Methods for nucleic acid manipulation
First Claim
1. A method for amplifying a target DNA, said method comprising the steps:
- i) forming a reaction mixture comprising said target DNA, bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence;
ii) maintaining the reaction mixture at a temperature below about 45°
C., wherein amplified target DNA is produced;
iii) and detecting the presence of amplified target DNA.
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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
20 Claims
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1. A method for amplifying a target DNA, said method comprising the steps:
- i) forming a reaction mixture comprising said target DNA, bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence;
ii) maintaining the reaction mixture at a temperature below about 45°
C., wherein amplified target DNA is produced;
iii) and detecting the presence of amplified target DNA. - View Dependent Claims (2, 3, 4, 5, 6, 7)
- i) forming a reaction mixture comprising said target DNA, bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence;
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8. A method for amplifying a target DNA, said method comprising the steps:
- i) forming a reaction mixture comprising said target DNA, bacteriophage UvsX protein, bacteriophage UvsY protein, two primers that anneal to the flanking ends of said target DNA, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence;
ii) maintaining the reaction mixture at a temperature below about 45°
C., wherein amplified target DNA is produced;
iii) and detecting the presence of amplified target DNA. - View Dependent Claims (9, 10, 11, 12, 13, 14)
- i) forming a reaction mixture comprising said target DNA, bacteriophage UvsX protein, bacteriophage UvsY protein, two primers that anneal to the flanking ends of said target DNA, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence;
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15. A method for amplifying a target DNA, said method comprising the steps:
- i) forming a reaction mixture comprising said target DNA, bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence;
ii) maintaining the reaction mixture at room temperature, wherein amplified target DNA is produced;
iii) and detecting the presence of amplified target DNA. - View Dependent Claims (16, 17, 18, 19, 20)
- i) forming a reaction mixture comprising said target DNA, bacteriophage UvsX protein, two primers that anneal to the flanking ends of said target DNA, a DNA polymerase, and nucleotides in an amount sufficient to support amplification of said target DNA sequence;
Specification