Fig 3. EvaGreen qPCR analysis using KlenTaq DNA polymerase expressing cellular reagents.

Indicated copies of synthetic Chlamydia trachomatis DNA template were amplified by PCR using 0.2 μL of pure commercial KlenTaq DNA polymerase (panels a, b, and c) or 2 x 10⁷ cells of KlenTaq cellular reagents (panels d, e, and f). Amplicon accumulation was assessed in real time by measuring increase in EvaGreen fluorescence. Panels a and d depict representative amplification curves generated using the “Abs quant” analysis in the LightCycler 96 software. Colors of the curve traces indicate starting numbers of template copies–black: 6x10⁶ template copies; blue: 6x10⁵ template copies; red; 6x10⁴ template copies; green: 6x10³ template copies; pink: 600 template copies; purple: 60 template copies; yellow: 6 template copies; and cyan: no templates. Taken together, these curves demonstrate the real-time kinetics of PCR amplification. Since EvaGreen is a non-specific DNA intercalating dye, the fidelity of amplicon generation was verified by determining their melting temperatures (panels b and e) using the “Tm calling” analysis protocol in the LightCycler 96 software. Color coding of the curves is the same as in panels a and d. The overlapping melting temperature peaks of amplicons generated from 6 x 10⁶ to 60 copies of templates are indicative of correctly amplified PCR products. Amplification curves observed in the presence of 0 to 6 template copies are non-specific as evident from their different melting temperatures peaks of these amplicons. Standard curve analyses performed using the “Abs quant” protocol in the LightCycler 96 software are depicted in panels c and f, respectively, and data for amplification efficiency, linearity, and error are tabulated as insets.