Methods for nucleic acid manipulation
First Claim
1. A method for amplifying a target DNA sequence comprising contacting said target DNA sequence with bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA sequence, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence;
- wherein said primers are present in a molar excess relative to said target DNA sequence; and
, detecting the presence of amplified target DNA 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.
35 Citations
10 Claims
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1. A method for amplifying a target DNA sequence comprising contacting said target DNA sequence with bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA sequence, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence;
- wherein said primers are present in a molar excess relative to said target DNA sequence; and
, detecting the presence of amplified target DNA sequence. - View Dependent Claims (2, 3, 4, 5)
- wherein said primers are present in a molar excess relative to said target DNA sequence; and
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6. A method for amplifying a target DNA sequence comprising contacting said target DNA sequence with bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA sequence, a DNA polymerase having proofreading activity, and nucleotides in an amount sufficient to support amplification of said target DNA sequence;
- wherein said primers are present in a molar excess relative to said target DNA sequence; and
, detecting the presence of amplified target DNA sequence. - View Dependent Claims (7, 8, 9, 10)
- wherein said primers are present in a molar excess relative to said target DNA sequence; and
Specification