Method for in vitro Recombination
The present invention relates to an in vitro method, using isolated protein reagents, for joining two double-stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest.
1 . A kit for in vitro joining a plurality of dsDNA molecules, where for each pair of dsDNA molecules to be joined, a distal region of a first DNA molecule and a proximal region of a second DNA molecule share a region of overlapping sequence homology, the kit comprising:
(a) an isolated enzyme having a 3′ or 5′ exonuclease activity;
(b) an isolated non strand-displacing DNA polymerase;
(c) a ligase which is compatible with the polymerase; and
(d) a crowding agent.
2 . The kit of claim 1 wherein the exonuclease is selected from the group consisting of: T7 exonuclease, lambda exonuclease, RedA of lambda phage, and RecE of prophage.
3 . The kit of claim 1 wherein the DNA polymerase is selected from the group consisting of: T7 DNA polymerase, T4 DNA polymerase, Taq DNA polymerase, DNA polymerase I, Klenow DNA polymerase, Phi29 DNA polymerase, Pfu polymerase, VentR DNA polymerase and Deep VentR DNA polymerase.
4 . The kit of claim 1 wherein the ligase is selected from the group consisting of: Taq DNA ligase, T4 DNA ligase, and E. coli DNA ligase.
5 . The kit of claim 1 wherein the crowding agent is PEG.
6 . The kit of claim 1 , where (a), (b), (c) and (d) are in different containers.
7 . The kit of claim 1 further comprising a solution, or components for making the solution which, when combined with the exonuclease and the dsDNA molecules to be joined, comprises about 5% PEG and/or Tris buffer.
8 . The kit of claim 1 , wherein two or more of components (a), (b), (c) and (d) are in the same container.
9 . The kit of claim 1 , wherein
(a) the enzyme having an exonuclease activity is T4 DNA polymerase;
(b) the polymerase is Taq DNA polymerase; and
(c) the ligase is Taq DNA ligase and
(d) the crowding agent is PEG.
10 . The kit of claim 9 wherein the PEG ranges from PEG-200 to PEG-20000.
11 . A kit for in vitro joining a plurality of dsDNA molecules, comprising:
(a) a vessel containing purified T4 DNA polymerase;
(b) a protein that enhances annealing of single-stranded DNAs; and
(c) a ligase that is compatible with the polymerase.