System for in vitro transposition

• US 5,965,443 A
• Filed: 09/09/1996
• Issued: 10/12/1999
• Est. Priority Date: 09/09/1996
• Status: Expired due to Term

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Goryshin, Igor Yu, Reznikoff, William S.
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): SANDALS, WILLIAM O
• Application Number:
  US08/814,877
• Time in Patent Office: 1,128 Days
• Field of Search: 435/6, 435/69.2, 435/193, 435/473, 530/350, 530/808, 536/23.1, 536/24.1
• US Class Current: 435/473
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 2800/90 Vectors containing a transposable element
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

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.

Claims

1. 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.

2. A Tn5 transposase as claimed in claim 1 wherein the mutation at position 54 is a substitution mutation.

3. A Tn5 transposase as claimed in claim 1 wherein the mutation at position 372 is a substitution mutation.

4. A Tn5 transposase as claimed in claim 3 wherein position 372 is a proline.

5. A Tn5 transposase as claimed in claim 1 further comprising a substitution mutation at position 56, wherein the mutant transposase lacks an inhibitor activity.

6. A Tn5 transposase as claimed in claim 5 wherein position 56 is alanine.

7. 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.

8. 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.

9. A system as claimed in claim 8 wherein the mutation at position 54 is a substitution mutation.

10. A system as claimed in claim 8 wherein the mutation at position 372 is a substitution mutation.

11. A system as claimed in claim 10 wherein position 372 is a proline.

12. A system as claimed in claim 8 wherein the transposase further comprises a substitution mutation at position 56, wherein the mutant transposase lacks an inhibitor activity.

13. A system as claimed in claim 12 wherein position 56 is an alanine.

14. 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.

15. 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.

16. A method as claimed in claim 15 wherein the mutation at position 54 is a substitution mutation.

17. A method as claimed in claim 15 wherein the mutation at position 372 is a substitution mutation.

18. A method as claimed in claim 17 wherein position 372 is a proline.

19. A method as claimed in claim 15 wherein the transposase further comprises a substitution mutation at position 56, wherein the mutant transposase lacks an inhibitor activity.

20. A method as claimed in claim 19 wherein position 56 is an alanine.

21. 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.