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 nucleic acid sequence comprising reacting said nucleic acid with two primers that are complementary to the flanking ends of said target nucleic acid sequence within a nucleic acid duplex, said reaction being carried out in the presence of a bacteriophage UvsX protein, bacteriophage UvsY protein, and a DNA polymerase having a proofreading/editing function, without previously denaturing said nucleic acid duplex, wherein said nucleic acid, primers and bacteriophase UvsX protein form a recombination intermediate and said polymerase combines with said recombination intermediate to form a polymerase complex.
2. The method according to claim 1 wherein said DNA polymerase is a gene product of a viral, bacteriophage, prokaryotic, or eukaryotic system.
3. The method according to claim 2 wherein said DNA polymerase is a gene product of bacteriophage T4.
4. The method according to claim 1 wherein said DNA polymerase is a holoenzyme complex.
5. The method according to claim 4 wherein said DNA polymerase holoenzyme complex is a gene product of a viral, bacteriophage, prokaryotic, or eukaryotic system.
6. The method according to claim 5 wherein said DNA polymerase holoenzyme complex comprises a polymerase enzyme, a clamp protein, and a clamp loader.
7. The method according to claim 5 wherein said DNA polymerase is a gene product of bacteriophage T4.
8. The method according to claim 1 wherein said method is carried out in the presence of a single stranded nucleic acid binding protein.
9. The method according to claim 1 wherein said method is carried out in the presence of a helicase.
10. The method according to claim 9 wherein said method is carried out in the presence of a helicase accessory factor.
11. The method according to claim 1 wherein said method is carried out in the presence of an ATP regeneration system.