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

a<p>The fidelity of DNA replication during PCR supplied with UDG and/or EndoIV was measured using a previously described assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027248#pone.0027248-Lundberg1" target="_blank">[29]</a>. Briefly, a 1.9-kb sequence encoding <i>lacIOZ</i>α was PCR -amplified using primers containing 5′<i>Eco</i>RI restriction sites and a 1-ng <i>lacIOZ</i>α 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 <i>Eco</i>RI, 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 <i>Escherichia coli</i> 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).</p>b<p>Template doublings (<i>d</i>) are determined using the equation 2<i>^d</i> = (amount of PCR product)/(amount of starting target).</p>c<p>Mutant frequencies (<i>mf</i>) are determined by dividing the total number of blue plaques (<i>lacI</i>– mutants) on the X-gal plates by the total number of plaques containing a functional <i>lacZ</i>α sequence (blue plaques on X-gal plus IPTG plates).</p>d<p>Error rates are calculated using the equation <i>ER</i> = <i>mf</i>/(<i>bp·d</i>), where <i>mf</i> is the mutation frequency, <i>bp</i> is the number of detectable sites in <i>lacI</i> ( = 349), and <i>d</i> is the number of template doublings.