Method for making insertional mutations using a Tn5 synaptic complex
AssigneeWisconsin Alumni Research Foundation
InventorsWilliam S. Reznikoff, Igor Y. Goryshin
Life of this patent
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Abstract
A method for making insertional mutations at random or quasi-random locations in the chromosomal or extra-chromosomal nucleic acid of a target cell includes the step of combining, in the target cell, cellular nucleic acid with a synaptic complex that comprises (a) a Tn5 transposase protein and (b) a polynucleotide that comprises a pair of nucleotide sequences adapted for operably interacting with Tn5 transposase and a transposable nucleotide sequence therebetween, under conditions that mediate transpositions into the cellular DNA. In the method, the synaptic complex is formed in vitro under conditions that disfavor or prevent the synaptic complexes from undergoing productive transposition.
Claims18
A method for making an insertional mutation at a random or quasi-random position in cellular nucleic acid in a target cell, the method comprising the step of:
- introducing into the target cell a synaptic complex that comprises (a) a Tn5 transposase protein and (b) a polynucleotide that comprises a pair of nucleotide sequences adapted for operably interacting with Tn5 transposase to form a synaptic complex and a transposable nucleotide sequence therebetween, under conditions that mediate transpositions into the cellular nucleic acid.
A method for forming a synaptic complex between (a) a Tn5 transposase protein and (b) a polynucleotide that comprises a pair of nucleotide sequences adapted for operably interacting with Tn5 transposase to form a synaptic complex and a transposable nucleotide sequence therebetween, the method comprising the step of combining (a) and (b) in vitro under conditions that disfavor polynucleotide strand transfer to form the synaptic complex.
A method for forming a library of insertional mutations at random or quasi-random positions in cellular nucleic acid in a plurality of target cells, the method comprising the steps of:
- introducing into the target cells a synaptic complex that comprises (a) a Tn5 transposase protein and (b) a polynucleotide that comprises a pair of nucleotide sequences adapted for operably interacting with Tn5 transposase to form a synaptic complex and a transposable nucleotide sequence therebetween, the transposable nucleotide sequence comprising a selectable marker, under conditions that mediate transpositions into the cellular nucleic acid;
- and
- screening for the cells that comprise the selectable marker.
Dependencies are inferred from the claim text.