USER cloning definition

The uracil-excision based cloning, or USER, is a method to clone DNA fragments into linearized vectors to make a stable hybridization product without using ligation enzymes (Figure 1). The method relies on the creation of 3' complementary overhangs generated on PCR fragments by placing a single uracil residue in each primer used to amplify the target DNA. The resulting PCR products are then treated with specific enzymes and cohesive ends anneal at designed melting temperatures.

Figure 1. USER cloning overview.

In addition to inserting DNA fragments into linearized vectors, this method is also used to introduce any type of mutations (insertions, deletions, mismatches) into genes or vectors, as well as to perform combinatorial assembly.

The main advantage of the USER cloning is its simplicity. There is minimal handling, there is no need for using ligation enzymes, and primer design is easier than other DNA assembly techniques as the overlap sequence can be shorter.