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Nucleic Acids Research 2004 32(22):e178; doi:10.1093/nar/gnh177
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Published online 15 December 2004

Nucleic Acids Research, Vol. 32 No. 22 © Oxford University Press 2004; all rights reserved

Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications

R. G. Rutledge*

Natural Resources Canada, 1055 du P.E.P.S, Sainte-Foy, Quebec, Canada G1V 4C7

* Tel: +1 418 648 2582; Fax: +1 418 648 5849; Email: Bob.Rutledge{at}NRCan.gc.ca

Received May 25, 2004; Revised October 15, 2004; Accepted November 24, 2004

Quantitative real-time PCR has revolutionized many aspects of genetic research, biomedical diagnostics and pathogen detection. Nevertheless, the full potential of this technology has yet to be realized, primarily due to the limitations of the threshold-based methodologies that are currently used for quantitative analysis. Prone to errors caused by variations in reaction preparation and amplification conditions, these approaches necessitate construction of standard curves for each target sequence, significantly limiting the development of high-throughput applications that demand substantive levels of reliability and automation. In this study, an alternative approach based upon fitting of fluorescence data to a four-parametric sigmoid function is shown to dramatically increase both the utility and reliability of quantitative real-time PCR. By mathematically modeling individual amplification reactions, quantification can be achieved without the use of standard curves and without prior knowledge of amplification efficiency. Combined with provision of quantitative scale via optical calibration, sigmoidal curve-fitting could confer the capability for fully automated quantification of nucleic acids with unparalleled accuracy and reliability.


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