Theoretical uncertainty of measurements using quantitative polymerase chain reaction
Theoretical uncertainty of measurements using quantitative polymerase chain reaction
复制标题
DOI：
10.1016/s0006-3495(96)79205-6
复制
发表时间：
1996-07-01
影响因子：
3.4
通讯作者：
Jacob, C
中科院分区：
文献类型：
--
作者：
Peccoud, J;Jacob, C
目前的定量聚合酶链反应（PCR）方案仅指示靶序列相对于标准的量，因为还没有估计扩增速率的方法可用。几位作者已经报道了对分离细胞进行PCR的变异性，但由于缺乏进行动力学数据采集的系统和适用于分析此类数据的统计方法，因此无法对其进行广泛研究。我们使用分支过程理论来模拟和分析定量动力学PCR数据。我们计算了单个分子的后代的概率分布。我们证明了放大率对这种分布的形状有严重的影响。对于高值的扩增率，分布有几个最大的概率。使用单个扩增轨迹来估计靶序列的初始拷贝数及其置信区间，前提是扩增在超过20个循环内完成。在扩增的早期阶段中可能的分子波动的结果是小拷贝数导致比大的初始拷贝数相对更大的间隔。置信区间幅度是使用定量PCR的测量的理论不确定度。我们期望这些结果适用于下一代热循环仪产生的数据，用于定量应用。
Current quantitative polymerase chain reaction (PCR) protocols are only indicative of the quantity of a target sequence relative to a standard, because no means of estimating the amplification rate is yet available. The variability of PCR performed on isolated cells has already been reported by several authors, but it could not be extensively studied, because of lack of a system for doing kinetic data acquisition and of statistical methods suitable for analyzing this type of data. We used the branching process theory to simulate and analyze quantitative kinetic PCR data. We computed the probability distribution of the offspring of a single molecule. We demonstrated that the rate of amplification has a severe influence on the shape of this distribution. For high values of the amplification rate, the distribution has several maxima of probability. A single amplification trajectory is used to estimate the initial copy number of the target sequence as well as its confidence interval, provided that the amplification is done over more than 20 cycles. The consequence of possible molecular fluctuations in the early stage of amplification is that small copy numbers result in relatively larger intervals than large initial copy numbers. The confidence interval amplitude is the theoretical uncertainty of measurements using quantitative PCR. We expect these results to be applicable to the data produced by the next generation of thermocyclers for quantitative applications.