Plasmid construction by homologous recombination
AssigneeZymoGenetics, Inc.
InventorChristopher K. Raymond
Life of this patent
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Abstract
Methods for preparing double stranded, circular DNA molecules comprising a region encoding a protein of interest are disclosed. One or more donor DNA fragments encoding the protein of interest are combined with an acceptor plasmid, a first DNA linker, and a second DNA linker in a Saccharomyces cerevisiae host cell whereby the donor DNA fragment is joined to the acceptor plasmid by homologous recombination of the donor DNA, acceptor plasmid, and linkers to form the closed, circular plasmid.
Claims14
A method for preparing a double stranded, circular DNA molecule comprising the steps of:
- (a) providing a double-stranded donor DNA fragment encoding a polypeptide of interest;
- (b) providing a double-stranded, linear acceptor plasmid having blunt first and second ends and comprising a selectable marker and replication sequence that are functional in Saccharamyces cerevisiae, wherein the acceptor plasmid is essentially free of DNA encoding the polypeptide of interest;
- (c) providing a first double-stranded DNA linker comprising a first segment identical in sequence to a first region of the acceptor plasmid and a second segment identical in sequence to a first region of the donor DNA fragment, wherein each of the first and second segments of the first linker is at least 10 bp in length;
- (d) providing a second double-stranded DNA linker comprising a first segment identical in sequence to a second region of the acceptor plasmid and a second segment identical in sequence to a second region of the donor DNA fragment, wherein each of the first and second segments of the second linker is at least 10 bp in length;
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- (e) combining the donor DNA fragment, acceptor plasmid, first DNA linker, and second DNA linker in a Saccharomnyces cerevisiae host cell whereby the donor DNA fragment is joined to the acceptor plasmid by homologous recombination of the donor DNA, acceptor plasmid, and linkers to form a closed, circular plasmid comprising a region encoding the polypeptide of interest.
A method for preparing a double stranded, circular DNA molecule comprising the steps of:
- (a) providing a plurality of overlapping, double-stranded donor DNA fragments which collectively encode a polypeptide of interest;
- (b) providing a double-stranded, linear acceptor plasmid having blunt first and second ends and comprising a selectable marker and replication sequence that are functional in Saccharamyces cerevisiae, wherein the acceptor plasmid is essentially free of DNA encoding the polypeptide of interest;
- (c) providing a first double-stranded DNA linker comprising a first segment identical in sequence to a first region of the acceptor plasmid and a second segment identical in sequence to a region of one of the donor DNA fragments, wherein each of the first and second segments of the first linker is at least 10 bp in length;
- (d) providing a second double-stranded DNA linker comprising a first segment identical in sequence to a second region of the acceptor plasmid and a second segment identical in sequence to a region of another of the donor DNA fragments, wherein each of the first and second segments of the second linker is at least 10 bp in length;
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- (e) combining the donor DNA fragments, acceptor plasmid, first DNA linker, and second DNA linker in a Saccharamyces cerevisiae host cell whereby the donor DNA fragments are joined to the acceptor plasmid by homologous recombination of the donor DNA fragments, acceptor plasmid and linkers to form a closed, circular plasmid comprising a region encoding the polypeptide of interest.
Dependencies are inferred from the claim text.