TABLE 3.
Effects of modifying Taq2 sequence on affinity for Taq polymerase
| Taq2 modification | Sequencea | Fold decrease in affinityb |
|---|---|---|
| Taq2 | 5′-gcctgcaggtcgactctagaCCCCAATTTGCGAACGTGCCT | NA |
| 3′-cggacgtccagctgagatctGGGGTTAAACGCTTGCACGGACAGC | ||
| Random | 5′-gcctgcaggtcgactctagaNNNNNNNNNNNNNNNNNNNNN | >30 |
| 3′-cggacgtccagctgagatctNNNNNNNNNNNNNNNNNNNNNNNNN | ||
| Modified 8-bp core | 5′-gcctgcaggtcgactctagaCCCCAATTTGCGCCATGTAAT | 8 |
| 3′-cggacgtccagctgagatctGGGGTTAAACGCGGTACATTACAGC | ||
| Modified noncore nucleotides | 5′-gcctgcaggtcgactctagaAAAACCGGGTATAACGTGCCG | >30 |
| 3′-cggacgtccagctgagatctTTTTGGCCCATATTGCACGGCACTA | ||
| Modified noncore/nonoverhang nucleotides | 5′-gcctgcaggtcgactctagaAAAACCGGGTATAACGTGCCT | 7 |
| 3′-cggacgtccagctgagatctTTTTGGCCCATATTGCACGGACAGC | ||
| −1 shift | 5′-cctgcaggtcgactctagaCCCCAATTTGCGAACGTGCCTG | >30 |
| 3′-ggacgtccagctgagatctGGGGTTAAACGCTTGCACGGACAGCC | ||
| 4-bp modification A | 5′-gcctgcaggtcgactctagaCCCCAATTTGCGCCATTGCCT | >30 |
| 3′-cggacgtccagctgagatctGGGTCAGGTTGTCTTTCGTATCAGC | ||
| 4-bp modification B | 5′-gcctgcaggtcgactctagaCCCCAATTTGCGAACGGTAAT | >30 |
| 3′-cggacgtccagctgagatctGGGTCAGGTGTGAGGGAGTATCAGC |
Lowercase letters are bases derived from the fixed region of the starting material, while bases derived from the random region are capitalized. “N” signifies that the base was random in the starting material (either A, T, C, or G), while “N” is the complement of that base in the template strand. Underlined regions highlight changes from the Taq2 sequence, while the region of Taq2 shared by all 3 sequences recovered from SELEX (Fig. 2) is underlined in Taq2.
Affinities measured in gel shift assays were measured with 2 nM primer-template and under the conditions described in Materials and Methods. The derived Kd values were useful for comparisons between these sequences but were not determined under conditions that would necessarily yield accurate Kd values. Therefore, they are referred to as apparent Kd values that are specific for the gel shift experiment (Kd,app,gelshift). Values listed are approximate changes in affinity compared with the Taq2 sequence (measured Kd,app,gelshift, 6.3 ± 0.1 nM; averages from at least 3 experiments ± SD), with higher numbers indicating fold lower affinity. NA, not applicable (reference sequence).