Error rate of PCR by DNA polymerase in the presence of UDG and/or endo IVa.

aThe fidelity of DNA replication during PCR supplied with UDG and/or EndoIV was measured using a previously described assay [29]. Briefly, a 1.9-kb sequence encoding lacIOZα was PCR -amplified using primers containing 5′EcoRI restriction sites and a 1-ng lacIOZα target. The PCR mixtures (50 µL) were subjected to predenaturation of 5 min at 95°C and 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 3 min, followed by a 5-min extension at 72°C. The amplified fragments were digested with EcoRI, purified by gel electrophoresis, and ligated into λgt10 arms. The ligation reactions were packaged, and the λ phage was used to infect an α-complementing Escherichia coli host strain. Aliquots of infected cells were plated on duplicate LB plates with top agar containing either X-gal (1 mg/mL) or X-gal plus IPTG (1.5 mM).bTemplate doublings (d) are determined using the equation 2d = (amount of PCR product)/(amount of starting target).cMutant frequencies (mf) are determined by dividing the total number of blue plaques (lacI– mutants) on the X-gal plates by the total number of plaques containing a functional lacZα sequence (blue plaques on X-gal plus IPTG plates).dError rates are calculated using the equation ER = mf/(bp·d), where mf is the mutation frequency, bp is the number of detectable sites in lacI ( = 349), and d is the number of template doublings.