Efficiency of the polymerase chain reaction

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, quantitativerealtimePCRisusedtoassesscopynumbersfromoverallyield. In this study the yield is analyzed as a function of several processes: (1) thermal damage of the template and polymerase occurring during the denaturing step,(2) competition existing 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. (C) 2010 Elsevier Ltd. All rights reserved.