(451d) A Quantitative Model of Error Accumulation during Pcr Amplification with Application to Gene Synthesis
AIChE Annual Meeting
2006
2006 Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Metabolic Engineering & Systems Biology Poster Session
Wednesday, November 15, 2006 - 3:15pm to 5:45pm
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. Below melting temperatures, the DNA molecules are mostly in the double stranded form, hence the hydrolytic attack on the DNA bases are sterically hindered. Two hydrolytic damage reactions are prominent at elevated temperatures; C deamination and A+G depurination. The rate constants of these thermal damage reactions follow Arrhenius kinetics, hence the rates increase rapidly with temperature.
The theory is successfully applied to the assembly of synthetic genes. Specifically, the theory is used to minimize errors during the synthesis of the eight genes of the influenza A virus. Results are presented on the synthesis of the important haemagglutinin and neuraminidase genes and the challenges to build the longer PA, PB1 and PB2 genes. The presentation concludes with results on packaging the genes into virus-like particles.