Figure 3.

The chemically-stable, reduced dG-Ap cross-link 5 blocks primer extension by ϕ29 DNA polymerase. The ³²P-labeled primers were extended by incubation of the DNA substrates with ϕ29 DNA polymerase (10 units) and the four dNTPs (1 mM in each) in Tris-HCl (50 mM, pH 7.5), MgCl₂ (10 mM), (NH₄)₂SO₄ (10 mM), DTT (4 mM), and bovine serum albumin (0.1 mg/mL) for 30 min at 24 °C. After reaction work-up, the primer extension products were analyzed by electrophoresis on a 20% denaturing polyacrylamide gel. Lane 1 is an iron-EDTA cleavage reaction on a synthetic standard of the full-length extension product (5′-³²P-GAT CAC AGT GAG TAC AAT AGA ATA GAT GAA CTA AGA CAT ATA), lane 2 is the 15 nt, 5′-³²P-labeled primer, lane 3 is the 5′-³²P-labeled full-length extension product, and lanes 4–8 depict the results of primer extension on templates C–G. The arrow corresponds to extension of the primer to the last base in the single-stranded region of the template. The slight difference in gel mobility observed for the iron-EDTA cleavage products and the primer extension products reflects the fact that the cleavage products generated by iron-EDTA possess 3′-phosphate termini,²⁶ while the primer extension products possess 3′-hydroxyl termini.