Rapid subcloning using site-specific recombination
First Claim
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1. A method for the recombination of nucleic acid constructs, comprising:
- a) providing;
i) a first nucleic acid construct comprising, in operable order, an origin of replication, a first sequence-specific recombinase target site, and a nucleic acid of interest;
ii) a second nucleic acid construct comprising, in operable order, an origin of replication, a regulatory element and a second sequence-specific recombinase target site adjacent to and downstream from said regulatory element; and
iii) a site-specific recombinase;
b) contacting said first and said second nucleic acid constructs with said site-specific recombinase under conditions such that said first and second nucleic acid constructs are recombined to form a third nucleic acid construct, wherein said nucleic acid of interest is operably linked to said regulatory element.
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Abstract
The present invention provides compositions, including vectors, and methods for the rapid subcloning of nucleic acid sequences in vivo and in vitro. In particular, the invention provides vectors used to contain a gene of interest that comprise a sequence-specific recombinase target site. These vectors are used to rapidly transfer the gene or genes of interest into any vector that contains a sequence-specific recombinase target site located downstream of a regulatory element so that the gene of interest may be regulated.
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Citations
36 Claims
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1. A method for the recombination of nucleic acid constructs, comprising:
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a) providing;
i) a first nucleic acid construct comprising, in operable order, an origin of replication, a first sequence-specific recombinase target site, and a nucleic acid of interest;
ii) a second nucleic acid construct comprising, in operable order, an origin of replication, a regulatory element and a second sequence-specific recombinase target site adjacent to and downstream from said regulatory element; and
iii) a site-specific recombinase;
b) contacting said first and said second nucleic acid constructs with said site-specific recombinase under conditions such that said first and second nucleic acid constructs are recombined to form a third nucleic acid construct, wherein said nucleic acid of interest is operably linked to said regulatory element. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method for the recombination of nucleic acid constructs, comprising:
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a) providing;
i) a vector;
ii) a linear nucleic acid molecule comprising a sequence complementary to at least a portion of said vector; and
iii) an E. coli host cell, wherein said host cell comprises an endogenous recombination system, a loss of function rec mutation, a suppressor, and a loss of function endogenous restriction modification system mutation; and
b) introducing said vector and said linear nucleic acid molecule into said host cell under conditions such that said linear nucleic acid molecule and said vector are recombined to form a recombinant nucleic acid construct. - View Dependent Claims (23, 24, 25)
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26. A method for the cloning of nucleic acid libraries, comprising:
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a) providing;
i) a plurality of first nucleic acid constructs comprising, in operable order, an origin of replication, a first sequence-specific recombinase target site, and a nucleic acid member from a nucleic acid library;
ii) a plurality of second nucleic acid constructs comprising, in operable order, an origin of replication, a regulatory element and a second sequence-specific recombinase target site adjacent to and downstream from said regulatory element; and
iii) a site-specific recombinase;
b) contacting said plurality of first and second nucleic acid constructs with said site-specific recombinase under conditions such that said plurality of first and second nucleic acid constructs are recombined to form a plurality of third nucleic acid constructs, wherein said nucleic acid members from said nucleic acid library are operably linked to said regulatory elements. - View Dependent Claims (27)
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28. A method for the directional cloning of a nucleic acid molecule, comprising:
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a) providing;
i) first and second portions of a regulatory element;
ii) a first nucleic acid molecule comprising said first portion of said regulatory element; and
iii) a second nucleic acid molecule comprising said second portion of said regulatory element; and
b) combining said first and said second nucleic acid molecules to produce a third nucleic acid molecule under conditions whereby an intact regulatory element is produced from the combination of said first and said second portions of said regulatory element, wherein the presence of said intact regulatory element in said third nucleic acid molecule indicates a direction of cloning of said first nucleic acid molecule with respect to said second nucleic acid molecule. - View Dependent Claims (29)
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30. A method for regulated recombination in host cells that constitutively express a recombinase, comprising:
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a) providing;
i) a host cell expressing a recombinase;
ii) a first nucleic acid construct comprising an origin of replication, a first site-specific recombinase site, a second site-specific recombinase site that differs in sequence from said first site-specific recombinase site such that said recombinase will not initiate recombination between said first and second site-specific recombinase sites, and a selectable marker gene between said first and second site-specific recombinase sites; and
iii) a second nucleic acid construct comprising an origin of replication, a third site-specific recombinase target site, and a fourth site-specific recombinase target site that differs in sequence from said third site-specific recombinase site such that said recombinase will not initiate recombination between said third and fourth site-specific recombinase sites; and
b) introducing said first and second nucleic acid constructs into said host cell under conditions such that said first and second nucleic acid constructs are recombined. - View Dependent Claims (31, 32, 33, 34)
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35. A method for the recombination of nucleic acid constructs, comprising:
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a) providing;
i) a first nucleic acid construct comprising a loxH site;
ii) a second nucleic acid construct comprising a loxH site; and
iii) a site-specific recombinase; and
b) contacting said first and said second nucleic acid constructs with said site-specific recombinase under conditions such that said first and second nucleic acid constructs are recombined. - View Dependent Claims (36)
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Specification