Efficient and rapid method for assembling and cloning double-stranded DNA fragments

1. A composition for joining a first set of double-stranded (ds) DNA molecules, the composition comprises:

(a) an isolated enzyme that exhibits a 3′ or 5′ exonuclease activity;

(b) a non strand-displacing DNA polymerase;

(c) a DNA ligase that is compatible with the DNA polymerase in (b); and

(d) a chaperone agent,

wherein the chaperone agent is selected from the group consisting of glycerol, sorbitol and betaine, wherein if the chaperone agent is glycerol, then a final concentration of glycerol from about 6 to 8.5% w/v is present in a final mixture comprising the first set of double-stranded (ds) DNA molecules and the components of the composition.

2. The composition of claim 1 , wherein the isolated enzyme that exhibits a 3′ or 5′ exonuclease activity comprises T4 DNA polymerase.

3. The composition of claim 1 , wherein the non strand-displacing DNA polymerase comprises Taq DNA polymerase.

4. The composition of claim 1 , wherein the DNA ligase comprises Taq DNA ligase.

5. The composition of claim 1 , wherein the chaperone agent is glycerol.

6. The composition of claim 1 , wherein the chaperone agent is sorbitol or betaine.

7. A kit for joining a first set of double-stranded (ds) DNA molecules, the kit comprises:

(a) an isolated enzyme having a 3 or 5 exonuclease activity;

(b) an isolated non strand-displacing DNA polymerase;

(c) an isolated ligase that is compatible with the isolated non strand-displacing polymerase of (b); and

(d) a reagent solution comprising a chaperone agent,

wherein the chaperone agent is selected from the group consisting of glycerol, sorbitol and betaine, wherein if the chaperone agent is glycerol, then a final concentration of glycerol from about 6 to 8.5% w/v is present in a final mixture comprising the first set of double-stranded (ds) DNA molecules and the components of the kit.

8. The kit of claim 7 , wherein the isolated enzyme that exhibits a 3′ or 5′ exonuclease activity comprises T4 DNA polymerase.

9. The kit of claim 7 , wherein the isolated non strand-displacing DNA polymerase comprises Taq DNA polymerase.

10. The kit of claim 7 , wherein the isolated ligase comprises Taq DNA ligase.

11. The kit of claim 7 , wherein the chaperone agent is glycerol.

12. The kit of claim 7 , wherein the chaperone agent is sorbitol or betaine.

13. The kit of claim 7 , wherein the reagent solution comprises a concentrated amount of the chaperone agent sufficient to enhance joining the first set of double-stranded (ds) DNA molecules in a final mixture comprising the first set of double-stranded (ds) DNA molecules and the components of the kit.

14. The kit of claim 13 , wherein the concentrated amount of the chaperone agent comprises an amount of sorbitol to provide a final concentration from about 0.25 M to about 1.50 M in the final mixture comprising the first set of double-stranded (ds) DNA molecules and the components of the kit.

15. The kit of claim 13 , wherein the reagent solution further comprises a concentrated amount of Tris pH7.5 buffer sufficient to support joining the first set of double-stranded (ds) DNA molecules in a final mixture comprising the first set of double-stranded (ds) DNA molecules and the components of the kit.

16. The kit of claim 7 , further comprising instructions.

17. The composition of claim 6 , wherein the composition provides a final concentration of sorbitol or betaine at least 0.25M in a final mixture comprising the first set of double-stranded (ds) DNA molecules and the components of the composition.

18. The kit of claim 12 , wherein the composition provides a final concentration of sorbitol or betaine at least 0.25M in a final mixture comprising the first set of double-stranded (ds) DNA molecules and the components of the kit present in a composition for joining a first set of double-stranded (ds) DNA molecules.