Experimental validation of a fundamental model for PCR efficiency

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.

Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (Scophthalmus maximus) gonad dataset

“…This efficiency varies from cycle to cycle. This theoretical study was validated in [ 35 ]. So, at first, a constant efficiency should not be assumed.…”

Introduction to the Gene Expression Analysis

“…The qPCR reaction involves three phases that may be represented as a sigmoidal curve [ 16 ]. The three phases are as follows [ 12 ] ( Fig.…”

Model-based Analysis for Real-time Label-free Ultraviolet Quantification of Ultrafast Plasmonic Polymerase Chain Reaction

“…To simplify the calibration process, the polymerase binding rates at annealing and elongation temperatures (60 and 72 º C) are considered equal (K C =K C * ). In Table  and Table , we used the same nomenclature as Booth et al [11,12]. Different experiments with varying initial DNA concentrations were undergone plasmonic amplification, and the amplification curves (transmittance vs. cycles, T Experimental ) were obtained experimentally ( Table ).…”

“…Other models have considered the cycle dependency of PCR efficiency by incorporating the melting, annealing, and elongation steps efficiencies into overall efficiency [8,9]. These models were used in qPCRs to analyze and detect parameters controlling or degrading denaturing, annealing, and extension efficiencies [10,11]. In this study, we use a mathematical model presented by Booth et al to investigate cycle to cycle efficiencies and PCR reagent concentrations of a real-time 260nm detection plasmonic PCR system [11,12].…”

“…These models were used in qPCRs to analyze and detect parameters controlling or degrading denaturing, annealing, and extension efficiencies [10,11]. In this study, we use a mathematical model presented by Booth et al to investigate cycle to cycle efficiencies and PCR reagent concentrations of a real-time 260nm detection plasmonic PCR system [11,12].…”