Peak scanner software version 1

A capillary electrophoresis-based assay was used to determine the polymerase incorporation activity of each PolD (33 (link)). A primer-template was prepared by annealing a 50mer 5′-FAM primer (5 μM) (5′-FAM-AGT GAA TTC GAG CTC GGT ACC CGG GGA TCC TCT AGA GTC GAC CTG CAG GT-3′) to a 67mer template (6.25 μM) (5′-AAG CAC GAA AGC AGG GTG CCT GCA GGT CGA CTC TAG AGG ATC CCC GGG TAC CGA GCT CGA ATT CAC T-3′) in 1× annealing buffer (20 mM Tris–HCl, 100 mM NaCl, pH 7.5) by heating to 95°C for 3 min followed by cooling to room temperature. 1 μl of each polymerase was added to 20 μl of 1× Thermopol buffer (20 mM Tris–HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1% Triton X-100, pH 8.8 at 25 °C) in a 96-well plate and six -fold serial dilutions in 1× Thermopol buffer were performed. An equal volume of 20 nM DNA (10 nM final), 2  μM dNTPs (1  μM final) was added to each PolD and the reactions were incubated at 65°C for 30 min followed by quenching with equal volume of 50 mM EDTA. Reaction products were separated by capillary electrophoresis using a 3730xl Genetic Analyzer (Applied Biosystems) and fluorescent peaks were analyzed using Peak Scanner software version 1.0 (Applied Biosystems). All assays were performed in triplicate to ensure experiment reproducibility.