Confirmation of the structure of pfRCT product via digestion by RNase H with the help of various Helpers. (A) Illustration of the position of various Helpers. “Helper-Ab” and “Helper-Sb” have the same sequences with the bubble in “T-B15.” “Helper-Ac” and “Helper-Sc” have the same sequences with a part of the duplex in “T-B15” (sequence information and length are shown in Supplemental Table S1). (B) The digestion products were analyzed by 10% PAGE. Lane 1: T-B15 template; lane 2: RCT product without RNase H; lane 3: product in lane 2 was treated by DNase I (to remove T-B15); lane 4: product in lane 3 was treated by RNase H without any Helper; lanes 5–9: product in lane 3 was digested by RNase H with the help of various Helpers; lane 10: size marker. The reaction conditions are shown in Materials and Methods. (C) Transcription products (8% dPAGE with 8 M urea) of “T-pro” (a 118-bp circular dsDNA template with promoter sequence). Lane 1: T-Pro template; lane 2: products obtained by RCT; lanes 3–5, products obtained by pfRCT with/without the aid of Helper (1/0 µM). The reaction conditions are shown in Materials and Methods.
