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US 2006/0051776 A1Published application
Gene manipulation method using homologous recombination
AssigneeSmithKline Beecham Corporation
InventorsDouglas DeMarini, Steven Sheardown
Life of this patent
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Abstract
Provided is a method of inserting an insert polynucleotide into a target nucleic acid having a first end and a second end by homologous recombination.
Claims19
A method of inserting an insert polynucleotide into a target nucleic acid having a first end and a second end by homologous recombination comprising:
- inserting into a recombination-competent cell the following first nucleic acids:
- one or more insert polynucleotides each comprising an insert segment, and
- at least one linker comprising (i) a substantially single-stranded recombination sequence, (ii) a recombination sequence of no more than 25 base pairs, or (iii) a combined length of no more than 45 nucleotides;
- and
- generating within the cell a nucleic acid containing the insert segments inserted between the first and second ends in an order defined by recombination sequences found in the target nucleic acid and the first nucleic acids.
2 more elements
A method of cloning or subcloning an insert nucleic acid comprising:
- isolating an insert polynucleotide comprising the (i) insert nucleic acid and, (ii) at least at one end of the insert nucleic acid, an additional polynucleotide portion;
- inserting into a recombination-competent cell the following first nucleic acids:
- the insert polynucleotide,
- a vector-related nucleic acid having a first end and a second end,
- at least one linker comprising (i) a substantially single-stranded recombination sequence, (ii) a recombination sequence of no more than 25 base pairs, or (iii) a combined length of no more than 45 nucleotides;
- and
- generating within the cell a vector containing the insert nucleic acid inserted between the first and second ends in an order defined by recombination sequences found in the first nucleic acids.
4 more elements
A method of cloning or subcloning an insert nucleic acid:
- isolating (i) a first insert polynucleotide comprising (a) a first insert segment that defines a portion of the insert nucleic acid and, at one or both ends of the first insert segment, (b) additional polynucleotide sequence, and (ii) one or more additional insert polynucleotides comprising additional insert segments that define portions of the insert nucleic acid, wherein the insert segments can be aligned to generate the insert nucleic acid;
- inserting into a recombination-competent cell the following first nucleic acids:
- the first insert polynucleotides and the additional insert polynucleotides,
- a vector-related nucleic acid having a first end and a second end,
- at least one linker comprising (i) a substantially single-stranded recombination sequence, (ii) a recombination sequence of no more than 25 base pairs, or (iii) a combined length of no more than 45 nucleotides;
- and
- generating within the cell a vector containing the insert nucleic acid inserted between the first and second ends in an order defined by recombination sequences found in the first nucleic acids.
4 more elements
Dependencies are inferred from the claim text.