Methods for nucleic acid manipulation
A method for replicating and amplifying a target nucleic acid sequence is described. A method of the invention involves the formation of a recombination intermediate without the prior denaturing of a nucleic acid duplex through the use of a recombination factor. The recombination intermediate is treated with a high fidelity polymerase to permit the replication and amplification of the target nucleic acid sequence. In preferred embodiments, the polymerase comprises a polymerase holoenzyme. In further preferred embodiments, the recombination factor is bacteriophage T4 UvsX protein or homologs from other species, and the polymerase holoenzyme comprises a polymerase enzyme, a clamp protein and a clamp loader protein, derived from viral, bacteriophage, prokaryotic, archaebacterial, or eukaryotic systems.
1. A composition for amplifying a target nucleic acid sequence comprising: (a) bacteriophage UvsX protein; (b) two primers that are complementary to the ends of said target nucleic acid sequence, said primers present in a molar excess relative to said target nucleic acid sequence; (c) a polymerase; and (d) nucleotides in an amount sufficient to support amplification of said target nucleic acid sequence.
2. A composition according to claim 1 , further comprising a UvsX protein accessory factor.
3. A composition according to claim 1 , wherein said polymerase is a polymerase holoenzyme.
4. A composition according to claim 3 , wherein said polymerase holoenzyme comprises a polymerase enzyme, a clamp protein, and a clamp loader protein.
5. A composition according to claim 1 , further comprising a single stranded nucleic acid binding protein.
6. A composition according to claim 1 , further comprising a helicase.
7. A method for amplifying a target nucleic acid comprising contacting said target nucleic acid sequence with the composition of claim 1 .
8. A composition for amplifying a target nucleic acid sequence comprising: (a) bacteriophage UvsX protein; (b) two primers that are complementary to the ends of said target nucleic acid sequence; (c) a prokaryotic polymerase; and (d) nucleotides in an amount sufficient to support amplification of said target nucleic acid sequence.
9. A composition according to claim 8 , further comprising a UvsX protein accessory factor.
10. A composition according to claim 8 , wherein said polymerase is a polymerase holoenzyme.
11. A composition according to claim 10 , wherein said polymerase holoenzyme comprises a polymerase enzyme, a clamp protein, and a clamp loader protein.
12. A composition according to claim 8 , further comprising a single stranded nucleic acid binding protein.
13. A composition according to claim 8 , further comprising a helicase.
14. A method for amplifying a target nucleic acid comprising contacting said target nucleic acid sequence with the composition of claim 8 .
15. A composition for amplifying a target nucleic acid sequence comprising: (a) bacteriophage UvsX protein; (b) a UvsX protein accessory factor; (c) two primers that are complementary to the ends of said target nucleic acid sequence; (c) a polymerase, and (d) nucleotides in an amount sufficient to support amplification of said target nucleic acid sequence.
16. A composition for amplifying a target DNA sequence comprising: (a) bacteriophage UvsX protein; (b) two primers that are complementary to the ends of said target nucleic acid sequence; and (c) a prokaryotic polymerase.
17. A composition according to claim 16 , further comprising a UvsX protein accessory factor.
18. A composition according to claim 16 , wherein said polymerase is a polymerase holoenzyme.
19. A composition according to claim 18 , wherein said polymerase holoenzyme comprises a polymerase enzyme, a clamp protein, and a clamp loader protein.
20. A composition according to claim 16 , further comprising a single stranded nucleic acid binding protein.
21. A composition according to claim 16 , further comprising a helicase.
22. A method for amplifying a target nucleic acid comprising contacting said target nucleic acid sequence with the composition of claim 16 .