Departments of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada.
Methods. 2010 Apr;50(4):313-22. doi: 10.1016/j.ymeth.2010.02.003. Epub 2010 Feb 6.
For real-time monitoring of PCR amplification of DNA, quantitative PCR (qPCR) assays use various fluorescent reporters. DNA binding molecules and hybridization reporters (primers and probes) only fluoresce when bound to DNA and result in the non-cumulative increase in observed fluorescence. Hydrolysis reporters (TaqMan probes and QZyme primers) become fluorescent during DNA elongation and the released fluorophore remains fluorescent during further cycles; this results in a cumulative increase in observed fluorescence. Although the quantification threshold is reached at a lower number of cycles when fluorescence accumulates, in qPCR analysis no distinction is made between the two types of data sets. Mathematical modeling shows that ignoring the cumulative nature of the data leaves the estimated PCR efficiency practically unaffected but will lead to at least one cycle underestimation of the quantification cycle (C(q) value), corresponding to a 2-fold overestimation of target quantity. The effect on the target-reference ratio depends on the PCR efficiency of the target and reference amplicons. The leftward shift of the C(q) value is dependent on the PCR efficiency and with sufficiently large C(q) values, this shift is constant. This allows the C(q) to be corrected and unbiased target quantities to be obtained.
为了实时监测 DNA 的 PCR 扩增，定量 PCR（qPCR）检测使用了各种荧光报告分子。DNA 结合分子和杂交报告分子（引物和探针）只有在与 DNA 结合时才会发出荧光，从而导致观察到的荧光非累积性增加。水解报告分子（TaqMan 探针和 QZyme 引物）在 DNA 延伸过程中发出荧光，并且释放的荧光团在进一步的循环中仍然保持荧光；这导致观察到的荧光累积增加。尽管荧光累积时达到定量阈值的循环数较少，但在 qPCR 分析中，两种数据集之间没有区别。数学模型表明，忽略数据的累积性质实际上不会影响估计的 PCR 效率，但至少会导致一个循环的定量循环（C(q)值）低估，相当于目标数量的 2 倍高估。对目标-参照比值的影响取决于目标和参照扩增子的 PCR 效率。C(q)值的左移取决于 PCR 效率，并且随着 C(q)值足够大，这种移位是恒定的。这允许校正 C(q)值并获得无偏的目标数量。
