Figure 1.
Long-range primer extension reactions on normal and single-uracil templates. (a) Denaturing polyacrylamide gel showing products of reactions in which a 31-mer primer was extended against a 119-nucleotide template (see Methods) containing either a deoxyuridine (U) or a deoxythymidine (T) 23 nucleotides from the 5′ end. Reactions were performed with DNA polymerases from Pyrococcus furiosus (Pfu), Pyrococcus woesei (Pwo), and Thermus aquaticus (Taq). All three polymerases produce full-length products on T templates, but the archaeal enzymes produce smaller major products on U templates. (b) Polyacrylamide gel showing the position of the major premature termination product from the primer extension reaction against the U template with Pyrococcus furiosus DNA polymerase (Pfu exo+), relative to a sequencing ladder of the segment of pUC19 used in the long-range primer extension reaction (see Methods). The sequence for the template strand is given, reading 3′→5′ from the bottom (shorter primer extension products) to the top (longer primer extension products). The position of the deoxyuridine (U) is indicated. The major Pfu products correspond to termination of the polymerase reaction 4–6 bases upstream of the template deoxyuridine. The position of the full-length product obtained from primer-extension against the T template (not shown) was consistent with termination at the end of the 119-base template.