Methods for nucleic acid manipulation
First Claim
1. A method for amplifying a target nucleic acid sequence comprising contacting said target nucleic acid sequence with a composition comprising a polymerase, bacteriophage T4 UvsX protein, and two primers that are complementary to the flanking ends of said target nucleic acid sequence.
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Abstract
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.
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Citations
12 Claims
- 1. A method for amplifying a target nucleic acid sequence comprising contacting said target nucleic acid sequence with a composition comprising a polymerase, bacteriophage T4 UvsX protein, and two primers that are complementary to the flanking ends of said target nucleic acid sequence.
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12. A method for amplifying a target nucleic acid sequence comprising contacting said target nucleic acid sequence with a composition comprising a polymerase, bacteriophage T4 UvsX protein, and two primers that are complementary to the flanking ends of said target nucleic acid sequence as a temperature of about 37°
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Specification