Fig. 9. Biochemical evaluation of nucleotide 15.

Gel image (PAGE 20%) shows the analysis of the products stemming from (A) PEX reactions with various DNA polymerases and the P1/T1 system and (B) TdT-mediated extension reactions with 5′-FAM-labeled primer P2. PEX reaction contained natural and modified triphosphates at 200 µM. The following quantities of polymerases were used: Phusion (2 U), Hemo Klen Taq (8 reactions), Taq (5 U), Bst (8 U), Q5 (2 U), Therminator (2 U), Vent (exo⁻) (2 U): 60 °C, 30 min; Dpo4 (2 U), Deep Vent (2 U): 55 °C, 30 min; Kf (exo⁻) (5 U): 37 °C, 30 min. Negative control (T−): No polymerase added to the mixtures or reactions with only dATP and dCTP or dATP, dCTP, and dTTP only. Positive control (T+): with all natural nucleotides and Taq polymerase. All reactions were incubated at adequate reaction temperatures for 1 h. TdT reaction mixtures contained TdT (10 U), triphosphate at various, given concentrations, Co²⁺ (0.25 mM) or Mn²⁺ (1 mM) cofactors, and were incubated at 37 °C for given reaction times. P represents unreacted, 5′-FAM-labeled primer.