Fig. 2
Screening approach and selection for identifying Taq pol variants with increased RT-PCR activity (A) Principle of the screening assay. RT and amplification of the cDNA is catalysed by different Taq pol variants. The applied TaqMan probe is 5’- fluorescently labelled with FAM (6-carboxyfluorescein) and 3’- labelled with BHQ1 (Black Hole Quencher 1). Product formation can be followed by monitoring the increase in fluorescence signal as the probe is digested by the Taq pol variants and fluorescence is no longer quenched by BHQ1. (B) Selection of promising Taq pol variants are shown as an example for the first expression plate (amplification curve for lysate containing the RT-Taq depicted in red and amplification curve for lysates containing different Taq pol variants depicted in blue). Selection of promising variants were based on lower Cq values than those of the RT-Taq (red curve) and amplification curves following a sigmoidal shape. (C) Frequency of each mutation (L459M, N483K, E507K, S515R, V586G, I614K, I638F and M747K) is depicted in active vs. inactive Taq pol variants. Left: 15 different Taq pol variants were included in the group of active variants and right: 8 Taq pol variants were included in the group of inactive variants. (B) Mutation level (0-fold, 1-fold, 2-fold…) is plotted against their frequency. Left: 15 different Taq pol variants were included in the group of active variants and right: 8 Taq pol variants were included in the group of inactive variants.
