Table 1. DNA sequencing properties of Pfu–Pol mutants.
| Category of enzyme | Mutant | Activity (%) | ddNTP:dNTP ratio optimised for DNA sequencing ladders | Fold improvement |
|---|---|---|---|---|
| Wild-type | Wild-type | 100 | 30:1 | |
| Alterations to highly | Q484A | <1 | Low activity | |
| conserved amino acids | K488A | 27 | Worse than wild-type | |
| in P-helix (shown in | N492Y | <1 | Low activity | |
| green in Figs 1 and 2) | N492H/K/G | ~40 (all) | Worse than wild-type | |
| Y495/I/D/C/S | n.d. | Similar to wild-type | ||
| Insertions into P-helixa | A(Y)N and TIN(Y)GVL | <1 (both) | Low activity | |
| Alterations to other | L479Y/W/P | 33/25/28 | Similar to wild-type | |
| amino acids in | A486Y | 26 | 1:5 | 150 |
| P-helix (see Fig. 2) | A486W | 20 | Worse than wild-type | |
| L490W | 123 | 1:3 | 90 | |
| L490Y | 78 | 5:1 | 6 | |
| S493Y | 100 | Similar to wild-type | ||
| F494Y/C/S/T/V | ~50 (all) | Similar to wild-type | ||
| G496P/S/A | 80/45/47 | Similar to wild-type | ||
| Y497F | 51 | Worse than wild-type | ||
| Y497W | 46 | 10:1 | 3 | |
| Y497A | 40 | 5:1 | 6 | |
| Alterations to Y410 | Y410A/F | 33/47 | Worse than wild-type | |
| Alterations to loop | Q472H | 82 | 5:1 | 6 |
| preceding the P-helix | ||||
| (see Fig. 2) | ||||
| Multiple mutations | Q472H-N492H | 100 | 5:1 | 6 |
| A486Y-L490W | 40 | 1:5 | 150 | |
| Q472H-A486Y-N492H | 25 | Worse than A486Y | ||
| Q472H-L490W-N492H | 43 | Worse than L490W |
N492H/K/G/Y indicates that N492 was changed to H, K, G and Y etc.
aA(Y)N has a Y inserted between A491 and N492 of the wild-type enzyme. In TIN(Y)GVL this sequence replaces the amino acids between 489 and 494 (LLANSF) in the wild-type enzyme. Activity was determined using the ‘activated calf thymus’ method (33) and the activity seen with the wild-type set to 100. The wild-type enzyme gives readable DNA sequencing gels at a 30:1 ddNTP:dNTP ratio, but unreadable gels at 5:1. Mutants described as similar to wild type have this property; mutants noted as worse give unreadable gels at the 30:1 ratio. For mutants with improved discrimination the number reported is the lowest ratio of ddNTP:dNTP at which readable sequencing gels were obtained. In these cases the ‘fold improvement’ (defined as [ddNTP/dNTP]wild type/[ddNTP:dNTP]mutant) is also given.