Open Access. Powered by Scholars. Published by Universities.®
Articles 1 - 1 of 1
Full-Text Articles in Medical Cell Biology
Effect Of Dna Damage On Pcr Amplification Efficiency With The Relative Threshold Cycle Method, Jan Sikorsky, Donald Primerano, Terry Fenger, James Denvir
Effect Of Dna Damage On Pcr Amplification Efficiency With The Relative Threshold Cycle Method, Jan Sikorsky, Donald Primerano, Terry Fenger, James Denvir
Donald A Primerano
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 …