Figure 5.

Prospective study. Optimized Bi-PASA reactions for single-base changes in the factor IX, FV-opp, and D1 genes. (M) Size standard (120 ng of φX174/HaeIII). (A) Optimation of the FIX gene Bi-PASA. Bi-PASA reactions shown were performed with a WT/M sample. PQ segment T_m = 73.6°C. The annealing temperature was set at 55°C. (Lanes 1–4 PCR with each P and Q primer at 0.1, 0.05, 0.025, and 0.0125 μm concentrations. Lane 2 was designated as the minimum optimal concentration for the PQ segment. (Lanes 5–10) Control reactions with P and Q primers at 0.05 μm concentrations and A and B primers at 0.1 μm concentrations. (Lanes 11–16) Final Bi-PASA reaction for the FIX gene after adjustment of the A primer concentration to 0.05 μm. (B) Bi-PASA reaction of FV-opp and D1 genes. In the initial experiments using the FV gene, the WT and M allele-specific primers were in the upstream and downstream directions, respectively. As a test of the robustness of the Bi-PASA reaction, new primers were designed such that the WT and M allele-specific primers were in the downstream and upstream directions, respectively. WT/WT, M/M, and WT/M templates were used for optimized Bi-PASA reactions for the FV-opp and D1 genes. The T_m values of the PQ segments of the FV-opp and D1 genes were 74.8°C and 81.4°C, respectively, and the annealing temperatures were 55°C and 60°C, respectively. Final primer concentrations for the FV-opp Bi-PASA reaction were 0.1 μm for the P primer, 0.05 μm for the Q primer, 0.1 μm for the B primer, and 0.1 μm for the A primer. Final primer concentrations for the D1 Bi-PASA reaction were 0.1 μm for the P primer, 0.1 μm for the Q primer, 0.1 μm for the B primer, and 0.1 μm for the A primer. (M) Size standard (120 ng of φX174/HaeIII). (Lanes 1–4) Bi-PASA reaction for FV-opp; (lanes 5–8) bi-PASA for D1.