In vitro recombination method

An in vitro method, using isolated proteins, for joining two double strand (ds) DNA molecules of interest, comprising:

• providing a first dsDNA molecule and a second dsDNA molecule which share a region of sequence identity at a terminal end on each DNA molecule,
• contacting the two dsDNA molecules with:
• (a) a purified 5′ exonuclease;
• (b) a purified DNA polymerase;
• and
• (c) a purified ligase,
• under conditions whereby:
• a 3′ single-stranded overhang is generated in each molecule by the exonuclease without the use of a restriction enzyme;
• the two single-stranded overhangs anneal to form a gapped molecule;
• the gaps are filled in by the polymerase;
• and
• nicks are sealed by the ligase, thereby joining the molecules and forming a substantially intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained, wherein none of the enzymatic reactions is actively terminated prior to beginning another of the reactions.