This dissertation reports on research that examined the assumption behind polymerase stop assays and their use in quantifying DNA damage, that single lesions are sufficient to block thermostable DNA polymerase progression.
This author of this dissertation describes research that used 90 base oligonucleotides containing normal or modified DNA bases which were amplified using real-time polymerase chain reactions (PCR), in order to test the assumption that single lesions are sufficient to block thermostable DNA polymerase progression. The author reports that data implied that PCR efficiency was influenced to differing degrees depending on which base lesion was present on the input oligonucleotide. The author also reports that the presence of two tandem 8-oxodGs substantially hindered amplification when compared with two 8-oxodGs separated by 13 bases which indicated that the position of lesions also influenced the PCR. The author used novel mathematical formulae, developed to report differences in exponential amplification as rates of damage bypass, to quantify variations in amplification. To determine the validity of those novel formulae, the author used DNA samples quantified previously using current polymerase stop methods. Results from the author’s research enabled the adaptation of real-time PCR for DNA damage quantification, identified DNA base legions as potential PCR mutagens, and has provided the basis for further refinement of polymerase stop assays as research and clinical tools to monitor DNA damage and repair.