Figure 5. The dA-Ap (8) cross-link 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 60 min at 24 °C. After reaction work-up, the primer extension products were subjected to electrophoretic analysis on a 20% denaturing polyacrylamide gel. Lane 1 is the 15 nt, 5′-³²P-labeled primer, lane 2 is the 5′-³²P-labeled full-length extension product, lanes 3–7 depict the results of primer extension reactions on substrates O–S, and lane 8 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). The arrow corresponds to extension of the primer to the last base in the single-stranded region of the template.