Examining the TSO annealing specificity and its relationship with template switching efficiency. (A) Outline of the experimental design that uses two sets of template switching reactions. As shown in the figure, in the first set of reactions (Scheme 1), a DNA/RNA duplex with 3′-d(AAA) overhang was used in combination with RNA TSOs of varying 3′ nt sequences. In the second set (Scheme 2), a blunt-ended DNA/RNA duplex was used in combination with the same set of TSOs. (B) Electrophoretic analysis of the template-switching products generated using the DNA/RNA duplex with 3′-d(AAA) overhang and (C) the blunt-ended DNA/RNA duplex. The template switching efficiency (TS%) is the average of three repeated experiments for each reaction shown in the figures.
