Figure 4.

Primer extension reactions. Pfu-Pol was used to extend a Cy5-labelled primer (24 bases in length) annealed to different templates (all 44 bases long) that place uracil (U), hypoxanthine (H), a stable abasic site (Ab), difuorobenzene (diFBz) and fluorobenzimidazole (FBzIm) either 6 (+6) or 10 (+10) bases ahead of the primer-template junction). Controls use a template that contains only canonical DNA bases, resulting in full extension of the primer. U and H stall replication 3-4 bases prior to encounter, resulting in truncated products (such stalling products are highlighted with a vertical arrow). The abasic site also gives shortened products but these are longer than those seen with U and H as, here, the polymerase stops directly at the abasic site. The positions of the starting primer and fully extended primer are shown with the horizontal arrows at the sides of the gel, labeled S and P, respectively. (A) Results seen with diFBz after 15 minutes; a stalled product (arrowed) is seen with diFBz+10 but not with diFBz+6. (B) Results found with diFBz after 2 minutes; here a stalled product (arrowed) can be seen with diFBz+6, although the main product runs with that produced by the abasic site. (C) Results seen with FBzIm after 15 minutes; no evidence of stalling is visible, rather extended products seen with this isostere correspond to those produced by the abasic site. Similar results were found at shorter times.