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. In combination:
(1) a composition for use in 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; and,
(2) a detection system for detecting the presence of said amplified target nucleic acid sequence.
2. The combination of claim 1 , said composition further comprising bacteriophage T4 UvsY protein.
3. The combination of claim 1 , wherein said polymerase is a polymerase holoenzyme.
4. The combination of claim 1 wherein said polymerase holoenzyme comprises a polymerase enzyme, a clamp protein, and a clamp loader protein.
5. The combination of claim 1 , said composition further comprising nucleotide triphosphates in amounts sufficient to permit amplification of said target nucleic acid sequence.
6. The combination of claim 1 , said composition further comprising said target nucleic acid sequence.
7. The combination of claim 1 , said composition further comprising bacteriophage T4 gene product 32 protein.
8. The combination of claim 7 , wherein said primers are present in a molar excess relative to said target nucleic acid sequence.
9. The combination of claim 1 , said composition further comprising a helicase.
10. The combination of claim 9 , wherein said helicase is bacteriophage T4 gene product 41 DNA helicase.
11. The combination of claim 9 wherein said helicase is bacteriophage T4 replicative helicase complex comprising bacteriophage T4 gene product 41 and gene product 59.