Table 3.
Influence of Target-Probe Flanking Sequence Mismatches on Hybridization for the Targets and Probes with Matching Codons: Reproducibility of Maximum Tm Across Triplicate Experiments
| Flanking sequence∗ |
Codon | Maximum Tm (replicate experiments), °C |
Mean, °C | SD, °C | CV, % | ||||
|---|---|---|---|---|---|---|---|---|---|
| Target | Probe | Target-probe mismatches, n | R1 | R2 | R3 | ||||
| 1 | 1 | 0 | AAA | 60.8 | 61.0 | 61.4 | 61.1 | 0.3 | 0.5 |
| 1 | 1 | 0 | AAC | 60.3 | 61.6 | 64.0 | 61.9 | 1.9 | 3.0 |
| 1 | 1 | 0 | AAG | 58.5 | 60.2 | 61.9 | 60.2 | 1.7 | 2.9 |
| 1 | 1 | 0 | AAT | 61.2 | 61.4 | 61.6 | 61.4 | 0.2 | 0.4 |
| 1 | 1 | 0 | AGA | 60.0 | 60.7 | 61.0 | 60.5 | 0.5 | 0.9 |
| 1 | 1 | 0 | AGC | 64.1 | 63.4 | 63.0 | 63.5 | 0.6 | 0.9 |
| 1 | 2 | 1 | AAA | 59.3 | 59.3 | 59.9 | 59.5 | 0.4 | 0.6 |
| 1 | 2 | 1 | AAC | 58.5 | 60.0 | 62.3 | 60.3 | 1.9 | 3.1 |
| 1 | 2 | 1 | AAG | 57.8 | 58.8 | 60.7 | 59.1 | 1.5 | 2.5 |
| 1 | 2 | 1 | AAT | 60.4 | 59.4 | 59.6 | 59.8 | 0.5 | 0.9 |
| 1 | 2 | 1 | AGA | 58.1 | 59.2 | 57.5 | 58.3 | 0.8 | 1.4 |
| 1 | 2 | 1 | AGC | 63.5 | 62.3 | 62.0 | 62.6 | 0.8 | 1.3 |
| 1 | 3 | 3 | AAA | 51.7 | 52.3 | 52.7 | 52.2 | 0.5 | 0.9 |
| 1 | 3 | 3 | AAC | 51.8 | 53.2 | 55.2 | 53.4 | 1.7 | 3.3 |
| 1 | 3 | 3 | AAG | 50.5 | 52.8 | 53.3 | 52.2 | 1.5 | 2.9 |
| 1 | 3 | 3 | AAT | 53.8 | 51.8 | 52.5 | 52.7 | 1.0 | 1.9 |
| 1 | 3 | 3 | AGA | 50.8 | 51.8 | 52.6 | 51.8 | 0.9 | 1.7 |
| 1 | 3 | 3 | AGC | 56.9 | 56.6 | 55.5 | 56.4 | 0.7 | 1.3 |
| 2 | 1 | 1 | AAA | 59.3 | 60.9 | 60.4 | 60.2 | 0.8 | 1.4 |
| 2 | 1 | 1 | AAC | 61.8 | 62.4 | 62.0 | 62.1 | 0.3 | 0.5 |
| 2 | 1 | 1 | AAG | 59.5 | 60.6 | 61.3 | 60.5 | 0.9 | 1.6 |
| 2 | 1 | 1 | AAT | 58.5 | 59.8 | 60.2 | 59.5 | 0.9 | 1.5 |
| 2 | 1 | 1 | AGA | 60.4 | 59.0 | 57.7 | 59.1 | 1.3 | 2.8 |
| 2 | 1 | 1 | AGC | 61.2 | 59.8 | 61.0 | 60.7 | 0.8 | 1.3 |
| 2 | 2 | 0 | AAA | 60.9 | 62.1 | 62.5 | 61.8 | 0.8 | 1.3 |
| 2 | 2 | 0 | AAC | 63.9 | 64.5 | 63.7 | 64.0 | 0.4 | 0.6 |
| 2 | 2 | 0 | AAG | 61.3 | 62.5 | 63.4 | 62.4 | 1.1 | 1.7 |
