Figure 3.

Time course of short-range primer extension and effect of an abasic site in the template. (a) Denaturing polyacrylamide gels showing the short-range primer extension products produced by Pfu (exo⁻) on a template containing a single uracil positioned 10 bases from the end of the primer-template duplex region. The time of the incubation was varied as shown and in all cases a truncated product is produced. The first lane (primer) contains just the primer strand as a marker. The second lane (no dU) shows the maximum length 44-mer product. (b) Denaturing polyacrylamide gels showing the short-range primer extension products produced by Pfu (exo⁻) on a template containing either a stable abasic site (Ab+10) or a single uracil (dU+10) positioned 10 bases from the end of the primer-template duplex region. The first two lanes contain markers 24 (primer size) and 34 bases in length. With Ab+10 two terminated products, 35 and 36 bases long, are produced. These correspond to incorporation of a dNTP at the abasic site followed by the random non-template-directed addition of two further nucleotides. As the polymerase used lacks a functional 3′-5′ exonuclease, these nontemplate additions persist, so that the full-length product is longer than the 44-mer (marker, and small amount of full-length 44-mer produced in the dU+10 case). With the dU+10 template, the terminated product is shorter, clearly indicating a stop preceding the dU rather than in the vicinity of the dU.