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 system for replicating a target nucleic acid sequence within a nucleic acid molecule, the system comprising:
a reaction vessel containing a reaction mixture, wherein the reaction mixture includes:
(a) a target nucleic acid molecule including a target nucleic acid sequence;
(b) a UvsX recombination factor;
(c) two primers that bind to the flanking ends of the target nucleic acid sequence;
(d) a DNA polymerase; and
(e) nucleotides in an amount sufficient to support replication of the target nucleic acid sequence.
2. The system according to claim 1 , wherein the system further includes a detector configured to detect the product produced via replication of the target nucleic acid sequence.
3. The system according to claim 2 , wherein the detector is configured to detect fluorescence.
4. The system according to claim 1 wherein said DNA polymerase is a gene product of a viral, bacteriophage, prokaryotic, or eukaryotic system.
5. The system according to claim 1 wherein said DNA polymerase is a holoenzyme complex.
6. The system according to claim 1 wherein said uvsX recombination factor is a gene product of a bacteriophage.
7. The system according to claim 1 , wherein said DNA polymerase is a product of a viral, bacteriophage, prokaryotic, or eukaryotic system.
8. The system according to claim 1 wherein the reaction vessel also includes a single stranded nucleic acid binding protein.
9. The system according to claim 1 wherein the reaction vessel also includes a helicase.
10. The system according to claim 1 wherein the reaction vessel also includes an accessory factor configured to stabilize the UvsX recombination factor.
11. The system according to claim 1 wherein the reaction vessel also includes an ATP regeneration system.
12. The system according to claim 1 , wherein the target nucleic acid molecule is double stranded.
13. The system according to claim 1 , wherein the target nucleic acid sequence is embedded within the longer target nucleic acid molecule.