A multi-enzyme model for pyrosequencing
A multi-enzyme model for pyrosequencing
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DOI：
10.1093/nar/gnh159
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Griffin, PB
中科院分区：
文献类型：
--
作者：
Agah, A;Aghajan, M;Griffin, PB
焦磷酸测序是一种基于合成测序的DNA测序技术，能够快速实时测定序列。这项技术在一个试管中使用四个酶促反应来监测DNA合成。核苷酸被迭代地加入到反应中，在掺入的情况下，焦磷酸盐（PPi）被释放。PPi触发一系列反应，产生光，这与DNA的数量和结合核苷酸的数量成正比。产生的光被检测系统以峰值信号的形式检测和记录，这反映了反应中所有四种酶的活性。我们开发了模拟方法来模拟酶的动力学。这些模拟为焦磷酸测序四酶系统提供了一个完整的模型，基于该模型，可以根据酶和底物的浓度预测峰的高度和形状。仿真结果与实验数据吻合较好。基于这些模拟，可以确定链中的限速步骤，并可以计算出四种酶在焦磷酸测序中的k - m和k（cat）。
Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid real-time sequence determination. This technique employs four enzymatic reactions in a single tube to monitor DNA synthesis. Nucleotides are added iteratively to the reaction and in case of incorporation, pyrophosphate (PPi) is released. PPi triggers a series of reactions resulting in production of light, which is proportional to the amount of DNA and number of incorporated nucleotides. Generated light is detected and recorded by a detector system in the form of a peak signal, which reflects the activity of all four enzymes in the reaction. We have developed simulations to model the kinetics of the enzymes. These simulations provide a full model for the Pyrosequencing four-enzyme system, based on which the peak height and shape can be predicted depending on the concentrations of enzymes and substrates. Simulation results are shown to be compatible with experimental data. Based on these simulations, the rate-limiting steps in the chain can be determined, and K-M and k(cat) of all four enzymes in Pyrosequencing can be calculated.