Figure 1. A diagram of the Hot Fusion process for single fragment cloning.

Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand DNA molecules. Phusion DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.