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US 6,270,969 B1Granted
Recombinational cloning using engineered recombination sites
AssigneeInvitrogen Corporation
InventorsJames L. Hartley, Michael A. Brasch
Life of this patent
Estimating the term…
Abstract
Recombinational cloning is provided by the use of nucleic acids, vectors and methods, in vitro and in vivo, for moving or exchanging segments of DNA molecules using engineered recombination sites and recombination proteins to provide chimeric DNA molecules that have the desired characteristic(s) and/or DNA segment(s).
Claims130
An in vitro method for apposing an expression signal and a gene or partial gene comprising:
- (a) mixing in vitro a first nucleic acid molecule comprising said expression signal and at least a first recombination site, and a second nucleic acid molecule comprising said gene or partial gene and at least a second recombination site;
- and
- (b) incubating said mixture in vitro in the presence of at least one recombination protein under conditions sufficient to cause recombination of at least said first and second recombination sites thereby apposing said expression signal and said gene or partial gene such that expression of said gene or partial gene can be controlled by said expression signal.
A method for apposing an expression signal and a gene or partial gene comprising:
- (a) mixing a first nucleic acid molecule comprising said expression signal and at least a first recombination site, and a second nucleic acid molecule comprising said gene or partial gene and at least a second recombination site;
- and
- (b) contacting said mixture with at least one recombination protein under conditions sufficient to cause recombination of at least said first and second recombination sites thereby apposing said expression signal and said gene or partial gene,
- wherein at least one of said recombination sites comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NOs:
- 1-16, or a nucleic acid sequence complementary thereto.
2 more elements
A method for apposing an expression signal and a gene or partial gene comprising:
- (a) mixing a first nucleic acid molecule comprising said expression signal and at least a first recombination site, and a second nucleic acid molecule comprising said gene or partial gene and at least a second recombination site;
- and
- (b) contacting said mixture with at least one recombination protein under conditions sufficient to cause recombination of at least said first and second recombination sites thereby apposing said expression signal and said gene or partial gene,
- wherein at least one of said recombination sites comprises a mutation which removes one or more stop codons from said recombination site.
1 more element
A method for apposing an expression signal and a gene or partial gene comprising:
- (a) mixing a first nucleic acid molecule comprising said expression signal and at least a first recombination site, and a second nucleic acid molecule comprising said gene or partial gene and at least a second recombination site;
- and
- (b) contacting said mixture with at least one recombination protein under conditions sufficient to cause recombination of at least said first and second recombination sites thereby apposing said expression signal and said gene or partial gene,
- wherein at least one of said recombination sites comprises a mutation which avoids hairpin formation.
1 more element
An in vitro method for apposing an expression signal and a gene or partial gene comprising:
- (a) mixing in vitro
- (i) a first nucleic acid molecule comprising said expression signal, and a first recombination site and a second recombination site, wherein said first and second recombination sites do not recombine with each other;
- and
- (ii) a second nucleic acid molecule comprising said gene or partial gene, and a third recombination site and a fourth recombination site, wherein said third and fourth recombination sites do not recombine with each other;
- and
- (b) contacting said mixture in vitro with at least one recombination protein under conditions sufficient to cause recombination of said first and third recombination sites and/or said second and fourth recombination sites, thereby apposing said expression signal and said gene or partial gene such that expression of said gene or partial gene can be controlled by said expression signal and such that a single protein is expressed upon expression of said gene or partial gene.
3 more elements
An in vitro method for apposing an expression signal and a gene or partial gene comprising:
- (a) mixing in vitro
- (i) a first nucleic acid molecule comprising said expression signal, and a first recombination site and a second recombination site, wherein said first and second recombination sites do not recombine with each other;
- and
- (ii) a second nucleic acid molecule comprising said gene or partial gene, and a third recombination site and a fourth recombination site, wherein said third and fourth recombination sites do not recombine with each other;
- and
- (b) contacting said mixture in vitro with at least one recombination protein under conditions sufficient to cause recombination of said first and third recombination sites and/or said second and fourth recombination sites, thereby apposing said expression signal and said gene or partial gene.
3 more elements
An in vitro method for apposing an expression signal and a gene or partial gene, comprising:
- (a) inserting, by direct ligation cloning, a DNA segment comprising said gene or partial gene into a first nucleic acid molecule comprising at least a first recombination site, thereby producing a second nucleic acid molecule comprising said gene or partial gene and at least said first recombination site;
- (b) mixing in vitro said second nucleic acid molecule with a third nucleic acid molecule comprising said expression signal and at least a second recombination site;
- and
- (c) incubating said mixture in vitro in the presence of at least one recombination protein under conditions sufficient to cause recombination of at least said first and said second recombination sites, thereby producing a fourth nucleic acid molecule wherein said expression signal and said gene or partial gene have been apposed such that expression of said gene or partial gene can be controlled by said expression signal.
1 more element
Dependencies are inferred from the claim text.