Table 1. Changes in melting temperature (ΔTm) for DNA duplexes formed by TO-labelled ODN1–4 (A) and changes in fluorescence emission (Fds/Fss) before and after formation of matched and mismatched DNA duplexes (B). Tm, Fss, Fds were obtained in a buffer containing 10 mM phosphate 200 mM NaCl at pH 7.0. DNA concentrations for Tm studies were 3.0 μM of ODN1–4 and 3.3 μM of target strand. For Fds/Fss 0.25 μM of ODN1–4 and 0.28 μM of target were used. Tm values are an average of 4 measurements, fluorescence data were measured at least as a duplicate. TOQ/B6: λex = 484 nm, λem = 510 nm, slit ex = 7 nm, slit em = 7 nm at 20 °C.
| 1A | |||||||||||
| Sequence | ODN1 |
ODN2 |
ODN3 |
ODN4 |
Average ΔTm | Average ΔTm m–mm c | |||||
| dGCATX[combining low line]TTACG |
dGCAAX[combining low line]ATACG |
dGCACX[combining low line]CTACG |
dGCAGX[combining low line]GTACG |
||||||||
| X = | Duplex | ΔTm | ΔTm m–mm c | ΔTm | ΔTm m–mm c | ΔTm | ΔTm m–mm c | ΔTm | ΔTm m–mm c | ||
| PA-TOQ6 | Match a | 1.3 | 15.7 | 6.0 | 10.5 | 7.1 | 11.5 | 6.3 | 12.5 | 5.2 | 12.6 |
| Mismatch b | 2.7 | 9.8 | 9.4 | 10.5 | 8.1 | ||||||
| PA-TOB6 | Match a | 6.6 | 14.7 | 12.8 | 12.5 | 14.6 | 13.3 | 12.3 | 14.4 | 11.6 | 13.7 |
| Mismatch b | 9.0 | 14.6 | 15.1 | 14.6 | 13.3 | ||||||
| AE-TOQ6 | Match a | 7.7 | 11.9 | 10.8 | 12.8 | 9.3 | 8.6 | 9.5 | 12.7 | 9.3 | 11.5 |
| Mismatch b | 12.9 | 12.3 | 14.5 | 13.5 | 13.3 | ||||||
| AE-TOB6 | Match a | 6.3 | 11.2 | 11.3 | 12.9 | 7.9 | 10.3 | 8.9 | 12.0 | 8.6 | 11.6 |
| Mismatch b | 12.2 | 12.7 | 11.4 | 13.6 | 12.5 | ||||||
| C6-TOQ6 | Match a | 2.3 | 7.6 | 4.8 | 5.6 | 4.7 | 0.5 | 4.0 | 5.4 | 4.0 | 4.8 |
| Mismatch b | 11.8 | 13.5 | 18.0 | 15.3 | 14.7 | ||||||
| C6-TOB6 | Match a | 8.4 | 13.3 | 9.9 | 10.4 | 11.5 | 9.7 | 10.3 | 12.0 | 10.0 | 11.4 |
| Mismatch b | 12.2 | 13.8 | 15.6 | 15.0 | 14.2 | ||||||
| Average values | Match | 5.4 | 12.4 | 9.3 | 10.8 | 9.2 | 9.0 | 8.6 | 11.5 | ||
| Mismatch | 10.1 | 12.8 | 14.0 | 13.8 | |||||||
| T m of T* | Match | 41.6 | 17.1 | 39.6 | 14.3 | 47.6 | 13.8 | 49.1 | 16.7 | ||
| Mismatch | 24.5 | 25.3 | 33.8 | 32.4 | |||||||
| 1B | |||||||||||
| Sequence | ODN1 |
ODN2 |
ODN3 |
ODN4 |
Average Fds/Fss | Average Im/Imm | |||||
| dGCATX[combining low line]TTACG |
dGCAAX[combining low line]ATACG |
dGCACX[combining low line]CTACG |
dGCAGX[combining low line]GTACG |
||||||||
| X = | Duplex | F ds/Fss d | I m/Imm e | F ds/Fss | I m/Imm e | F ds/Fss | I m/Imm e | F ds/Fss | I m/Imm e | ||
| PA-TOQ6 | Match | 1.0 | 1.1 | 1.4 | 1.2 | 1.5 | 0.7 | 0.9 | 0.9 | 1.2 | 1.0 |
| Mismatch | 0.9 | 1.3 | 2.6 | 1.3 | 1.5 | ||||||
| PA-TOB6 | Match | 3.7 | 3.4 | 5.5 | 1.8 | 5.3 | 1.3 | 2.7 | 2.1 | 4.3 | 2.1 |
| Mismatch | 1.1 | 3.8 | 3.3 | 1.3 | 2.4 | ||||||
| AE-TOQ6 | Match | 2.8 | 2.0 | 7.4 | 2.7 | 4.1 | 3.5 | 1.3 | 2.1 | 3.9 | 2.6 |
| Mismatch | 1.5 | 3.2 | 1.5 | 0.6 | 1.7 | ||||||
| AE-TOB6 | Match | 0.8 | 0.9 | 1.0 | 0.8 | 1.9 | 1.4 | 0.8 | 1.5 | 1.1 | 1.1 |
| Mismatch | 0.9 | 1.2 | 1.3 | 0.6 | 1.0 | ||||||
| C6-TOQ6 | Match | 1.0 | 1.5 | 3.7 | 1.3 | 3.1 | 3.4 | 1.4 | 2.1 | 2.3 | 2.1 |
| Mismatch | 0.5 | 2.5 | 0.9 | 0.6 | 1.1 | ||||||
| C6-TOB6 | Match | 6.6 | 3.3 | 8.6 | 1.9 | 6.9 | 2.8 | 3.0 | 3.8 | 6.3 | 3.0 |
| Mismatch | 2.3 | 5.5 | 2.6 | 1.1 | 2.9 | ||||||
| Average values | Match | 2.7 | 2.0 | 4.6 | 1.6 | 3.8 | 2.2 | 1.7 | 2.1 | ||
| mismatch | 1.2 | 2.9 | 2.0 | 0.9 | |||||||
aIn comparison to the matched unmodified duplex.
bIn comparison to the mismatched unmodified duplex.
cDifference between Tm of matched (m) and mismatched (mm) target in °C, * = Tm value of unmodified matched and mismatched duplexes.
dRatio of integrated fluorescence intensity for Fds and Fss of ODN probes with matched or mismatched targets.
eRatio of fluorescence intensity of matched (Im) and mismatched (Imm) intensity of probes-targets duplexes at λem, max.