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US 5,925,545 AGranted
System for in vitro transposition
AssigneeWisconsin Alumni Research Foundation
InventorsWilliam S. Reznikoff, Igor Yu Goryshin, Hong Zhou
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.
Claims14
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 an 18 or 19 base pair flanking DNA sequence comprising nucleotide A at position 10, nucleotide T at position 11, and nucleotide A at position 12;
- and
- a target DNA molecule into which the transposable element can transpose.
1 more element
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 an 18 or 19 base pair flanking DNA sequence comprising nucleotide A at position 10, nucleotide T at position 11, and nucleotide A at position 12, 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.
1 more element
Dependencies are inferred from the claim text.