(516m) A Theoretical Assessment of Polymerase Fidelity and Thermal Damage during Pcr Amplification

The in vitro amplification of target DNA by the polymerase chain reaction produces thermally damaged copies. 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. Viljoen et al., (2005) and Griep et al., (2006) developed a macroscopic model of PCR kinetics which is based on the probabilistic kinetic approach described by Ninio (1987) . The principle idea of Ninio's approach is to track a single enzyme/template complex over time and to determine its average behavior. The main results of the analysis macrokinetics model are expressions for the average extension rate and the error frequency. 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. Since many polymerases fail to bypass apurinic or cytosine deaminated sites, thermal damage has serious affects on overall yields, especially in the case of long targets. A quantitative model of thermal damage is presented to determine the amount of depurination and deamination that occurs during amplification. The reaction rates of depurination and deamination differ for double stranded DNA and single stranded DNA. Therefore the analysis includes a model for dsDNA/ssDNA transition. It is shown that thermal damage is not symmetric; a distinction is made between the accruement of damage on the template and extending strands. Error frequencies do not scale linearly with target length and damage increases nonlinearly with progressively longer targets. It is concluded that, unless a polymerase is used that can process DNA with apurinic sites, and in lieu of repair enzyme activity, yields could decrease to less than 0.04% of the theoretical values for the 10 kb targets that have been investigated. The time DNA is exposed to elevated temperatures is the most critical factor in thermal damage, and proper thermal management should be part of a carefully designed experiment.