Efficient pfRCT by using a dsDNA template with fewer mismatches and symmetric sequences. (A) Sequence of bubbles. The newly designed bubble (only five mismatches) in T-B15S is completely symmetric (palindromic). (B) Schematic illustration of pfRCT with the aid of a DNA Helper to improve the cleavage efficiency of RNase H. For T-B15S, only one 15-nt ssDNA Helper is added. For T-B15, two 15-nt DNA oligonucleotides with the same sequences as the bubble are required. Once the ssRNA is transcribed, it hybridizes with the ssDNA Helper and is cleaved by RNase H. Accordingly, the transcription and truncation proceed simultaneously without interference with each other. (C) Electrophoresis analysis (10% PAGE) of pfRCT products in the absence or presence of ssDNA Helpers. Lanes 1 and 4: dsDNA templates; lanes 2 and 5: pfRCT for T-B15/T-B15S without Helper; lanes 3 and 6: pfRCT for T-B15/T-B15S with Helper. Other conditions: [Template] = 50 nM, [each NTP] = 2 mM; [each Helper] = 1 µM.
