| Use hot start |
| Use TD PCR (enhances specificity and sensitivity) |
| Optimize primer design |
| ↓ Mg++ |
| ↓ dNTP (also favors higher fidelity) |
| Optimize pH |
| ↓ Taq polymerase
|
| ↓ Cycle segment lengths |
| ↓ Number of cycles |
| ↑ Annealing temperature |
| ↓ Inhibitors |
| ↑ Ramp speed |
| Add and optimize enhancer(s) |
| ↓ Primer concentration |
| ↓ Primer degeneracy |
| ↑ Template denaturation efficiency |
|
| Adjusting conditions in the direction opposite that listed above usually favors increased sensitivity (i.e., more product)
and the concomitant risk of non-specific amplification. The aim is to strike a balance between these two opposing tendencies.
↑ and ↓ signify increase and decrease, respectively.
|
