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. Author manuscript; available in PMC: 2009 Sep 15.
Published in final edited form as: Anal Biochem. 2008 May 20;380(2):164–173. doi: 10.1016/j.ab.2008.05.018

Figure 4.

Figure 4

Optimization of designs for real-time PCR. (A) Optimized thrombin-sensing conformation-switching aptamer sequences. ThrA3 and ThrA7 were designed based on ThrX3 and Thr7, respectively, as described in the text. Arrows indicate sequence changes. (B) Activation of ligation in the presence of thrombin. Ligation assays were carried out as described in Figure S1, except that the ligation time in the presence of 500 nM thrombin was varied between 20min and 70min. (C) Amplified ligation signals with different primer sets. Ligations were carried out for 20min with ThrA7 in the presence or absence of thrombin. An aliquot from the ligation reaction in the presence of 500 nM thrombin was used for real-time PCR with various combinations of the primers t.F1, t.F2, t.R1, and t.R2. The Ct difference between amplification in the presence and absence of thrombin (BSA alone) is indicated. Error bars were derived from three determinations.