Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process
Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process
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DOI：
10.1186/1471-2105-8-409
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发表时间：
2007-10-23
影响因子：
3
通讯作者：
Portier, Chris J.
中科院分区：
文献类型：
--
作者：
Smith, Marjo V.;Miller, Chris R.;Portier, Chris J.
背景资料：自从真实的时间PCR被首次开发以来，已经尝试了几种方法来估计RT-PCR反应中的初始模板量。虽然最初仅使用对应于指数复制的早期热循环，但最近已经努力在PCR中使用所有循环。这些努力包括拟合经验S形曲线和更精细的机制模型，以探索每个周期中发生的化学反应。更精细的机械模型需要更多的参数比可以从一个单一的扩增，而经验模型提供很少的洞察力，很难定制到特定reactors.Results：我们直接估计的扩增子的初始量使用一个简化的机械模型的基础上在退火步骤的PCR的化学反应。其基本模型包括Taqman探针的酶切作用下的DNA复制和先前合成的相反方向的DNA链之间的再退火。通过模拟Taqman探针消化的量并将其与观察到的荧光相匹配，可以估计荧光染料分子的数量与观察到的荧光发射之间的转换因子，沿着估计扩增子的绝对初始量和再退火的速率参数。该模型被应用到几个PCR反应与已知量的扩增子，并显示出相当不错的工作。模型的扩展版本允许通过向模型添加1个以上参数来复制扩增子而不释放荧光染料。在初始引物浓度超过初始探针浓度的大多数情况下，额外的过程是有帮助的。用于将算法应用于数据的软件可以在http://www.niehs.nih.gov/research/resources/software/pcranalyzer/Conclusion下载：我们提出了基于机械的模型可以适合于来自单个PCR扩增的观察的原理的证明。在不使用标准品溶液的情况下很好地估计了扩增子的初始量。使用来自2个PCR的扩增子的预测初始量的比率显示出良好的工作，即使当扩增子的绝对量在各个PCR中被低估时。
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 http://www.niehs.nih.gov/research/resources/software/pcranalyzer/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.