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

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 o...

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Autores principales: Smith, Marjo V, Miller, Chris R, Kohn, Michael, Walker, Nigel J, Portier, Chris J
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2007
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2194744/
https://www.ncbi.nlm.nih.gov/pubmed/17956631
http://dx.doi.org/10.1186/1471-2105-8-409
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author Smith, Marjo V
Miller, Chris R
Kohn, Michael
Walker, Nigel J
Portier, Chris J
author_facet Smith, Marjo V
Miller, Chris R
Kohn, Michael
Walker, Nigel J
Portier, Chris J
author_sort Smith, Marjo V
collection PubMed
description 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 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.
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spelling pubmed-21947442008-01-14 Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process Smith, Marjo V Miller, Chris R Kohn, Michael Walker, Nigel J Portier, Chris J BMC Bioinformatics Methodology Article 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 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. BioMed Central 2007-10-23 /pmc/articles/PMC2194744/ /pubmed/17956631 http://dx.doi.org/10.1186/1471-2105-8-409 Text en Copyright © 2007 Smith et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology Article
Smith, Marjo V
Miller, Chris R
Kohn, Michael
Walker, Nigel J
Portier, Chris J
Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process
title Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process
title_full Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process
title_fullStr Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process
title_full_unstemmed Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process
title_short Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process
title_sort absolute estimation of initial concentrations of amplicon in a real-time rt-pcr process
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2194744/
https://www.ncbi.nlm.nih.gov/pubmed/17956631
http://dx.doi.org/10.1186/1471-2105-8-409
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