Abstract for presentation at 11th International Congress of Human Genetics

A quatitative model of errer accumulation during PCR amplification

  • Magda Theron, Department of Human Genetics, University of the Free State and NHLS, Bloemfontein, South Africa
  • Dr Nerina van der Merwe, Department of Human Genetics, NHLS, South Africa
  • Miss Elsje Pienaar, Department of Chemical Engineering, University of Nebraska, Lincoln, NE, United States
  • Mr Michael Nelson, Megabase Research Products, Lincoln, NE, United States
  • Prof Hendrik Viljoen, Department of Chemical Engineering, University of Nebraska, Lincoln, NE, United States

    • The amplification of target DNA by the polymerase chain reaction (PCR) produces copies which may contain errors. Two sourcesd 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 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.

    Conference Organiser - ICMS Pty Ltd