Since polymerase stop assays used to quantify DNA damage assume that single lesions are sufficient to block polymerase progression, to test the effect of specific lesions on PCR amplification efficiency, the authors amplified synthetic 90 base oligonucleotides containing normal or modified DNA bases using real-time PCR and determined the relative threshold cycle amplification efficiency of each template.
The authors found that although the amplification efficiencies of templates containing a single 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) were not significantly perturbed, the presence of a single 8-oxo-7,8-dihydro-2′-deoxyadenosine, abasic site, or a cis–syn thymidine dimer dramatically reduced amplification efficiency. In addition, while templates containing two 8-oxodGs separated by 13 bases amplified as well as the unmodified template, the presence of two tandem 8-oxodGs substantially hindered amplification. From these findings, the authors conclude that the reduction in polymerase progression is dependent on the type of damage and the relative position of lesions within the template. (Published abstract provided)