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US 6,787,316 B2Granted
DNA cloning method
AssigneeEuropaisches Laboratorium fur Molekularbiologie (EMBL)
InventorsFrancis Stewart, Youming Zhang, Frank Buchholz
Life of this patent
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Abstract
The invention relates to methods for cloning DNA molecules using recE/recT-mediated homologous recombination mechanism between at least two DNA molecules where one DNA molecule is a circular or linear DNA molecule and the second DNA molecule is a circular DNA molecule, and the second DNA molecule contains two regions with sequence homology to the first DNA molecule. Competent cells and vectors are also described.
Claims29
A method for cloning DNA molecules in prokaryotic cells comprising the steps of:
- a) providing a prokaryotic host cell capable of performing homologous recombination,
- b) contacting in said host cell a circular first DNA molecule which is capable of being replicated in said host cell with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, under conditions for promoting homologous recombination between said first and second DNA molecules, and
- c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred,
- wherein a second DNA molecule is introduced into the host cell in a form which allows recombination without further modification, and wherein the homologous recombination occurs by recE and recT mediated gene recombination,
- wherein the recE gene is a nucleic acid molecule selected from the group consisting of:
- (i) the nucleic acid sequence from position 1320 (ATG) to 2159 (GAC) of SEQ ID No. 2,
- (ii) a nucleic acid encoding a recE polypeptide encoded by the nucleic acid sequence of (i) within the degeneracy of the genetic code, and
- (iii) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequence from (i) and/or (ii).
5 more elements
A method for cloning DNA molecules in prokaryotic cells comprising the steps of:
- a) providing a prokaryotic host cell capable of performing homologous recombination,
- b) contacting in said host cell a circular first DNA molecule which is capable of being replicated in said host cell with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, under conditions for promoting homologous recombination between said first and second DNA molecules, and
- c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred,
- wherein a second DNA molecule is introduced into the host cell in a form which allows recombination without further modification, and wherein the homologous recombination occurs by recE and recT mediated gene recombination,
- wherein the recT gene is a nucleic acid molecule selected from the group consisting of:
- (i) the nucleic acid sequence from position 2155 (ATG) to 2961 (GM) of SEQ ID No. 4,
- (ii) a nucleic acid encoding a recT polypeptide encoded by the nucleic acid sequence of (i) within the degeneracy of the genetic code, and
- (iii) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequences from (i) and/or (ii).
5 more elements
A method for cloning DNA molecules in prokaryotic cells comprising the steps of:
- a) providing a prokaryotic host cell capable of performing homologous recombination, wherein the host cell expresses a recBC inhibitor gene,
- b) contacting in said host cell a circular first DNA molecule which is capable of being replicated in said host cell with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, under conditions for promoting homologous recombination between said first and second DNA molecules, and
- c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred,
- wherein a second DNA molecule is introduced into the host cell in a form which allows recombination without further modification, and wherein the homologous recombination occurs by recE and recT mediated gene recombination,
- wherein the recBC inhibitor gene is a nucleic acid molecule selected from the group consisting of:
- (i) the nucleic acid sequence from position 3588 (ATG) to 4002 (GTA) of SEQ ID No. 11,
- (ii) a nucleic acid encoding a recBC inhibitor polypeptide encoded by the nucleic acid sequence of (i) within the degeneracy of the genetic code, and
- (iii) a nucleic acid sequence which hybridizes under stringent conditions as defined above with the nucleic acid sequence of (i) and/or (ii).
5 more elements
Dependencies are inferred from the claim text.