Fig 4. Effect of betaine, recombinase and nucleotide addition on LAMP amplification.

(A) Different combinations of RecA and betaine were used in LAMP reactions containing 1 ng target DNA and 1 mM ATP and the time needed for each sample to reach the fluorescence threshold of 0.5 (T_threshold) in real-time LAMP was determined (n = 10). (B) RecA and/or ATP were added into reactions containing 0.4 M betaine. Reactions without RecA and ATP were used as control (n = 3). “+” and “-” below each bar indicated the present and absent of the component respectively. (C) Additional 1 mM ATP, dATP, dTTP, dCTP or dGTP was added into reactions containing 0.4 M betaine. Reactions without additional ATP and dNTP were used as controls. (D) Different amounts of ATP were added into reactions containing 0.4 M betaine (n = 3). To investigate interplay between dNTPs and Mg²⁺, 4.8 mM (dark blue), 8.8 mM (red) or 12.8 mM (green) total dNTPs was added into reactions containing 0.4 M betaine and either (E) 8 mM Mg²⁺ or (F) 10 mM Mg²⁺ respectively. (G) Two sets of reactions containing 0.4 M betaine and either 0 mM (black) or 2 mM (grey) ATP were prepared. Additional swarm and/or loop primers along with standard LAMP primers were added into each set of reactions. Standard LAMP primers alone were used as controls (n = 4). All T_threshold data was analysed using one-way ANOVA with a post-hoc Tukey’s multiple comparison of means test (p value < 0.05). Error bars represent standard deviation.