Methods for nucleic acid manipulation
First Claim
1. A composition for amplifying a target nucleic acid sequence comprising:
- (a) bacteriophage UvsX protein;
(b) two primers that are complementary to the ends of said target nucleic acid sequence, said primers present in a molar excess relative to said target nucleic acid sequence;
(c) a polymerase; and
(d) nucleotides in an amount sufficient to support amplification 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.
17 Citations
22 Claims
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1. A composition for amplifying a target nucleic acid sequence comprising:
- (a) bacteriophage UvsX protein;
(b) two primers that are complementary to the ends of said target nucleic acid sequence, said primers present in a molar excess relative to said target nucleic acid sequence;
(c) a polymerase; and
(d) nucleotides in an amount sufficient to support amplification of said target nucleic acid sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7)
- (a) bacteriophage UvsX protein;
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8. A composition for amplifying a target nucleic acid sequence comprising:
- (a) bacteriophage UvsX protein;
(b) two primers that are complementary to the ends of said target nucleic acid sequence;
(c) a prokaryotic polymerase; and
(d) nucleotides in an amount sufficient to support amplification of said target nucleic acid sequence. - View Dependent Claims (9, 10, 11, 12, 13, 14)
- (a) bacteriophage UvsX protein;
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15. A composition for amplifying a target nucleic acid sequence comprising:
- (a) bacteriophage UvsX protein;
(b) a UvsX protein accessory factor;
(c) two primers that are complementary to the ends of said target nucleic acid sequence;
(c) a polymerase, and (d) nucleotides in an amount sufficient to support amplification of said target nucleic acid sequence.
- (a) bacteriophage UvsX protein;
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16. A composition for amplifying a target DNA sequence comprising:
- (a) bacteriophage UvsX protein;
(b) two primers that are complementary to the ends of said target nucleic acid sequence; and
(c) a prokaryotic polymerase. - View Dependent Claims (17, 18, 19, 20, 21, 22)
- (a) bacteriophage UvsX protein;
Specification