Kinetics of the DNA polymerase pyrococcus kodakaraensis

Computational Biology and Chemistry

Whitney

et al. 2008

Self Cite

107

A theoretical analysis is presented with experimental confirmation to conclusively demonstrate the critical role that annealing plays in efficient PCR amplification of GC-rich templates. The analysis is focused on the annealing of primers at alternative binding sites (competitive annealing) and the main result is a quantitative expression of the efficiency (η) of annealing as a function of temperature (TA), annealing period tA) and template composition. The optimal efficiency lies in a narrow region of TA and tA for GC-rich templates and a much broader region for normal GC templates. To confirm the theoretical findings, the following genes have been PCR amplified from human cDNA template: ARX, and HBB (with 78.72% and 52.99% GC respectively). Theoretical results are in excellent agreement with the experimental findings. Optimum annealing times for GC-rich genes lie in the range of 3 to 6 seconds and depend on annealing temperature. Annealing times greater than 10 seconds yield smeared PCR amplified products. The non-GC-rich gene did not exhibit this sensitivity to annealing times. Theory and experimental results show that, shorter annealing times are not only sufficient but also necessary for efficient PCR amplification of GC-rich templates.

Section: Methodssupporting

confidence: 53%

“…The activity of Taq pol. has been used (Innis et al, 1988; Gelfand and White, 1990) although higher rates have been observed for KOD (Griep et al, 2006a, 2006b). The diffusion coefficient and characteristic length are calculated as D = 4 ×10 −11 m 2 / s and λ = 1.2 ×10 −8 m .…”

Section: Mathematical Model Of Annealingmentioning

confidence: 99%

A fundamental study of the PCR amplification of GC-rich DNA templates

Mamedov

Computational Biology and Chemistry

Whitney

et al. 2008

Self Cite

107

“…For a selected amplicon, the minimum elongation times have been measured at different elongation temperatures and dNTP pool compositions. The amplicon that has been used in the study is a 52% GC-rich, 2517-base pair fragment of pUC19 (Griep et al (2005b) …”

Section: Kinetic Parameter Valuesmentioning

confidence: 99%

A quantitative model of error accumulation during PCR amplification

Computational Biology and Chemistry

Theron

Nelson³

et al. 2006

Self Cite

The amplification of target DNA by the polymerase chain reaction (PCR) produces copies which may contain errors. Two sources of errors are associated with the PCR process: (1) editing errors that occur during DNA polymerase-catalyzed enzymatic copying and (2) errors due to DNA thermal damage. In this study a quantitative model of error frequencies is proposed and the role of reaction conditions is investigated. The errors which are ascribed to the polymerase depend on the efficiency of its editing function as well as the reaction conditions; specifically the temperature and the dNTP pool composition. Thermally induced errors stem mostly from three sources: A+G depurination, oxidative damage of guanine to 8-oxoG and cytosine deamination to uracil. The post-PCR modifications of sequences are primarily due to exposure of nucleic acids to elevated temperatures, especially if the DNA is in a single-stranded form. The proposed quantitative model predicts the accumulation of errors over the course of a PCR cycle. Thermal damage contributes significantly to the total errors; therefore consideration must be given to thermal management of the PCR process.

“…The parameters that do not change are: D i = 1×10 5 copies, β = 5, k C = 15 (μM s) −1 (Mamedov et al, 2008), η d = 1, η dE = 0.99, l ext =400 nt, P i = 6×10 12 copies - i.e. 10 picomole, reaction volume is 25 μL (Griep et al, 2006) and the maximum cycle number is 40. We use the simple form of the overall efficiency, i.e.…”

Section: Resultsmentioning

confidence: 99%

Efficiency of the polymerase chain reaction

Booth

Chemical Engineering Science

TerMaat

et al. 2010

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.