Figure 5.

Effect of Mn⁺² on deoxynucleotidylation and oligonucleotide elongation. (A) The effect of Mn⁺². Left panel. The template used was the TO89. The reactions shown in lanes 1, 2 and 3, contained only [α³²P]-dCTP precursor. The others were supplemented with the three non-radioactive dNTPs (1 μM each; lanes 4 and 5) or dGTP (‘dG’), dATP (‘dA’) and ddTTP (‘ddT’; lane 6). The reactions in lanes 2 and 5, Mg²⁺ (7 μM) was substituted with Mn²⁺ (1 μM), and that in lane 3 contained no divalent metal ions. Right panel. The same samples were heat denatured before electrophoresis. The different forms of products (1, 2, 3, 4, x) present in the gels are marked. (B) The effect of ddCTP. ddCTP (‘ddC’) was added in different (0, 20 and 330 nM) in the presence of Mg⁺² (lanes 1–3) or Mn²⁺ (lanes 4–6). No non-radioactive dGTP, dATP or dTTP (‘dT’) was added. (C) The predicted secondary structure formed by TO89. The nt at the 5′ end of TO89 and TO84 are indicated by the arrows.