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
PCR results for RNaseP and Cyp1B1, showing replicates. Observations from the RNaseP and Cyp1B1 data sets are shown in linear scale, with the 40 cycle cutoff marked by the vertical line. The horizontal axes show cycle number.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: PCR results for RNaseP and Cyp1B1, showing replicates. Observations from the RNaseP and Cyp1B1 data sets are shown in linear scale, with the 40 cycle cutoff marked by the vertical line. The horizontal axes show cycle number.

Mentions: Even when the basic model is used with the data of only 40 cycles (2nd column), the estimates of the initial concentration of amplicon are of the right order of magnitude (although substantially underestimated), ranging from 57% to 72% of the true value. The estimates are somewhat better for the larger initial amounts, (63 – 72% of the true value) than for the smallest initial amounts (57 – 59% of the true value), suggesting that there is information carried in the later cycles of a PCR. Figure 2A shows that the measured fluorescence at the 40th cycle is further from the horizontal asymptote for the lowest initial amounts of amplicon so that there are fewer information-carrying observations for the lower dilutions.


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)

PCR results for RNaseP and Cyp1B1, showing replicates. Observations from the RNaseP and Cyp1B1 data sets are shown in linear scale, with the 40 cycle cutoff marked by the vertical line. The horizontal axes show cycle number.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: PCR results for RNaseP and Cyp1B1, showing replicates. Observations from the RNaseP and Cyp1B1 data sets are shown in linear scale, with the 40 cycle cutoff marked by the vertical line. The horizontal axes show cycle number.
Mentions: Even when the basic model is used with the data of only 40 cycles (2nd column), the estimates of the initial concentration of amplicon are of the right order of magnitude (although substantially underestimated), ranging from 57% to 72% of the true value. The estimates are somewhat better for the larger initial amounts, (63 – 72% of the true value) than for the smallest initial amounts (57 – 59% of the true value), suggesting that there is information carried in the later cycles of a PCR. Figure 2A shows that the measured fluorescence at the 40th cycle is further from the horizontal asymptote for the lowest initial amounts of amplicon so that there are fewer information-carrying observations for the lower dilutions.

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