A quantitative model of error accumulation during PCR amplification.
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.
Pienaar
E
E
Department of Chemical Engineering, University of Nebraska, Lincoln, NE 68588-0643, USA.
Theron
M
M
Nelson
M
M
Viljoen
H J
HJ
D001483:Base Sequence; D019295:Computational Biology; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D007700:Kinetics; D015233:Models, Statistical; D008969:Molecular Sequence Data; D009010:Monte Carlo Method; D016133:Polymerase Chain Reaction / Q000379:methods / Q000706:statistics & numerical data*; D013816:Thermodynamics
D004247:DNA; D004259:DNA-Directed DNA Polymerase