### Abstract

Recently a theoretical analysis of PCR efficiency has been published by Booth et al. (2010). The PCR yield is the product of three efficiencies: (i) the annealing efficiency is the fraction of templates that form binary complexes with primers during annealing, (ii) the polymerase binding efficiency is the fraction of binary complexes that bind to polymerase to form ternary complexes and (iii) the elongation efficiency is the fraction of ternary complexes that extend fully. Yield is controlled by the smallest of the three efficiencies and control could shift from one type of efficiency to another over the course of a PCR experiment. Experiments have been designed that are specifically controlled by each one of the efficiencies and the results are consistent with the mathematical model. The experimental data has also been used to quantify six key parameters of the theoretical model. An important application of the fully characterized model is to calculate initial template concentration from real-time PCR data. Given the PCR protocol, the midpoint cycle number (where the template concentration is half that of the final concentration) can be theoretically determined and graphed for a variety of initial DNA concentrations. Real-time results can be used to calculate the midpoint cycle number and consequently the initial DNA concentration, using this graph. The application becomes particularly simple if a conservative PCR protocol is followed where only the annealing efficiency is controlling.

Original language | English (US) |
---|---|

Pages (from-to) | 1783-1789 |

Number of pages | 7 |

Journal | Chemical Engineering Science |

Volume | 66 |

Issue number | 8 |

DOIs | |

State | Published - Apr 15 2011 |

### Fingerprint

### Keywords

- Biological and biomolecular engineering
- Enzyme
- Kinetics
- Mathematical modeling
- Molecular biology
- PCR efficiency

### ASJC Scopus subject areas

- Chemistry(all)
- Chemical Engineering(all)
- Industrial and Manufacturing Engineering

### Cite this

*Chemical Engineering Science*,

*66*(8), 1783-1789. https://doi.org/10.1016/j.ces.2011.01.029

**Experimental validation of a fundamental model for PCR efficiency.** / Louw, Tobias M.; Booth, Christine S.; Pienaar, Elsje; TerMaat, Joel R.; Whitney, Scott E.; Viljoen, Hendrik J.

Research output: Contribution to journal › Article

*Chemical Engineering Science*, vol. 66, no. 8, pp. 1783-1789. https://doi.org/10.1016/j.ces.2011.01.029

}

TY - JOUR

T1 - Experimental validation of a fundamental model for PCR efficiency

AU - Louw, Tobias M.

AU - Booth, Christine S.

AU - Pienaar, Elsje

AU - TerMaat, Joel R.

AU - Whitney, Scott E.

AU - Viljoen, Hendrik J

PY - 2011/4/15

Y1 - 2011/4/15

N2 - Recently a theoretical analysis of PCR efficiency has been published by Booth et al. (2010). The PCR yield is the product of three efficiencies: (i) the annealing efficiency is the fraction of templates that form binary complexes with primers during annealing, (ii) the polymerase binding efficiency is the fraction of binary complexes that bind to polymerase to form ternary complexes and (iii) the elongation efficiency is the fraction of ternary complexes that extend fully. Yield is controlled by the smallest of the three efficiencies and control could shift from one type of efficiency to another over the course of a PCR experiment. Experiments have been designed that are specifically controlled by each one of the efficiencies and the results are consistent with the mathematical model. The experimental data has also been used to quantify six key parameters of the theoretical model. An important application of the fully characterized model is to calculate initial template concentration from real-time PCR data. Given the PCR protocol, the midpoint cycle number (where the template concentration is half that of the final concentration) can be theoretically determined and graphed for a variety of initial DNA concentrations. Real-time results can be used to calculate the midpoint cycle number and consequently the initial DNA concentration, using this graph. The application becomes particularly simple if a conservative PCR protocol is followed where only the annealing efficiency is controlling.

AB - Recently a theoretical analysis of PCR efficiency has been published by Booth et al. (2010). The PCR yield is the product of three efficiencies: (i) the annealing efficiency is the fraction of templates that form binary complexes with primers during annealing, (ii) the polymerase binding efficiency is the fraction of binary complexes that bind to polymerase to form ternary complexes and (iii) the elongation efficiency is the fraction of ternary complexes that extend fully. Yield is controlled by the smallest of the three efficiencies and control could shift from one type of efficiency to another over the course of a PCR experiment. Experiments have been designed that are specifically controlled by each one of the efficiencies and the results are consistent with the mathematical model. The experimental data has also been used to quantify six key parameters of the theoretical model. An important application of the fully characterized model is to calculate initial template concentration from real-time PCR data. Given the PCR protocol, the midpoint cycle number (where the template concentration is half that of the final concentration) can be theoretically determined and graphed for a variety of initial DNA concentrations. Real-time results can be used to calculate the midpoint cycle number and consequently the initial DNA concentration, using this graph. The application becomes particularly simple if a conservative PCR protocol is followed where only the annealing efficiency is controlling.

KW - Biological and biomolecular engineering

KW - Enzyme

KW - Kinetics

KW - Mathematical modeling

KW - Molecular biology

KW - PCR efficiency

UR - http://www.scopus.com/inward/record.url?scp=79952239324&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79952239324&partnerID=8YFLogxK

U2 - 10.1016/j.ces.2011.01.029

DO - 10.1016/j.ces.2011.01.029

M3 - Article

C2 - 21822325

AN - SCOPUS:79952239324

VL - 66

SP - 1783

EP - 1789

JO - Chemical Engineering Science

JF - Chemical Engineering Science

SN - 0009-2509

IS - 8

ER -