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. 2013 Nov 13;42(3):1747–1756. doi: 10.1093/nar/gkt1098

Figure 4.

Figure 4.

The availability of primers signals the lagging strand polymerase to cycle. (A) Optimized rolling circle reactions were carried out as described except 120 nM synthetic 15-mer primers substituted for primase and GTP, CTP and UTP. ddGTP was added to the designated concentrations at the same time radiolabeled nucleotide was added. Okazaki fragments incorporating α-[32P] dGTP (5000 cpm/pmol) were monitored by alkaline agarose gel electrophoresis. Lengths were determined with a 20-kb cutoff to exclude leading strand products. (B) In the presence or absence of 4 µM ddGTP, optimized rolling circle reactions were conducted as in (A) except α-[32P] dCTP was added at the same time with ATP and dNTPs. The 3 min elongation step in the presence of non-radioactive dNTPs before the addition of radioactive nucleotide was skipped so that the products would be short enough for accurate length quantification. Leading strand products incorporating α-[32P] dCTP (20 000 cpm/pmol) were monitored by alkaline agarose gel electrophoresis. The products of 20, 30 and 40 s were used to calculate the rate of leading strand synthesis. (C) The amount of lagging strand synthesis in the presence of 1 µM ddGTP was quantified.