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.
Claims16
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, wherein the sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1 and the sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2 flanking the DNA sequence of interest are arranged in a directly repeated orientation;
- (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 that does not contain bacterial sequences, except the 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, wherein the circular DNA construct comprising the DNA sequence of interest does not contain bacterial sequences, except the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:3, and wherein the circular DNA construct comprising the DNA sequence of interest allows integration of the DNA sequence of interest into a target genomic DNA sequence of a host cell by intermolecular recombination mediated by phage lambda integrase, said target genomic sequence comprising a 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 sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2, wherein the sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1 and the sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2 flanking the DNA sequence of interest are arranged in a directly repeated orientation;
- (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 that does not contain bacterial sequences, except the 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.
Dependencies are inferred from the claim text.