Waterfall Christy M, Eisenthal Robert, Cobb Benjamin D
Molecular Sensing plc, Challeymead Business Park, Bradford Road, Melksham, Wiltshire SN12 8LH, UK.
Biochem Biophys Res Commun. 2002 Dec 20;299(5):715-22. doi: 10.1016/s0006-291x(02)02750-x.
A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR.
本文提出了一种在快速循环PCR过程中估算Taq DNA聚合酶动力学参数的新方法。构建了一个模型，该模型使用简化的S形函数来表示PCR过程中的底物积累，并结合描述米氏酶高底物抑制的通用方程。PCR进程曲线被视为一系列独立反应，其中每个循环的初始速率都被精确测量。通过等位基因特异性PCR（AS-PCR）扩增获得动力学参数，以研究错配对扩增的影响。获得了高度相关性，这为底物抑制是PCR后期出现平台期的主要原因提供了证据。
