Homologous recombination-based DNA cloning methods and compositions

1. A method of cloning a donor DNA molecule into an acceptor vector at a predetermined location, the method comprising:

a) preparing an extended donor DNA molecule by adding to the 5′-end and the 3′-end of the donor DNA molecule a first sequence and a second sequence, respectively, wherein each of the first and second sequences, independently, is at least 12 nucleotides in length and is at least 90% identical to a first region and a second region of the acceptor vector, respectively;

b) providing a reaction mixture comprising:

i) the acceptor vector;

ii) the extended donor DNA molecule; and

iii) an enzyme cocktail comprising an exonuclease having only 3′- or 5′-exonucleolytic single-strand degradation activity, and a single-stranded DNA binding protein;

c) incubating the reaction mixture to obtain an intermediate product;

d) transforming a cell with the intermediate product to obtain a transformed cell; and

e) culturing the transformed cell under conditions to produce a recombinant DNA molecule comprising the donor DNA located between the first region and the second region.

2. The method according to claim 1 , wherein the acceptor vector is a circular vector or a linearized vector.

3. The method according to claim 2 , wherein the first region and the second region are present at the 3′-end and the 5-end of the linearized vector, respectively.

4. The method according to claim 1 , wherein the first and the second sequences are 100% identical to the first and the second regions, respectively.

5. The method according to claim 1 , wherein the extended donor DNA molecule is prepared by a polymerase chain reaction (PCR), using a first PCR primer comprising the first sequence and a second PCR primer comprising the complement of the second sequence.

6. The method according to claim 1 , wherein the exonuclease is selected from the group consisting of Escherichia coli exonuclease I, Escherichia coli exonuclease III, Escherichia coli exonuclease VII, bacteriophage lambda exonuclease, and bacteriophage T7-exonuclease Gene 6, or a combination thereof.

7. The method according to claim 1 , wherein the single-stranded DNA binding protein is selected from the group consisting of extreme thermostable single-strand DNA binding protein (ET SSB), RecA, T4 Gene 32 Protein, Thermus thermophilus RecA (Tth RecA) and Escherichia coli single-strand DNA binding protein (SSB), or a combination thereof.

8. The method according to claim 7 , wherein the enzyme cocktail comprises a combination selected from the group consisting of Escherichia coli exonuclease I and RecA; Escherichia coli exonuclease III and RecA; Escherichia coli exonuclease VII and RecA; bacteriophage lambda exonuclease and RecA; bacteriophage T7-exonuclease Gene 6 III and RecA; Escherichia coli exonuclease I and Tth RecA; Escherichia coli exonuclease III and Tth RecA; Escherichia coli exonuclease VII and Tth RecA; bacteriophage lambda exonuclease and Tth RecA; bacteriophage T7-exonuclease Gene 6 III and Tth RecA; Escherichia coli exonuclease I and ET SSB; Escherichia coli exonuclease I and T4 Gene 32 Protein; Escherichia coli exonuclease I and Escherichia coli SSB; Escherichia coli exonuclease III and ET SSB; Escherichia coli exonuclease III and T4 Gene 32 Protein; Escherichia coli exonuclease III and Escherichia coli SSB; Escherichia coli exonuclease VII and ET SSB; Escherichia coli exonuclease VII and T4 Gene 32 Protein; and Escherichia coli exonuclease VII and Escherichia coli SSB.

9. The method according to claim 1 , wherein the reaction mixture comprises about 1 to about 100 mg/l of the exonuclease and about 1 to about 100 mg/l of the single-stranded DNA binding protein.

10. The method according to claim 1 , wherein the reaction mixture further comprises about 1 to about 10 g/l of tris(hydroxymethyl)aminomethane (Tris), about 0.1 to about 10 g/l of NaCl, about 0.1 to about 10 g/l of ethylenediaminetetraacetic acid (EDTA), about 0.1 to about 10 g/l of MgCl 2 , about 10 to about 200 g/l of glycerol, about 10 to about 50 g/l of bovine serum albumin (BSA), about 0.1 to about 10 g/l of adenosine-5′-triphosphate (ATP), about 0.1 to about 10 g/l of dithiothreitol (DTT), and wherein the pH of the reaction mixture is about 5.0 to about 9.0.

11. The method according to claim 1 , wherein in step (c), the incubation is conducted at a temperature of about 10° C. to about 38° C. for about 15 minutes to about 60 minutes.

12. The method according to claim 1 , wherein the cell is an Escherichia coli cell and the acceptor vector is a plasmid comprising an origin of replication that directs replication of the plasmid in the Escherichia coli cell.

13. The method according to claim 1 , wherein the transformed cell expresses a marker gene from the recombinant DNA molecule that allows the selection or screening of the transformed cell.