Site-specific dna recombination
AssigneeNanyang Technological University
InventorsHarshyaa Makhija, Peter Droge
Life of this patent
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Abstract
The present invention provides a method of stably integrating a DNA sequence of interest into a target genomic DNA sequence of a host cell, wherein the target genomic DNA sequence comprises a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1, as well as a kit for use in said method. Also provided is a method of generating a circular DNA construct essentially consisting of a DNA sequence of interest and a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:3.
Claims22
A method of stably integrating a DNA sequence of interest into a target genomic DNA sequence of a host cell, wherein the target genomic DNA sequence comprises a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1, the method comprising the steps of:
- (i) providing a bacterial plasmid comprising the DNA sequence of interest flanked on a first end by a first nucleotide sequence comprising a sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1 and on a second end by a second nucleotide sequence comprising a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2;
- (ii) subjecting the bacterial plasmid to conditions that allow intramolecular recombination between the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1 and the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2 in the presence of a phage lambda integrase, to obtain a first circular DNA construct essentially consisting of the DNA sequence of interest and a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:3 and a second circular DNA construct comprising the remainder sequence of the bacterial plasmid and a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:4;
- (iii) linearizing the second circular DNA construct by means of an endonuclease activity;
- (iv) isolating the first circular DNA construct comprising the DNA sequence of interest;
- (v) introducing the first circular DNA construct into the host cell, said cell further comprising a phage lambda integrase;
- and
- (vi) subjecting the host cell to conditions that allow integration of the DNA sequence of interest into the target genomic DNA sequence of the host cell, wherein said integration is mediated by the phage lambda integrase.
A method of generating a circular DNA construct essentially consisting of a DNA sequence of interest and a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:3, the method comprising the steps of:
- providing a bacterial plasmid comprising the DNA sequence of interest flanked on a first end by a first nucleotide sequence comprising a sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1 and on a second end by a second nucleotide sequence comprising a sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2;
- (ii) subjecting the bacterial plasmid to conditions that allow intramolecular recombination between the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1 and the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2 in the presence of a phage lambda integrase, to obtain a first circular DNA construct essentially consisting of the DNA sequence of interest and a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:3 and a second circular DNA construct comprising the remainder sequence of the bacterial plasmid and the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:4;
- (iii) linearizing the second circular DNA construct by means of an endonuclease activity;
- and
- (iv) isolating the first circular DNA construct comprising the DNA sequence of interest.
A kit for use in a method of stably integrating a DNA sequence of interest into a target genomic DNA sequence of a host cell, wherein the target genomic DNA sequence comprises a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1, the method comprising the steps of:
- providing a bacterial plasmid comprising the DNA sequence of interest flanked on a first end by a first nucleotide sequence comprising a sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1 and on a second end by a second nucleotide sequence comprising a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2;
- (ii) subjecting the bacterial plasmid to conditions that allow intramolecular recombination between the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1 and the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2 in the presence of a phage lambda integrase, to obtain a first circular DNA construct essentially consisting of the DNA sequence of interest and a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:3 and a second circular DNA construct comprising the remainder sequence of the bacterial plasmid and a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:4;
- (iii) linearizing the second circular DNA construct by means of an endonuclease activity;
- (iv) isolating the first circular DNA construct comprising the DNA sequence of interest;
- (v) introducing the first circular DNA construct into the host cell, said cell further comprising a phage lambda integrase;
- and
- (vi) subjecting the host cell to conditions that allow integration of the DNA sequence of interest into the target genomic DNA sequence of the host cell, wherein said integration is mediated by the phage lambda integrase,
- wherein the kit comprises any one or more components selected from the group consisting of (a) a bacterial plasmid comprising the DNA sequence of interest of step (i), (b) a phage lambda integrase mediating the intramolecular recombination of step (ii), (c) an endonuclease linearizing the second circular DNA construct of step (iii), (d) a means for isolating the first circular DNA construct of step (iv), (e) a means for introducing the first circular DNA construct into the host cell of step (v), (f) a phage lambda integrase mediating the genomic integration of step (vi) or an expression plasmid encoding the phage lambda integrase, and (g) a means for determining the genomic integration events of step (vi).
Dependencies are inferred from the claim text.