FAQ: How can I reduce nonspecific or non-template amplification?
Isothermal amplification reactions can be extremely rapid and robust, but commonly exhibit nonspecific amplification. This can show up as false positives and must be controlled for reliable diagnostic amplification. Design and optimization of primers for methods such as LAMP are typically the most effective method for improving specificity, but other reaction parameters can also be optimized. For example, reaction temperature can be used to improve reaction efficiency and specificity; reactions using Bst, Large Fragment likely have an optimum temperature of approximately 65°C, but a range of 50–66°C can be used for optimization. For Bst 2.0, this range extends to 72°C depending on application, and the additional increase in temperature may prove beneficial in improving specificity. Similarly, we recommend 4 U–8 U of polymerase in most reactions (25 µL volume), but for robust applications, such as LAMP and SDA, this can be lowered to 0.5 U in cases with rapid amplification but high nonspecific activity. We recommend titrating down the amount of polymerase for these situations.
An alternative source of nonspecific amplification occurs during reaction setup. DNA polymerases exhibit low- or room-temperature primer extension activity on improperly annealed primers and extendable primer secondary structures. Specificity for PCR applications has been addressed by the creation of “Hot Start” DNA polymerases, where activity is suppressed until reactions are heated to the denaturation temperature of PCR reactions. For isothermal applications, these temperatures would denature the enzymes, thus NEB developed a “Warm Start” version of Bst 2.0. All polymerase activity is inhibited below 45°C, enabling setup at room temperature or in warm environments without ice. When reactions are heated for amplification, the WarmStart® aptamer releases from Bst 2.0 and amplification can proceed without any loss of efficiency. For high-throughput, diagnostic, and field applications of isothermal amplification, we recommend Bst 2.0 WarmStart® for improved specificity and reproducibility.