| 2 | 2 | 0 | AAT | 60.0 | 61.2 | 61.9 | 61.0 | 1.0 | 1.6 |
| 2 | 2 | 0 | AGA | 61.8 | 60.5 | 58.6 | 60.3 | 1.6 | 2.6 |
| 2 | 2 | 0 | AGC | 63.1 | 61.3 | 63.1 | 62.5 | 1.0 | 1.7 |
| 2 | 3 | 2 | AAA | 57.1 | 58.6 | 58.6 | 58.1 | 0.9 | 1.5 |
| 2 | 3 | 2 | AAC | 60.1 | 60.8 | 60.4 | 60.5 | 0.3 | 0.5 |
| 2 | 3 | 2 | AAG | 58.4 | 59.1 | 59.8 | 59.1 | 0.7 | 1.2 |
| 2 | 3 | 2 | AAT | 56.5 | 57.7 | 57.6 | 57.3 | 0.7 | 1.2 |
| 2 | 3 | 2 | AGA | 58.5 | 56.6 | 56.5 | 57.2 | 1.1 | 2.0 |
| 2 | 3 | 2 | AGC | 59.9 | 58.7 | 59.9 | 59.5 | 0.7 | 1.2 |
| 3 | 1 | 3 | AAA | 57.1 | 59.0 | 55.3 | 57.1 | 1.8 | 3.2 |
| 3 | 1 | 3 | AAC | 56.9 | 57.0 | 57.8 | 57.2 | 0.5 | 0.9 |
| 3 | 1 | 3 | AAG | 54.8 | 57.8 | 57.7 | 56.8 | 1.7 | 3.0 |
| 3 | 1 | 3 | AAT | 54.1 | 53.0 | 60.7 | 55.9 | 4.1 | 7.4 |
| 3 | 1 | 3 | AGA | 54.8 | 54.8 | 58.3 | 56.0 | 2.0 | 3.5 |
| 3 | 1 | 3 | AGC | 55.7 | 57.4 | 61.4 | 58.2 | 2.9 | 5.0 |
| 3 | 2 | 2 | AAA | 59.5 | 61.2 | 57.9 | 59.5 | 1.7 | 2.8 |
| 3 | 2 | 2 | AAC | 59.4 | 59.8 | 60.1 | 59.7 | 0.4 | 0.6 |
| 3 | 2 | 2 | AAG | 58.2 | 59.8 | 60.7 | 59.6 | 1.2 | 2.1 |
| 3 | 2 | 2 | AAT | 57.2 | 55.4 | 62.7 | 58.4 | 3.8 | 6.5 |
| 3 | 2 | 2 | AGA | 58.3 | 58.3 | 60.6 | 59.1 | 1.3 | 2.3 |
| 3 | 2 | 2 | AGC | 58.6 | 60.4 | 64.5 | 61.2 | 3.0 | 4.9 |
| 3 | 3 | 0 | AAA | 63.2 | 61.9 | 58.3 | 61.1 | 2.6 | 4.2 |
| 3 | 3 | 0 | AAC | 63.2 | 63.7 | 63.7 | 63.6 | 0.3 | 0.5 |
| 3 | 3 | 0 | AAG | 62.0 | 63.3 | 64.3 | 63.2 | 1.2 | 1.8 |
| 3 | 3 | 0 | AAT | 61.5 | 59.5 | 65.6 | 62.2 | 3.1 | 4.9 |
| 3 | 3 | 0 | AGA | 61.7 | 60.8 | 63.7 | 62.1 | 1.5 | 2.4 |
| 3 | 3 | 0 | AGC | 62.0 | 63.8 | 68.6 | 64.8 | 3.4 | 5.3 |
CV, coefficient of variation; Tm, melting temperature.
∗
In all cases maximum Tm occurred when the target codon matched the probe codon. The flanking sequence used for each of the three probe sets is shown in Table 1. Flanking sequence 1 differs from flanking sequence 2 by one nucleotide; flanking sequence 2 differs from flanking sequence 3 by two nucleotides; and flanking sequence 1 differs from flanking sequence 3 by three nucleotides.