Mathematical model of real-time PCR kinetics
Mathematical model of real-time PCR kinetics
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DOI：
10.1002/bit.20617
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发表时间：
2005-11-05
影响因子：
3.8
通讯作者：
Roth, CM
中科院分区：
文献类型：
--
作者：
Gevertz, JL;Dunn, SM;Roth, CM
几种实时聚合酶链式反应(RTPCR)定量技术目前被用来以荧光强度的形式从RTPCR数据中确定单个基因的表达水平。在这些定量技术中，大多数都假定RT-PCR的效率是恒定的。然而，我们对RTPCR数据的分析表明，即使在RTPCR的指数阶段，反应的效率也不是恒定的，而是循环数的函数。为了更好地理解这一行为的机理，我们建立了一个关于PCR过程的退火期和延长期的数学模型。利用该模型，我们可以模拟一系列反应周期中的聚合酶链式反应过程。因此，该模型允许我们在给定一组初始条件和参数值的情况下，在任何周期数预测RTPCR的效率，这些初始条件和参数值大多可以从生物物理数据中估计出来。该模型预测，当产品浓度达到足以与底物-模板退火剂竞争的模板-模板再退火剂浓度时，循环效率急剧下降；这一行为与现有的实验数据一致。这个模型提供了对RTPCR的定量理解，可以让我们开发更准确的方法来从RTPCR数据中量化基因表达水平。(C)2005年威利期刊公司。
Several real-time PCR (rtPCR) quantification techniques are currently used to determine the expression levels of individual genes from rtPCR data in the form of fluorescence intensities. In most of these quantification techniques, it is assumed that the efficiency of rtPCR is constant. Our analysis of rtPCR data shows, however, that even during the exponential phase of rtPCR, the efficiency of the reaction is notconstant, but is instead a function of cycle number. In order to understand better the mechanisms belying this behavior, we have developed a mathematical model of the annealing and extension phases of the PCR process. Using the model, we can simulate the PCR process over a series of reaction cycles. The model thus allows us to predict the efficiency of rtPCR at any cycle number, given a set of initial conditions and parameter values, which can mostly be estimated from biophysical data. The model predicts a precipitous decrease in cycle efficiency when the product concentration reaches a sufficient level for template-template reannealing to compete with primer-template annealing; this behavior is consistent with available experimental data. The quantitative understanding of rtPCR provided by this model can allow us to develop more accurate methods to quantify gene expression levels from rtPCR data. (c) 2005 Wiley Periodicals, Inc.