Figure 4.

HPLC analysis of nucleotides incorporation into poly(dG)–poly(dC). (A) Size-dependent HPLC separation of the products of polymerase synthesis. Polymerase extension assay was performed as described in Materials and Methods with 0.2 μM (dG)₁₀–(dC)₁₀ and 20 μg/ml of Klenow exo⁻ at 37°C. Polymerization reaction was started by addition of the enzyme. Aliquots of 50 μl were withdrawn from the assay mixture before (black curve) and 30 (red curve), 60 (green curve) and 120 (blue curve) min after the addition of the enzyme and loaded on TSKgel G-DNA-PW column (7.8 × 300 mm). Elution was performed with 20 mM Tris–Acetate buffer, pH 7.0, at a flow rate of 0.5 ml/min. (B) Anion-exchange HPLC separation of nucleotides. Nucleotide peaks from corresponding size-exclusion separation (A) were collected and loaded on an anion-exchange PolyWax LP column (4.6 × 200 mm). Elution was performed using a 30 min linear K-Pi, pH 7.4, gradient between 0.02 and 0.5 M in the presence of 10% acetonitrile at a flow rate of 0.8 ml/min. Elution was followed at 260 nm.