Alvarez M J, Depino A M, Podhajcer O L, Pitossi F J
Institute for Biochemical Research, Campomar Foundation, CONICET, Patricias Argentinas 435, Buenos Aires, 1405, Argentina.
Anal Biochem. 2000 Dec 1;287(1):87-94. doi: 10.1006/abio.2000.4823.
Competitive PCR is a highly sensitive method for specific DNA quantification. Despite the lack of studies related to the accuracy of the method it has been widely used. Here we present a simulation model for competitive PCR, which takes into account the efficiency decay as a linear relationship of the total product yield. The model helped us to study the kind and magnitude of errors that arise from quantitative and semiquantitative competitive PCR protocols and to find ways to minimize them. The simulation data suggest that differences in amplification efficiency between target and standard templates induce stronger biases in quantitative than in semiquantitative competitive PCR. Quantitative competitive PCR can only be used when both efficiencies are equal. In contrast, semiquantitative competitive PCR can be used even when the target is amplified with a higher efficiency than the standard, since under such conditions the method tends to underestimate the differences in initial DNA content. These predictions have been confirmed with experimental data and show that the estimation of the amplification efficiencies is a prerequisite for the use of quantitative and semiquantitative competitive PCR. A simple method for this estimation is also presented.
竞争性聚合酶链反应（PCR）是一种用于特定DNA定量的高灵敏度方法。尽管缺乏关于该方法准确性的研究，但它已被广泛使用。在此，我们提出了一种竞争性PCR的模拟模型，该模型将效率衰减视为总产物产量的线性关系。该模型帮助我们研究定量和半定量竞争性PCR方案中出现的误差类型和大小，并找到将其最小化的方法。模拟数据表明，靶标模板和标准模板之间扩增效率的差异在定量竞争性PCR中比在半定量竞争性PCR中引起的偏差更大。只有当两种效率相等时，才能使用定量竞争性PCR。相比之下，即使靶标比标准扩增效率更高，也可以使用半定量竞争性PCR，因为在这种情况下，该方法往往会低估初始DNA含量的差异。这些预测已通过实验数据得到证实，表明扩增效率的估计是使用定量和半定量竞争性PCR的先决条件。还提出了一种用于此估计的简单方法。
