Modified tn5-transposase, set for transposition, the method of transposition, a fragment of dna and the genetic design of a mobile dna sequence

 

The invention relates to genetic engineering. In the application described set for transposition in vitro, including DNA donor, which is a mobile genetic element, flanked by a pair of sequences repeats outer ends of the bacterial transposon Tn5 DNA target, in which a mobile genetic element can be transferred, and a modified Tn5 transposase. Presents a modified Tn5-transposase, which is less likely compared with Tn5-transposase wild-type adopts an inactive multi-dimensional formula. Modified the transposase has a substitution at position 54, 372 Tn5-model wild-type and optionally in position 56. For transposition in vitro combine the DNA molecule, the donor molecule with the DNA target and a modified Tn5-transposase. The described DNA fragment encoding a modified Tn5 transposase. Genetic design of a mobile DNA sequence comprises at its 5' and 3' ends of the flanking sequence 5'-CTGTCTCTTATACACATCT-3' or 5'-CTGTCTCTTATACAGATCT-3', compatible with the modified TP5-transposases. The invention allows to increase the reaction rate of transposition and to refuse the use of an external energy source. 5 C. and 12 C.le="text-align:center; margin-top:2mm;">Claims

1. Modified TP5-transposase, which comprises a substitution at position 54 TP5-a model of the wild type, which is due to the fact that modified the transposase binds with greater avidity sequence repeats outer end TP5 than TP5-wild-type transposase, and a substitution at position 372 TP5-a model of the wild type, which is due to the fact that modified the transposase less likely than transposases wild type accepts inactive multidimensional form.

2. Modified TP5-transposase under item 1, wherein the substitution at position 54 is a lysine.

3. Modified TP5-transposase under item 1 or 2, wherein the substitution at position 372 is a Proline.

4. Modified TP5-transposase according to any one of the preceding paragraphs, characterized in that modified the transposase further includes a mutation, characterized by the substitution at position 56 a model of the wild type.

5. Modified TP5-transposase under item 4, wherein the substitution at position 56 is an alanine.

6. Set for transposition in vitro cell DNA sequences, including modifitsirovannoe the th DNA moreover, the DNA sequence is flanked at its 5'- and 3'-end sequences of repeats external end that is compatible with the TP5 transposase, and a molecule of target DNA, which may be transported to a mobile genetic element.

7. Set on p. 6, characterized in that the DNA molecule donor flanked at its 5'- and 3'-end flanking a DNA sequence, which consists of 18 or 19 base pairs and comprises the nucleotide in position 10, the nucleotide T at position 11 and the nucleotide in position 12.

8. Set under item 7, wherein the flanking sequence comprises the nucleotide in position 4, selected from the group including a or T; nucleotide at position 15 is selected from the group comprising G or C, the nucleotide at position 17, selected from the group including a or T; nucleotide at position 18 is selected from the group comprising G or C.

9. Set under item 7 or 8, characterized in that the flanking sequence has the sequence 5'-CTGTCTCTTATACACATCT-3' or 5'-CTGTCTCTTATACAGATCT-3'.

10. The method of transposition in vitro, including the Association of the DNA donor, which includes the mobile of interest DNA sequence, flanked on its 5'- and 3'-end sequences of repeats Neubau of PP.1-5.

11. The method according to p. 10, characterized in that interest, the DNA sequence is flanked at its 5'- and 3'-end flanking a DNA sequence, which consists of 18 or 19 base pairs and comprises the nucleotide in position 10, the nucleotide T at position 11 and the nucleotide in position 12.

12. The method according to p. 11, wherein the flanking sequence comprises the nucleotide in position 4, selected from the group including a or T; nucleotide at position 15 is selected from the group comprising G or C, the nucleotide at position 17, selected from the group including a or T; nucleotide at position 18 is selected from the group comprising G or C.

13. The method according to p. 11, wherein the flanking sequence has the sequence 5'-CTGTCTCTTATACACATCT-3' or 5'-CTGTCTCTTATACAGATCT-3'.

14. A DNA fragment encoding a modified TP5 transposase according to any one of paragraphs.1-5.

15. The DNA fragment under item 14, characterized in that TP5-transposase in position 54 is a lysine residue, at position 372 - residue Proline.

16. The DNA fragment under item 15, characterized in that has a nucleotide sequence represented in SEQ ID NO:1.

17. Genetic design of a mobile DNA sequence, which includes at its 5th TP5-transposase under item 1.

Priority items:

09.09.1996 on PP.1-6, 10, 14-16;

02.05.1997 on PP.7-9, 11-13, 17.

 

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