Direct dsRNA preparation by promoter-free RCT and RNase H cleavage using one circular dsDNA template with a mismatched bubble

Bubble size and sequence greatly affect the pfRCT efficiency. (A) Sequence information for bubbles (red letters). The direction of the upper sequence from left to right is 5′ → 3′. Other regions of the template are the same with T-B15 shown in Figure 1B. (B) Results of pfRCT. Samples were visualized on an 8% denaturing PAGE (8 M urea). Lanes 1,3,5,7,9,11,13, and 15: dsDNA templates only; lanes 2,4,6,8,10,12,14, and 16: pfRCT results; lane 17: marker of dsDNA.