Format

Send to

Choose Destination
See comment in PubMed Commons below
Nucleic Acids Res. 2004 Dec 15;32(22):e178.

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

Author information

1
Natural Resources Canada, 1055 du P.E.P.S, Sainte-Foy, Quebec, Canada G1V 4C7.

Abstract

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.

PMID:
15601990
PMCID:
PMC545475
DOI:
10.1093/nar/gnh177
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Silverchair Information Systems Icon for PubMed Central
    Loading ...
    Support Center