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 of determining the presence of a target nucleic acid sequence in a sample comprising contacting said sample with a polymerase, bacteriophage T4 UvsX protein, and two primers that are complementary to the flanking ends of said target nucleic acid sequence; and detecting the presence of amplified target nucleic acid sequence.
2. The method of claim 1 , carried out at a constant temperature.
3. The method of claim 1 , carried out at a temperature below about 45° C.
4. The method of claim 1 , carried out in the presence of bacteriophage T4 UvsY protein.
5. The method of claim 1 , carried out in the presence of bacteriophage T4 gene product 32 protein.
6. The method of claim 1 , wherein said primers are present in a molar excess relative to said target nucleic acid sequence.
7. The method of claim 1 , carried out in the presence of a helicase.
8. The method of claim 7 , wherein said helicase is a bacteriophage T4 gene product 41 DNA helicase.
9. The method of claim 7 , wherein said helicase if bacteriophage T4 replicative helicase complex, comprising bacteriophage T4 gene product and gene product 59.
10. The method of claim 1 , carried out in the presence of nucleotide triphosphates in amounts sufficient to permit amplification of said target nucleic acid sequence.
11. The method of claim 1 , carried out at a temperature of about 37° C.
12. A method of determining the presence of a target nucleic acid sequence in a sample comprising contacting said sample with a polymerase, bacteriophage T4 UvsX protein, and two primers that are complementary to the flanking ends of said target nucleic acid sequence; and detecting the presence of amplified target nucleic acid sequence at a temperature of about 37° C.