USER enzymes

There are three main enzymes involved in the USER cloning method:

• High-fidelity DNA polymerase: Amplifies the uracil-containing DNA in a PCR. Available enzymes in the market are genetically-improved DNA polymerases based on the Pfu DNA polymerase, which is originally found in Pyrococcus furiosus. This enzyme is used in USER cloning due to its superior thermal stability and proofreading properties, which ensures fewer errors in the PCR product than using other polymerases. Some examples are PfuTurbo CX Hotstart, PfuX7, or Phusion U Hotstart.

• Uracil DNA glycosylase: Cleaves the glycosidic bond between the uracil and the ribose. Its action leads to the removal of the uracil from the DNA strand due to the weakening of the hydrogen bonds between uracil and adenine.

• Endonuclease VIII: Recognizes the abasic site left after the action of the uracil DNA glycosylase, cleaving the phosphodiester bond on the DNA single strand. This promotes the removal of the upstream DNA sequence due to the weakening of the remaining hydrogen bonds, and consequently the generation of the 3' overhangs.