescence can thus be calculated. As far as probe hydrolysis quantitatively reflects amplicon synthesis, this, in turn, makes it possible to convert measured fluorescence levels in the exponential phase into concentrations of produced amplicon. It follows that the absolute target cDNA concentration initially engaged in the qPCR can be directly estimated from the fluorescence data, with no need to refer to any calibration with known concentrations of target DNA.


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