Means and methods for cloning nucleic acid sequences
AssigneeRijksuniversiteit Groningen
InventorsEric Robin Geertsma, Berend Poolman
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Abstract
The invention provides means and methods for efficiently cloning nucleic acid sequences of interest in micro-organisms that are less amenable to conventional nucleic acid manipulations, as compared to, for instance, E.coli. The present invention enables high-throughput cloning (and, preferably, expressing) of a nucleic acid of interest in a first kind of micro-organism, while an initial (preferably high throughput) cloning of the nucleic acid of interest is done in a second kind of micro-organism.
Claims16
1-26. (canceled)
A method for cloning a nucleic acid sequence of interest in a first kind of micro-organism, the method comprising:
- inserting said nucleic acid of interest into a nucleic acid vehicle, which vehicle comprises an origin of replication of a second kind of micro-organism;
- cloning said vehicle in a culture of said second kind of micro-organism;
- isolating cloned vehicle comprising said nucleic acid sequence of interest;
- substituting the vehicle's origin of replication of a second kind of micro-organism by an origin of replication of said first kind of micro-organism;
- and
- cloning the resulting vehicle in a culture of said first kind of micro-organism.
A method according to claim 1, wherein said resulting vehicle is essentially devoid of elements derived from said second kind of micro-organism
A method according to claim 1, comprising:
- providing a first nucleic acid vehicle which comprises an origin of replication of said second kind of micro-organism;
- introducing a nucleic acid sequence of interest into said first vehicle;
- providing a second nucleic acid vehicle which comprises an origin of replication of said first kind of micro-organism;
- and
- substituting a part of said first vehicle, which part comprises said origin of replication of said second micro-organism but not said nucleic acid of interest, by a part of said second vehicle, said part comprising said origin of replication of said first microorganism.
A method according to claim 3, wherein said part of said first vehicle has different, non-palindromic overhangs that are not compatible with each other and that are compatible with the overhangs of said part of said second vehicle.
A method according to claim 3, wherein said parts are obtained by cleavage of said vehicles by SfiI.
A method according to claim 1, wherein said nucleic acid of interest is inserted into said first vehicle and cloned in a culture of said second kind of micro-organism using a method selected from the group consisting of a ligation independent cloning (LIC) procedure, Gateway, Univector Plasmid-fusion System (UPS) and LIC variants such as Enzyme-Free Cloning (EFC) and Sequence and Ligation Independent Cloning (SLIC).
A method according to claim 1, wherein said first micro-organism is a micro-organism other then Escherichia coli, preferably a recalcitrant micro-organism.
A method according to claim 1, further comprising allowing expression of said nucleic acid of interest by a culture of said first kind of micro-organism.
A method according to claim 1, further comprising obtaining an expression product of said nucleic acid sequence of interest.
A cloning assay wherein a nucleic acid of interest is cloned and expressed by a method according to claim 1.
A kit of parts comprising:
- a first nucleic acid vehicle which comprises an origin of replication of a second kind of micro-organism;
- and
- a second nucleic acid vehicle which comprises an origin of replication of a first kind of micro-organism;
- wherein each vehicle comprises at least two recombinase sites and/or restriction enzyme cleavage sites and wherein said recombinase sites and/or restriction enzyme cleavage sites are preferably not present within said origins of replication.
A kit of parts according to claim 11, wherein each vehicle comprises at least two restriction enzyme cleavage sites which, upon cleaving, yield two different, non-palindromic overhangs that are not compatible with each other.
A kit of parts according to claim 11, wherein said first vehicle and said second vehicle comprise the same kind of restriction enzyme cleavage sites which, upon cleaving, yield two different, non-palindromic overhangs that are not compatible with each other.
A kit of parts according to claim 11, wherein said restriction enzyme cleavage sites are SfiI cleavage sites.
A kit of parts according to claim 11, wherein said second vehicle comprises at least part of an origin of replication of a micro-organism other then Escherichia coli, preferably an origin of replication of a recalcitrant micro-organism.
Dependencies are inferred from the claim text.