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 method for amplifying a target DNA sequence comprising:
forming, within a reaction vessel, a reaction mixture comprising said target DNA sequence, a bacteriophage UvsX protein, a DNA polymerase, two primers that bind to the flanking ends of said target DNA sequence, and a buffer system compatible with said bacteriophage UvsX protein and the DNA polymerase; and
detecting the presence of said amplified target DNA sequence.
2. The method of claim 1 , wherein said DNA polymerase is a prokaryotic polymerase.
3. The method of claim 1 , wherein said primers are present in a molar excess relative to said target DNA sequence.
4. The method of claim 1 , wherein said reaction mixture further comprises an ATP regeneration system.
5. The method of claim 1 , wherein said reaction mixture further comprises a single stranded nucleic acid binding protein.
6. The method of claim 1 , wherein said reaction mixture further comprises a bacteriophage UvsY protein.
7. The method of claim 1 , wherein said reaction mixture further comprises nucleotides in an amount sufficient to support amplification of said target DNA sequence.
8. A method for amplifying a target DNA sequence comprising:
forming, within a reaction vessel, a reaction mixture comprising said target DNA sequence, a bacteriophage UvsX protein, a DNA polymerase having proofreading activity, two primers that bind to the flanking ends of said target DNA sequence, and a buffer system compatible with said bacteriophage UvsX protein and the DNA polymerase; and
detecting the presence of said amplified target DNA sequence.
9. The method of claim 8 , wherein said DNA polymerase is a prokaryotic polymerase.
10. The method of claim 8 , wherein said primers are present in a molar excess relative to said target DNA sequence.
11. The method of claim 8 , wherein said reaction mixture further comprises an ATP regeneration system.
12. The method of claim 8 , wherein said reaction mixture further comprises a single stranded nucleic acid binding protein.
13. The method of claim 8 , wherein said reaction mixture further comprises a bacteriophage UvsY protein.
14. The method of claim 8 , wherein said reaction mixture further comprises nucleotides in an amount sufficient to support amplification of said target DNA sequence.