Methods for nucleic acid manipulation
First Claim
1. A method for replicating and amplifying a target nucleic acid sequence comprising:
- a) reacting a primer that is complementary to a target sequence within a nucleic acid duplex with the nucleic acid duplex in the presence of a recombination factor to form a recombination intermediate, without previously denaturing said nucleic acid duplex;
b) admixing a polymerase with said recombination intermediate to form a polymerase complex, whereby the polymerase replicates the target 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
37 Claims
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1. A method for replicating and amplifying a target nucleic acid sequence comprising:
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a) reacting a primer that is complementary to a target sequence within a nucleic acid duplex with the nucleic acid duplex in the presence of a recombination factor to form a recombination intermediate, without previously denaturing said nucleic acid duplex;
b) admixing a polymerase with said recombination intermediate to form a polymerase complex, whereby the polymerase replicates the target sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 26, 27, 28, 29, 30, 31)
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21. A method for reproducing and amplifying a target nucleic acid sequence within a nucleic acid duplex at a temperature below about 45°
- C. comprising;
a) catalytically inserting a primer into said target nucleic acid sequence without previously denaturing said nucleic acid duplex in whole or in part to form a recombination intermediate;
b) admixing said recombination intermediate with a polymerase to form a polymerase complex, whereby said polymerase replicates the target nucleic acid sequence. - View Dependent Claims (22, 23, 24, 25, 32)
- C. comprising;
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33. A method of creating a library of nucleic acid sequences comprising:
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a) incubating a first double-stranded nucleic acid with an enzyme with exonuclease activity to form a plurality of single stranded DNA regions having random sizes;
b) treating said plurality of single stranded DNA regions with a recombination factor to form a plurality of pretreated single stranded DNA regions;
c) adding a second double-stranded nucleic acid to the plurality of pretreated single stranded DNA regions to form a plurality of three stranded crossover junctions;
d) incubating said plurality of three stranded crossover junctions with a helicase to form a plurality of Holliday junctions; and
e) resolving said plurality of Holliday junctions by incubation with an endonuclease. - View Dependent Claims (34, 35, 36, 37)
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Specification