Construction of a circular dsDNA template involving a bubble of mismatched duplex and the transcription results. (A) Construction strategy. After hybridization, the nicks are sealed by Taq DNA ligase. The topology of prepared circular dsDNA (T-B15) is controlled by changing the ligation temperature. (B) dPAGE analysis (8%, 8 M urea) of ligation. Lane 1, the short ssDNA AS-a and AS-b to construct C-AS. Lane 2: the circular ssDNA (C-AS). Lanes 4 and 6: the hybridization between C-AS and two complementary short ssDNA (S-a and S-b). The products were hybridized in the T4 Dnl buffer (lane 4) and Taq Dnl buffer (lane 6), respectively. Lanes 8 and 10: products obtained by sealing nicks with T4 Dnl (37°C) and Taq Dnl (65°C), respectively. Lanes 3,5,7,9, and 11: digestion of the products in lanes 2,4,6,8, and 10 using Exonuclease I and Exonuclease III. (C) dPAGE analysis (8%, 8 M urea) of transcription. Lane 1: T-B15; lane 2: conventional RCT (without RNase H). “Tr” refers to “Transcription”; lanes 3–5: pfRCT at various concentrations of RNase H; lane 6: the product in lane 5 was treated by ShortCut RNase III (digest dsRNA to short dsRNA fragments of 18–25 bp); lane 7: Ultralow range DNA Ladder (ULR). Transcription conditions: [T-B15 (prepared in 65°C)] = 50 nM, [T7 RNAP] = 2 U/µL, [RNase Inhibitor] = 2 U/µL, [NTP]each = 2 mM, 1× transcription buffer, 37°C for 4 h. For pfRCT, RNase H was present.
