Booth Christine S, Pienaar Elsje, Termaat Joel R, Whitney Scott E, Louw Tobias M, Viljoen Hendrik J
Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln Lincoln, NE 68588-0643.
Chem Eng Sci. 2010 Sep 1;65(17):4996-5006. doi: 10.1016/j.ces.2010.05.046.
The polymerase chain reaction (PCR) has found wide application in biochemistry and molecular biology such as gene expression studies, mutation detection, forensic analysis and pathogen detection. Increasingly quantitative real time PCR is used to assess copy numbers from overall yield. In this study the yield is analyzed as a function of several processes: (1) thermal damage of the template and polymerase occurs during the denaturing step, (2) competition exists between primers and templates to either anneal or form dsDNA, (3) polymerase binding to annealed products (primer/ssDNA) to form ternary complexes and (4) extension of ternary complexes. Explicit expressions are provided for the efficiency of each process, therefore reaction conditions can be directly linked to the overall yield. Examples are provided where different processes play the yield-limiting role. The analysis will give researchers a unique understanding of the factors that control the reaction and will aid in the interpretation of experimental results.
聚合酶链反应（PCR）已在生物化学和分子生物学中得到广泛应用，如基因表达研究、突变检测、法医分析和病原体检测。越来越多地使用定量实时PCR来评估总产量中的拷贝数。在本研究中，产量被分析为几个过程的函数：（1）在变性步骤中模板和聚合酶发生热损伤，（2）引物和模板之间存在退火或形成双链DNA的竞争，（3）聚合酶与退火产物（引物/单链DNA）结合形成三元复合物，以及（4）三元复合物的延伸。为每个过程的效率提供了明确的表达式，因此反应条件可以直接与总产量相关联。给出了不同过程起产量限制作用的示例。该分析将使研究人员对控制反应的因素有独特的理解，并有助于解释实验结果。
