TABLE 2.
Effects of modifying Taq1 sequence on affinity for Taq polymerase
| Taq1 modifications | Sequencea | Fold decrease in affinityb |
|---|---|---|
| Taq1 (no modification) | 5′-gcctgcaggtcgactctagaCCCAGTCCACACTAAAGCATA | NA |
| 3′-cggacgtccagctgagatctGGGTCAGGTGTGATTTCGTATCTGT | ||
| Random | 5′-gcctgcaggtcgactctagaNNNNNNNNNNNNNNNNNNNNN | >5 |
| 3′-cggacgtccagctgagatctNNNNNNNNNNNNNNNNNNNNNNNNN | ||
| Modified T3Pcore | 5′-gcctgcaggtcgactctagaCCCAGTCCACCAGCCCTCATA | >5 |
| 3′-cggacgtccagctgagatctGGGTCAGGTGGTCGGGAGTATCTGT | ||
| Modified sequences surrounding the T3Pcore | 5′-gcctgcaggtcgactctagaAAACTGAACAACTAAAGCATA | 2 |
| 3′-cggacgtccagctgagatctTTTGACTTGTTGATTTCGTATCTGT | ||
| Full T3 RNAP promoter sequence | 5′-gcctgcaggtcgactctagaAATTAACCCTCACTAAAGGGAG | >5 |
| 3′-cggacgtccagctgagatctTTAATTGGGAGTGATTTCCCTCTGTC | ||
| Modification of sequences 5′ of the T2Pcore | 5′-gcctgcaggtcgactctagaAAACTGAACAACTAAAGCATA | 2 |
| 3′-cggacgtccagctgagatctTTTGACTTGTTGATTTCGTATCTGT | ||
| 4-bp modification A | 5′-gcctgcaggtcgactctagaCCCAGTCCAACAGAAAGCATA | 1 |
| 3′-cggacgtccagctgagatctGGGTCAGGTTGTCTTTCGTATCTGT | ||
| 4-bp modification B | 5′-gcctgcaggtcgactctagaCCCAGTCCACACTCCCTCATA | 3 |
| 3′-cggacgtccagctgagatctGGGTCAGGTGTGAGGGAGTATCTGT | ||
| −1 shift | 5′-cctgcaggtcgactctagaCCCAGTCCACACTAAAGCATAG | >5 |
| 3′-ggacgtccagctgagatctGGGTCAGGTGTGATTTCGTATCTGTC | ||
| T3Pcore to T7Pcore | 5′-gcctgcaggtcgactctagaCCCAGTCCACACTATAGCATA | >5 |
| 3′-cggacgtccagctgagatctGGGTCAGGTGTGATATCGTATCTGT |
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 Taq1 sequence, while the region of Taq1 corresponding to the T3 RNAP core is underlined in Taq1.
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 relative to the Taq1 sequence (measured Kd,app,gelshift, 36 ± 6 nM; averages from at least 3 experiments ± SD), with higher numbers indicating fold lower affinity. NA, not applicable (reference sequence).