PCR反应的动力学模拟和相关因素对扩增结果的影响

Kinetic Simulation and Amplification Effects Resulted From Various Treatments for Polymerase Chain Reaction(PCR)

PCR技术建立二十年以来,它已经成为生物学实验室不可缺少的一部分。但因为其影响因素多、反应过程比较复杂,到今天为止它还是一个具有某种程度经验性的方法。我们从PCR反应的基本动力学过程分析出发,通过每一轮扩增循环的分析描述了产物的积累过程,在此基础上建立了反应的迭代动力学模型。通过该模型我们模拟了反应使用的模板量、扩增循环数、酶的使用量、酶的半衰期、以及每一轮扩增中变性时间对扩增结果的影响。进一步,我们对模板使用量、扩增循环数、酶的使用量对扩增结果的影响进行了实验验证和探讨;混合模板对扩增结果的影响也同时做了探讨。模拟和实验的结果表明:常规的PCR实验中使用中等偏低的模板量,选择较好的聚合酶同时使用合适的酶量,通过较低循环数的扩增就能得到较为满意的结果。PCR实验的成败最大的限制因素是实验准备过程中引物的设计,而实验过程中的调整优化只能起辅助作用。

PCR technology has been one important part of modem biological laboratories after it was established about 20 years ago. How to obtain a good result, however, relies on the operator＇ s experience to an extent, because it is possessed complicated kinetic process and influenced by various factors. Starting from the analysis of the reaction kinetics, we depicted the process of product accumulation for the PCR amplification. Furthermore, we constructed an iterative model for simulating the PCR. We evaluated effect of various factors on PCR amplification, i. e. , the amount of template, cycling numbers, amount of polymerase, the half-life of polymerase, and denaturation time with each amplification cycle. Finally, we performed various PCR experiments with different treatments respectively, including amounts of template, cycling number, amounts of polymerase, and the template mixture. The results indicated that excellent results could be obtained with moderate amount of template, with polymerase in higher quality, and with moderate cycle times. The key factor that determined results of various PCR is primers, but various adjustments for PCR operation possess of accessorial effects only.