Efficiency of the polymerase chain reaction

Abstract

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.

Enzymological description of multitemplate PCR—Shrinking amplification bias by optimizing the polymerase–template ratio

“…Several causes of bias in multitemplate PCR setups have been reported: competition between templates (Sykes et al, 1992), competition of complementary template strands with primers (Suzuki and Giovannoni, 1996), insufficient denaturation efficiency (Booth et al, 2010), primer mismatch, different primer annealing temperatures (Booth et al, 2010), different primer elongation efficiencies (Booth et al, 2010) (usually due to different amplicon length and different GC content: Baskaran et al, 1996;Arezi et al, 2003), polymerase error rates, and high cycle numbers (Patin et al, 2013).…”

Multi-template polymerase chain reaction

“…In practice this means that minor differences in composition or concentration of inhibitors as well as slight variations in other PCR components can drastically change the amplification efficiency of the same template. Thus, dissimilar amplification efficiencies of templates within a mixed sample may lead to a biased (and even false) outcome of the original sample composition; for a review see [46].…”

“…However, when double-stranded DNA molecules separate into single-stranded DNA under denaturing conditions, they become more susceptible to hydrolytic attack, oxidation and depurination and thus, increase the potential for polymerase-induced errors [26], [46], [57]. A second common type of DNA damage is spontaneous base release.…”

Experimental validation of a fundamental model for PCR efficiency

“…The cycle efficiency is the product of the individual efficiencies of the denaturing, annealing, polymerase binding and elongation steps (Booth et al 2010). As the reaction progresses the efficiency decreases resulting in the characteristic sigmoidal real-time curve (Kainz, 2000; Schnell, 1997; Schnell and Mendoza, 1997; Stolovitzky and Cecchi, 1996).…”

“… Recently a theoretical analysis of PCR efficiency has been published by Booth et al, (2010). The PCR yield is the product of three efficiencies: (i) the annealing efficiency is the fraction of templates that form binary complexes with primers during annealing, (ii)the polymerase binding efficiency is the fraction of binary complexes that bind to polymerase to form ternary complexes and (iii)the elongation efficiency is the fraction of ternary complexes that extend fully.…”