In vitro recombination methods using 5' exonuclease

A method of generating a recombinant DNA molecule comprising:

• contacting a 5′ exonuclease and at least two DNA molecules comprising a first linear double stranded DNA molecule and a second linear double stranded DNA molecule at a first incubation temperature and for a first incubation time sufficient to create a 3′ overhang on the first DNA molecule and the second DNA molecule, wherein
• the first DNA molecule and the second DNA molecule are substantially homologous at terminal ends for at least about 15 up to about 50 bases;
• inactivating the 5′ exonuclease at a second incubation temperature for a second incubation time sufficient to inactivate the 5′ exonuclease without substantially denaturing the at least two DNA molecules;
• annealing homologous ends of the first DNA molecule and the second DNA molecule at a third incubation temperature for a third incubation time sufficient to form an annealed complex;
• and
• contacting the annealed complex, at least one thermostable polymerase, and a plurality of dNTPs during the third incubation time to allow extension of a 3′ end of the first DNA molecule or the second DNA molecule, thereby filling a gap corresponding to exonuclease removal, to form a substantially non-gapped annealed complex;
• wherein the substantially non-gapped annealed complex is capable of transformation into a host cell without further in vitro processing.