Figure 3.

In silico simulations for probe set selection. A: Distribution of predicted melting temperature (T_m) for 81,126 sequences with three 34-bp probe sets (PS1, PS2, and PS3). Sequences included the 13,521 distinct low- and middle-income country 203-bp database sequences that encompassed codon 103 each with a different one of the six reported codons at position 103 (AAA, AAC, AAG, AAT, AGA, AGC). For each of the 81,126 (13,521 × 6) sequences, max_T_m_PS1-3 indicated the maximum T_m of PS1, PS2, and PS3. The temperature used for initial hybridization was 55°C. Dashed lines are at 55°C, the temperature at which binding is considered necessary for probe capture. B: Distribution of predicted ΔT_m between probes complementary to AAC, AAT, and AGC versus probes complementary to AAA for each of the three probe sets (PS1, PS2, and PS3). Because the probes within a probe set shared the same flanking sequence, the ΔT_m was caused entirely by the central mismatch at the codon 103 position. Dashed lines are at a ΔT_m of 2°C, the minimum temperature considered necessary for distinguishing between the finding of two different targets to the same probe.