Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications
AUTOR(ES)
Rutledge, R. G.
FONTE
Oxford University Press
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=545475Documentos Relacionados
- High-Throughput Real-Time Reverse Transcription-PCR Quantitation of Hepatitis C Virus RNA
- Modeling of 5′ Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica
- PCR candidate region mismatch scanning: adaptation to quantitative, high-throughput genotyping
- Real-Time Quantitative PCR for Detection of Helicobacter pylori
- Real-Time PCR for Quantitative Detection of Toxoplasma gondii