Experimental Validation of a Fundamental Model for PCR Efficiency.
Experimental Validation of a Fundamental Model for PCR Efficiency.
复制标题
PCR效率的基本模型的实验验证。
DOI：
10.1016/j.ces.2011.01.029
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发表时间：
2011-04-15
影响因子：
4.7
通讯作者：
Viljoen HJ
中科院分区：
文献类型：
--
作者：
Louw TM;Booth CS;Pienaar E;Termaat JR;Whitney SE;Viljoen HJ
最近，一篇关于PCR效率的理论分析已经由。PCR产率是三个效率的产物：(i)退火效率是模板在退火过程中与引物形成二元配合物的比例，（ii）聚合酶结合效率是二元配合物与聚合酶结合形成三元配合物的比例，（iii）延伸效率是三元配合物完全延伸的比例。产量由三种效率中最小的一种控制，在PCR实验过程中，控制可以从一种效率转变为另一种效率。设计了由各效率控制的实验，实验结果与数学模型一致。实验数据也被用来量化理论模型的六个关键参数。完全表征模型的一个重要应用是从实时PCR数据计算初始模板浓度。给定PCR方案，理论上可以确定中点周期数（模板浓度为最终浓度的一半），并绘制各种初始DNA浓度的图表。实时结果可用于计算中点周期数，从而计算初始DNA浓度，使用此图。如果遵循仅控制退火效率的保守PCR协议，则应用变得特别简单。
Recently a theoretical analysis of PCR efficiency has been published by. 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.
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