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. 2022 Jun 1;5:68. doi: 10.1038/s42004-022-00685-5

Fig. 1. Blocking enzymatic synthesis with a 3′-phosphorylated primer.

Fig. 1

Gel image (PAGE 20%) shows the result of the PEX reactions with 3′-phosphorylated primer P1 and template T1 with natural dNTPs and different DNA polymerases. Natural triphosphates were at a final concentration of 200 µM. The following quantities of polymerases and reaction conditions 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, 15 min and 30 min; Dpo4 (2 U), Deep Vent (2 U): 55 °C, 15 min and 30 min; Kf (exo−) (5 U): 37 °C, 15 min and 30 min. Negative control (T−): No polymerase added to the mixtures or reactions only with 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. P represents unreacted, 5′-FAM-labeled primer.