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.
Article
Effect of DNA damage on PCR amplification efficiency with the relative threshold cycle method
Biochemistry and Microbiology
Document Type
Article
Publication Date
10-1-2004
Disciplines
Abstract
Citation Information
Sikorsky, J.A., Primerano, D.A., Fenger, T.W., and Denvir, J., Effect of DNA damage on PCR amplification efficiency with the relative threshold cycle method. Biochem. Biophys. Res. Commun., 2004, vol. 323, no. 3, pp. 823–830.
A definitive version was subsequently published in Sikorsky, J.A., Primerano, D.A., Fenger, T.W., and Denvir, J., Effect of DNA damage on PCR amplification efficiency with the relative threshold cycle method. Biochem. Biophys. Res. Commun., 2004, vol. 323, no. 3, pp. 823–830. http://dx.doi.org/10.1016/j.bbrc.2004.08.168,
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