System for in vitro transposition
AssigneeWisconsin Alumni Research Foundation
InventorsWilliam S. Reznikoff, Igor Yu Goryshin
Life of this patent
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Abstract
A system for in vitro transposition includes a donor DNA that includes a transposable element flanked by a pair of bacterial transposon Tn5 outside end repeat sequences, a target DNA into which the transposable element can transpose, and a modified Tn5 transposase having higher binding avidity to the outside end repeat sequences and being less likely to assume an inactive multimer form than wild type Tn5 transposase.
Claims21
A mutant Tn5 transposase modified relative to a wild type Tn5 transposase, the mutant transposase comprising:
- a mutation at position 54;
- and
- a mutation at position 372,
- the mutant transposase having greater avidity for Tn5 outside end repeat sequences of a donor DNA and a lesser ability to form multimers than wild type Tn5 transposase.
A mutant Tn5 transposase modified relative to a wild type Tn5 transposase, the mutant transposase comprising a mutation at position 54 ;
- and
- a mutation at position 372,
- the mutant transposase having greater avidity for Tn5 outside end repeat sequences of a donor DNA and a lesser ability to form multimers than wild type Tn5 transposase wherein position 54 is a lysine.
A system for transposing a transposable DNA sequence in vitro, the system comprising:
- a mutant Tn5 transposase modified relative to a wild type Tn5 transposase, the mutant transposase comprising a mutation at position 54, and a mutation at position 372, the mutant transposase having greater avidity for Tn5 outside end repeat sequences of a donor DNA and a lesser ability to form multimers than wild type Tn5 transposase;
- a donor DNA molecule comprising the transposable DNA sequence, the DNA sequence being flanked at its 5'-and 3' ends by the Tn5 outside end repeat sequences;
- and
- a target DNA molecule into which the transposable element can transpose.
A system for transposing a transposable DNA sequence in vitro, the system comprising:
- a mutant Tn5 transposase modified relative to a wild type Tn5 transposase, the mutant transposase comprising a mutation at position 54, and a mutation at position 372, the mutant transposase having greater avidity for Tn5 outside end repeat sequences of a donor DNA and a lesser ability to form multimers than wild type Tn5 transposase;
- a donor DNA molecule comprising the transposable DNA sequence, the DNA sequence being flanked at its 5'-and 3' ends by the Tn5 outside end repeat sequences;
- and
- a target DNA molecule into which the transposable element can transpose
- wherein position 54 is a lysine.
A method for in vitro transposition, the method comprising the steps of:
- combining a donor DNA molecule that comprises a transposable DNA sequence of interest, the DNA sequence of interest being flanked at its 5'- and 3'-ends by Tn5 outside end repeat sequences, with a target DNA molecule and a mutant Tn5 transposase modified relative to wild type Tn5 transposase in a suitable reaction buffer at a temperature below a physiological temperature until the modified transposase binds to the outside end repeat sequences;
- and
- raising the temperature to a physiological temperature for a period of time sufficient for the enzyme to catalyze in vitro transposition,
- wherein the mutant transposase comprises a mutation at position 54 and a mutation at position 372, the mutant transposase having greater avidity for Tn5 outside end repeat sequences of a donor DNA and a lesser ability to form multimers than wild type Tn5 transposase.
A method for in vitro transposition, the method comprising the steps of:
- combining a donor DNA molecule that comprises a transposable DNA sequence of interest, the DNA sequence of interest being flanked at its 5'- and 3'-ends by Tn5 outside end repeat sequences, with a target DNA molecule and a mutant Tn5 transposase modified relative to wild type Tn5 transposase in a suitable reaction buffer at a temperature below a physiological temperature until the modified transposase binds to the outside end repeat sequences;
- and
- raising the temperature to a physiological temperature for a period of time sufficient for the enzyme to catalyze in vitro transposition,
- wherein the mutant transposase comprises a mutation at position 54 and a mutation at position 372, the mutant transposase having greater avidity for Tn5 outside end repeat sequences of a donor DNA and a lesser ability to form multimers than wild type Tn5 transposase wherein position 54 is a lysine.
Dependencies are inferred from the claim text.