Limits...
Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process.

Smith MV, Miller CR, Kohn M, Walker NJ, Portier CJ - BMC Bioinformatics (2007)

Bottom Line: The model is applied to several PCR reactions with known amounts of amplicon and is shown to work reasonably well.An expanded version of the model allows duplication of amplicon without release of fluorescent dye, by adding 1 more parameter to the model.Using the ratio of the predicted initial amounts of amplicon from 2 PCRs is shown to work well even when the absolute amounts of amplicon are underestimated in the individual PCRs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Constella Group, Suite 200, 2605 Meridian Pky, Durham, NC, USA. msmith@constellagroup.com

ABSTRACT

Background: Since real time PCR was first developed, several approaches to estimating the initial quantity of template in an RT-PCR reaction have been tried. While initially only the early thermal cycles corresponding to exponential duplication were used, lately there has been an effort to use all of the cycles in a PCR. The efforts have included both fitting empirical sigmoid curves and more elaborate mechanistic models that explore the chemical reactions taking place during each cycle. The more elaborate mechanistic models require many more parameters than can be fit from a single amplification, while the empirical models provide little insight and are difficult to tailor to specific reactants.

Results: We directly estimate the initial amount of amplicon using a simplified mechanistic model based on chemical reactions in the annealing step of the PCR. The basic model includes the duplication of DNA with the digestion of Taqman probe and the re-annealing between previously synthesized DNA strands of opposite orientation. By modelling the amount of Taqman probe digested and matching that with the observed fluorescence, the conversion factor between the number of fluorescing dye molecules and observed fluorescent emission can be estimated, along with the absolute initial amount of amplicon and the rate parameter for re-annealing. The model is applied to several PCR reactions with known amounts of amplicon and is shown to work reasonably well. An expanded version of the model allows duplication of amplicon without release of fluorescent dye, by adding 1 more parameter to the model. The additional process is helpful in most cases where the initial primer concentration exceeds the initial probe concentration. Software for applying the algorithm to data may be downloaded at http://www.niehs.nih.gov/research/resources/software/pcranalyzer/

Conclusion: We present proof of the principle that a mechanistically based model can be fit to observations from a single PCR amplification. Initial amounts of amplicon are well estimated without using a standard solution. Using the ratio of the predicted initial amounts of amplicon from 2 PCRs is shown to work well even when the absolute amounts of amplicon are underestimated in the individual PCRs.

Show MeSH
Model fit. The observations and predicted values are shown corresponding to one of the amplifications using RnaseP. For this fit, the true initial amount of amplicon was 1.667e-7 nmol/L (in 50 μl solution) and the estimated parameter values as follows: the estimated concentration of amplicon was 1.767e-7 nmol/L, the estimated re-annealing rate constant, 1.1680e4; the estimated rate constant for unmarked duplication, 266.21; and the estimated conversion factor, 2.9392e-2. The predicted values correspond to the equilibrium values of the solution to the 1st equation in the extended model, the wiqE. See Appendix.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2194744&req=5

Figure 1: Model fit. The observations and predicted values are shown corresponding to one of the amplifications using RnaseP. For this fit, the true initial amount of amplicon was 1.667e-7 nmol/L (in 50 μl solution) and the estimated parameter values as follows: the estimated concentration of amplicon was 1.767e-7 nmol/L, the estimated re-annealing rate constant, 1.1680e4; the estimated rate constant for unmarked duplication, 266.21; and the estimated conversion factor, 2.9392e-2. The predicted values correspond to the equilibrium values of the solution to the 1st equation in the extended model, the wiqE. See Appendix.)

Mentions: The model was applied to 3 data sets described in the method section. The 3 data sets amplified a total of 3 gene sequences, RnaseP, Cyp1B1 and Actin. In each case, the initial concentrations of the amplicon were known, with amplifications run on several known dilutions of the original concentration. Tables 1 through 4 in the body of the paper compare the estimated amount of amplicon with the known concentration. Figure 1 shows an example of the model fit to an amplification run of the RNaseP data set.


Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process.

Smith MV, Miller CR, Kohn M, Walker NJ, Portier CJ - BMC Bioinformatics (2007)

Model fit. The observations and predicted values are shown corresponding to one of the amplifications using RnaseP. For this fit, the true initial amount of amplicon was 1.667e-7 nmol/L (in 50 μl solution) and the estimated parameter values as follows: the estimated concentration of amplicon was 1.767e-7 nmol/L, the estimated re-annealing rate constant, 1.1680e4; the estimated rate constant for unmarked duplication, 266.21; and the estimated conversion factor, 2.9392e-2. The predicted values correspond to the equilibrium values of the solution to the 1st equation in the extended model, the wiqE. See Appendix.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2194744&req=5

Figure 1: Model fit. The observations and predicted values are shown corresponding to one of the amplifications using RnaseP. For this fit, the true initial amount of amplicon was 1.667e-7 nmol/L (in 50 μl solution) and the estimated parameter values as follows: the estimated concentration of amplicon was 1.767e-7 nmol/L, the estimated re-annealing rate constant, 1.1680e4; the estimated rate constant for unmarked duplication, 266.21; and the estimated conversion factor, 2.9392e-2. The predicted values correspond to the equilibrium values of the solution to the 1st equation in the extended model, the wiqE. See Appendix.)
Mentions: The model was applied to 3 data sets described in the method section. The 3 data sets amplified a total of 3 gene sequences, RnaseP, Cyp1B1 and Actin. In each case, the initial concentrations of the amplicon were known, with amplifications run on several known dilutions of the original concentration. Tables 1 through 4 in the body of the paper compare the estimated amount of amplicon with the known concentration. Figure 1 shows an example of the model fit to an amplification run of the RNaseP data set.

Bottom Line: The model is applied to several PCR reactions with known amounts of amplicon and is shown to work reasonably well.An expanded version of the model allows duplication of amplicon without release of fluorescent dye, by adding 1 more parameter to the model.Using the ratio of the predicted initial amounts of amplicon from 2 PCRs is shown to work well even when the absolute amounts of amplicon are underestimated in the individual PCRs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Constella Group, Suite 200, 2605 Meridian Pky, Durham, NC, USA. msmith@constellagroup.com

ABSTRACT

Background: Since real time PCR was first developed, several approaches to estimating the initial quantity of template in an RT-PCR reaction have been tried. While initially only the early thermal cycles corresponding to exponential duplication were used, lately there has been an effort to use all of the cycles in a PCR. The efforts have included both fitting empirical sigmoid curves and more elaborate mechanistic models that explore the chemical reactions taking place during each cycle. The more elaborate mechanistic models require many more parameters than can be fit from a single amplification, while the empirical models provide little insight and are difficult to tailor to specific reactants.

Results: We directly estimate the initial amount of amplicon using a simplified mechanistic model based on chemical reactions in the annealing step of the PCR. The basic model includes the duplication of DNA with the digestion of Taqman probe and the re-annealing between previously synthesized DNA strands of opposite orientation. By modelling the amount of Taqman probe digested and matching that with the observed fluorescence, the conversion factor between the number of fluorescing dye molecules and observed fluorescent emission can be estimated, along with the absolute initial amount of amplicon and the rate parameter for re-annealing. The model is applied to several PCR reactions with known amounts of amplicon and is shown to work reasonably well. An expanded version of the model allows duplication of amplicon without release of fluorescent dye, by adding 1 more parameter to the model. The additional process is helpful in most cases where the initial primer concentration exceeds the initial probe concentration. Software for applying the algorithm to data may be downloaded at http://www.niehs.nih.gov/research/resources/software/pcranalyzer/

Conclusion: We present proof of the principle that a mechanistically based model can be fit to observations from a single PCR amplification. Initial amounts of amplicon are well estimated without using a standard solution. Using the ratio of the predicted initial amounts of amplicon from 2 PCRs is shown to work well even when the absolute amounts of amplicon are underestimated in the individual PCRs.

Show MeSH