Figure 5.

Primer annealing is slow but not rate limiting while enzyme binding is fast. (A) Schematic of reaction to measure primer annealing kinetics. The hairpin template HT-1 is shown in black and the FAM-LR primer is shown in blue. Bst-LF polymerase is shown in grey surface representation from pdbid: 7k5t. (B) Time course of primer annealing, monitored by extension of LR. A solution of 0.1, 0.2, 0.3 or 0.4 μM HT-1 and 0.2, 0.4, 0.6, 0.8 μM Bst-LF polymerase was mixed with 50 nM FAM-LR and 400 μM dATP, dCTP and dTTP to start the reaction. At various times the reaction was quenched with EDTA and products were analyzed by capillary electrophoresis. Each concentration of template DNA is shown as a different color of data points from red at the lowest to yellow at the highest concentration. Data at each concentration are shown fit to a single exponential function. (C) Observed rate of primer annealing versus concentration of template DNA. The rates are shown fit to a linear function, giving a second order rate constant for primer binding of approximately 0.2 μM⁻¹ s⁻¹. (D) Schematic of reaction to measure polymerase binding to annealed primer/template kinetics. The hairpin template annealed to the FAM-LR primer are shown in black and blue, respectively. Bst-LF polymerase is shown in grey surface representation from pdbid: 7k5t. (E) Time course of polymerase binding to primer/template monitored by primer extension. A solution of 0.1–0.4 μM Bst-LF polymerase was mixed with 50 nM HT-1/FAM-LR and 400 μM dATP, dCTP and dGTP to start the reaction. At various times the reaction was quenched with EDTA and products were analyzed by capillary electrophoresis. Data at a single enzyme concentration are shown since the time course of product formation does not change with increasing enzyme concentration on this timescale. Data are shown fit to a single exponential burst equation.