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 within a nucleic acid molecule, comprising:
providing a target nucleic acid molecule containing a target nucleic acid sequence;
forming, within a reaction vessel, a reaction mixture containing the target nucleic acid molecule, a UvsX recombination factor, two primers that bind to flanking ends of the target nucleic acid sequence, a polymerase, and nucleotides in an amount sufficient to support amplification of the target nucleic acid sequence; and
forming an amplification product by amplifying the target nucleic acid sequence within the reaction vessel.
2. The method according to claim 1 , further including contacting the amplification product with a fluorescent molecule.
3. The method according to claim 1 , wherein the method further includes detecting the amplification product.
4. The method according to claim 3 , further including contacting the amplification product with a fluorescent molecule.
5. The method according to claim 3 , wherein the detecting step includes detecting fluorescence.
6. The method according to claim 1 wherein said polymerase is a holoenzyme complex.
7. The method according to claim 6 , wherein said holoenzyme complex is a gene product of a viral, bacteriophage, prokaryotic, or eukaryotic system.
8. The method according to claim 1 wherein the reaction vessel also includes a single stranded nucleic acid binding protein.
9. The method according to claim 1 wherein the reaction vessel also includes a helicase.
10. The method according to claim 1 wherein the reaction vessel also includes an ATP regeneration system.
11. The method according to claim 1 , further including separating the amplification product from the reaction mixture.
12. The method according to claim 1 , further including analyzing the amplification product to confirm the identity of the target nucleic acid sequence.
13. The method according to claim 1 wherein the target nucleic acid molecule is double stranded.
14. The method according to claim 1 , wherein the target nucleic acid sequence is embedded within the longer target nucleic acid molecule.