TABLE 4.
Binding parameters at 23°C and 60°C for selected Taq sequences
| Assay temp and construct namea | Kd (pM)b | koff (min−1)b | t1/2 (min)c |
|---|---|---|---|
| 23°C | |||
| Control (starting material) | 14 ± 8 | 0.034 ± 0.004 | 20 ± 2 |
| Taq1 | ND | ND | >480 |
| Taq2 | ND | ND | >480 |
| 60°C | |||
| Control (starting material) | ND | 0.159 ± 0.056 | 4.4 ± 1.2 |
| Taq1 | ND | 0.015 ± 0.003 | 46 ± 8 |
| Taq2 | ND | 0.0013 ± 0.0002 | 533 ± 71 |
| Taq1-core mod | ND | 0.124 ± 0.020 | 5.6 ± 0.8 |
| Taq2 (−1 shift) | ND | 0.198 ± 0.157 | 3.5 ± 1.5 |
a
Refer to Tables 1, 2, and 3 for construct information.
b
koff and Kd values were calculated using filter binding assays as described in Materials and Methods. Results were averages from at least 3 experiments ± SD. Kd values for Taq1 and Taq2 at 23°C were too low to measure with the assay, while values could not be accurately measured at 60°C due to temperature fluctuations that occur during application to the filter of the sample and washing of the filters. ND, not determined.
c
Half-life values (t1/2) for the binding of Taq to the construct were calculated from koff using the equation t1/2 = 0.693/koff.