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 T4 UvsX protein
(b) two primers that are complementary to the flanking ends of said target nucleic acid sequence, and
(c) a polymerase.
2. A composition according to claim 1 , further comprising bacteriophage T4 UvsY protein.
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 nucleotide triphosphates in amounts sufficient to permit amplification of said target nucleic acid sequence.
6. A composition according to claim 1 , further comprising said target nucleic acid sequence.
7. A composition according to claim 1 , further comprising bacteriophage T4 gene product 32 protein.
8. A composition according to claim 1 , wherein said primers are present in a molar excess relative to said target nucleic acid sequence.
9. A method for amplifying a target nucleic acid comprising contacting said target nucleic acid sequence with the composition of claim 1 .
10. A composition according to claim 1 , further comprising a helicase.
11. A composition according to claim 10 , wherein said helicase is bacteriophage T4 gene product 41 DNA helicase.
12. A composition according to claim 10 , wherein said helicase is bacteriophage T4 replicative helicase complex, comprising bacteriophage T4 gene product 41 and gene product 59.
13. A composition for amplifying a target nucleic acid sequence comprising:
(a) bacteriophage T4 UvsX protein
(b) two primers that are complementary to the flanking ends of said target nucleic acid sequence
(c) a polymerase, and
(d) a helicase.