1 Department of Mathematical Sciences, Montana State University , Bozeman, MT 59715 , USA.
2 Department of Chemical and Biological Engineering, Montana State University , Bozeman, MT 59715 , USA.
J R Soc Interface. 2019 May 31;16(154):20190143. doi: 10.1098/rsif.2019.0143. Epub 2019 May 29.
Isothermal DNA amplification reactions are a prevalent tool with many applications, ranging from analyte detection to DNA circuits. Exponential amplification reaction (EXPAR) is a popular isothermal DNA amplification method that exponentially amplifies short DNA oligonucleotides. A recent modification of this technique using an energetically stable looped template with palindromic binding regions demonstrated unexpected biphasic amplification and much higher DNA yield than EXPAR. This ultrasensitive DNA amplification reaction (UDAR) shows high-gain, switch-like DNA output from low concentrations of DNA input. Here we present the first mathematical model of UDAR based on four reaction mechanisms and show the model can reproduce the experimentally observed biphasic behaviour. Furthermore, we show that three of these mechanisms are necessary to reproduce biphasic experimental results. The reaction mechanisms are (i) positively cooperative multistep binding spurred by two trigger binding sites on the template; (ii) gradual template deactivation; (iii) recycling of deactivated templates into active templates; and (iv) polymerase sequestration. These mechanisms can potentially illuminate the behaviour of EXPAR as well as other nucleic acid amplification reactions.
等温 DNA 扩增反应是一种应用广泛的工具，从分析物检测到 DNA 电路都有涉及。指数扩增反应（EXPAR）是一种流行的等温 DNA 扩增方法，可对短 DNA 寡核苷酸进行指数扩增。最近对该技术的一种修改，使用具有回文结合区域的能量稳定环化模板，显示出出人意料的两相扩增和比 EXPAR 高得多的 DNA 产量。这种超灵敏的 DNA 扩增反应（UDAR）显示出从低浓度 DNA 输入获得高增益、类似开关的 DNA 输出。本文提出了第一个基于四个反应机制的 UDAR 数学模型，并表明该模型可以再现实验观察到的两相行为。此外，我们还表明，要再现两相实验结果，需要这三个机制。这些反应机制是：（i）模板上的两个触发结合位点促进的正协同多步结合；（ii）模板逐渐失活；（iii）失活模板循环回活模板；和（iv）聚合酶隔离。这些机制可能阐明 EXPAR 以及其他核酸扩增反应的行为。
