System for in vitro transposition

• US 5,925,545 A
• Filed: 05/02/1997
• Issued: 07/20/1999
• 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: Zhou, Hong, Goryshin, Igor Yu, Reznikoff, William S.
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): Sandals, William
• Application Number:
  US08/850,880
• Time in Patent Office: 809 Days
• Field of Search: 435/6, 435/69.2, 435/193, 435/172.3, 530/350, 530/808, 536/23.1
• US Class Current: 435/69.2
• 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 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.

2. The system as claimed in claim 1 wherein the flanking sequence further comprises a nucleotide at position 4 selected from the group consisting of A or T.

3. The system as claimed in claim 1 wherein the flanking sequence further comprises a nucleotide at position 15 selected from the group consisting of G or C.

4. The system as claimed in claim 1 wherein the flanking sequence further comprises a nucleotide at position 17 selected from the group consisting of A or T.

5. The system as claimed in claim 1 wherein the flanking sequence further comprises a nucleotide at position 18 selected from the group consisting of G or C.

6. The system as claimed in claim 1 wherein the flanking sequence has the sequence 5'"'"'-CTGTCTCTTATACACATCT-3'"'"' (SEQ ID NO:

8).

7. The system as claimed in claim 1 wherein the flanking sequence has the sequence 5'"'"'-CTGTCTCTTATACAGATCT-3'"'"' (SEQ ID NO:

9).

8. 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.

9. The system as claimed in claim 8 wherein the flanking sequence further comprises a nucleotide at position 4 selected from the group consisting of A or T.

10. The system as claimed in claim 8 wherein the flanking sequence further comprises a nucleotide at position 15 selected from the group consisting of G or C.

11. The system as claimed in claim 8 wherein the flanking sequence further comprises a nucleotide at position 17 selected from the group consisting of A or T.

12. The system as claimed in claim 8 wherein the flanking sequence further comprises a nucleotide at position 18 selected from the group consisting of G or C.

13. The system as claimed in claim 8 wherein the flanking sequence has the sequence 5'"'"'-CTGTCTCTTATACACATCT-3'"'"' (SEQ ID NO:

8).

14. The system as claimed in claim 8 wherein the flanking sequence has the sequence 5'"'"'-CTGTCTCTTATACAGATCT-3'"'"' (SEQ ID NO:

9).