Mathematics of quantitative kinetic PCR and the application of standard curves -: art. no. e93
Mathematics of quantitative kinetic PCR and the application of standard curves -: art. no. e93
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DOI：
10.1093/nar/gng093
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发表时间：
2003-08-15
影响因子：
14.9
通讯作者：
Côté, C
中科院分区：
文献类型：
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作者：
Rutledge, RG;Côté, C
荧光监测DNA扩增是实时聚合酶链式反应(Real-Time-PCR)技术的基础，它可以根据DNA扩增阈值产生的分数周期确定目标DNA浓度。绝对定量可以使用通过扩增已知数量的靶DNA而构建的标准曲线来实现。本研究对定量聚合酶链式反应的数学原理进行了详细的研究，并从几个基本方面阐述了阈值方法和标准曲线的应用。利用SYBER(R)Green I荧光标记技术，构建两对套式引物的5条重复标准曲线，检测其重复性和定量变异程度。基于这一分析，应用一条构造良好的单一标准曲线可以提供+/-6-21%的估计精度，这取决于达到阈值所需的循环次数。本文还提出了一种简化的绝对定量方法，定量尺度由阈值处的DNA质量决定。
Fluorescent monitoring of DNA amplification is the basis of real-time PCR, from which target DNA concentration can be determined from the fractional cycle at which a threshold amount of amplicon DNA is produced. Absolute quantification can be achieved using a standard curve constructed by amplifying known amounts of target DNA. In this study, the mathematics of quantitative PCR are examined in detail, from which several fundamental aspects of the threshold method and the application of standard curves are illustrated. The construction of five replicate standard curves for two pairs of nested primers was used to examine the reproducibility and degree of quantitative variation using SYBER(R) Green I fluorescence. Based upon this analysis the application of a single, well- constructed standard curve could provide an estimated precision of +/-6-21%, depending on the number of cycles required to reach threshold. A simplified method for absolute quantification is also proposed, in which quantitative scale is determined by DNA mass at threshold.