A mechanistic model of PCR for accurate quantification of quantitative PCR data.
BACKGROUND
Quantitative PCR (qPCR) is a workhorse laboratory technique for measuring the concentration of a target DNA sequence with high accuracy over a wide dynamic range. The gold standard method for estimating DNA concentrations via qPCR is quantification cycle () standard curve quantification, which requires the time- and labor-intensive construction of a standard curve. In theory, the shape of a qPCR data curve can be used to directly quantify DNA concentration by fitting a model to data; however, current empirical model-based quantification methods are not as reliable as standard curve quantification.


RESULTS
We have developed a two-parameter mass action kinetic model of PCR (MAK2) that can be fitted to qPCR data in order to quantify target concentration from a single qPCR assay. To compare the accuracy of MAK2-fitting to other qPCR quantification methods, we have applied quantification methods to qPCR dilution series data generated in three independent laboratories using different target sequences. Quantification accuracy was assessed by analyzing the reliability of concentration predictions for targets at known concentrations. Our results indicate that quantification by MAK2-fitting is as reliable as standard curve quantification for a variety of DNA targets and a wide range of concentrations.


CONCLUSIONS
We anticipate that MAK2 quantification will have a profound effect on the way qPCR experiments are designed and analyzed. In particular, MAK2 enables accurate quantification of portable qPCR assays with limited sample throughput, where construction of a standard curve is impractical.
Boggy
Gregory J
GJ
Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America. gboggy@umich.edu
Woolf
Peter J
PJ
D002138:Calibration; D004247:DNA / Q000235:genetics; D007700:Kinetics; D008962:Models, Theoretical; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards
D004247:DNA
Biotechnol Bioeng. 2005 Nov 5;92(3):346-55
16170827
BMC Bioinformatics. 2007 Oct 23;8:409
17956631
Anal Biochem. 2009 Jan 1;384(1):1-10
18812161
Nucleic Acids Res. 2004 Dec 15;32(22):e178
15601990
Nucleic Acids Res. 2001 May 1;29(9):e45
11328886
Biotechnology (N Y). 1993 Sep;11(9):1026-30
7764001
Biochem Biophys Res Commun. 2002 Jun 7;294(2):347-53
12051718
Biotechnol Bioeng. 2005 Sep 30;91(7):848-60
15986490
Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17807-12
17098862
Anal Biochem. 2002 Mar 1;302(1):52-9
11846375
BMC Bioinformatics. 2008 Apr 29;9:221
18445269
Bioinformatics. 2008 Jul 1;24(13):1549-51
18482995
BMC Biotechnol. 2008 May 08;8:47
18466619
Nucleic Acids Res. 2003 Oct 15;31(20):e122
14530455