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.
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