title: Effect of DNA damage on PCR amplification efficiency with the relative threshold cycle method.
abstract: 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, we 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. We found that while 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, we conclude that the reduction in polymerase progression is dependent on the type of damage and the relative position of lesions within the template.
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25704, USA.
authors.forename: Jan A
authors.initials: JA
authors.lastname: Sikorsky
authors.forename: Donald A
authors.initials: DA
authors.lastname: Primerano
authors.forename: Terry W
authors.initials: TW
authors.lastname: Fenger
authors.forename: James
authors.initials: J
authors.lastname: Denvir
mesh_terms: D020137:Base Pair Mismatch; D001483:Base Sequence; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D004249:DNA Damage / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013329:Structure-Activity Relationship
chemical_list: D009841:Oligonucleotides