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 contacting said target DNA sequence with bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA sequence, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence; wherein said primers are present in a molar excess relative to said target DNA sequence; and, detecting the presence of amplified target DNA sequence.
2. The method of claim 1 , wherein said polymerase is a prokaryotic polymerase.
3. The method of claim 1 , wherein said method is carried out in the presence of f an ATP regeneration system.
4. The method of claim 1 , wherein said method is carried out in the presence of a single stranded nucleic acid binding protein.
5. The method of claim 1 , wherein said method is carried out in the presence of of bacteriophage UvsY protein.
6. A method for amplifying a target DNA sequence comprising contacting said target DNA sequence with bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA sequence, a DNA polymerase having proofreading activity, and nucleotides in an amount sufficient to support amplification of said target DNA sequence; wherein said primers are present in a molar excess relative to said target DNA sequence; and, detecting the presence of amplified target DNA sequence.
7. The method of claim 6 , wherein said polymerase is a prokaryotic polymerase.
8. The method of claim 6 , wherein said method is carried out in the presence of an ATP regeneration system.
9. The method of claim 6 , wherein said method is carried out in the presence of a single stranded nucleic acid binding protein.
10. The composition of claim 6 , further comprising the addition of bacteriophage UvsY protein.