Figure 3.

Replacement of dGTP with dGDPNP leads to short Okazaki fragments followed by large gaps. An optimized rolling circle reaction with 20 mM template was carried out in the presence of 100 µM dGTP or the indicated concentrations of dGDPNP. Products were extracted with phenol–chloroform, precipitated by isopropanol and incubated with 100 µM dNTPs, 0.2 U Phusion polymerase and α-[³²P] dATP (12 000 cpm/pmol) at 72°C for 15 min. The products before and after the gap fill assay were monitored by alkaline agarose gel electrophoresis.