Method for ligating nucleic acids and molecular cloning

• US 20060166332A1
• Filed: 11/21/2005
• Published: 07/27/2006
• Est. Priority Date: 02/25/2000
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Stratagene California
• Inventors: Sorge, Joseph A., Carstens, Carsten-Peter
• Granted Patent: US 7,517,688 B2
• Time in Patent Office: Days
• US Class Current: 435/91.100
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

The invention provides cells and methods of circularizing linear DNA molecules. The cell is an isolated Escherichia coli cell which transiently expresses the Cre recombinase protein from an integrated Cre recombinase gene, and which is at least transiently repressed for RecBCD activity. The cells are used in a method of circularizing a linear DNA molecule comprising at least two loxP sites. The DNA molecule is introduced into the cells, and the linear DNA molecule is joined at said loxp sites.

Claims

1. An isolated Escherichia coli cell which transiently expresses the Cre recombinase protein from an integrated Cre recombinase gene, and which is at least transiently repressed for RecBCD activity.

2. The cell of claim 1, lacking a functional recB gene.

3. The cell of claim 1, lacking a functional recC gene.

4. The cell of claim 1, wherein said RecBCD activity is transiently repressed

5. The cell of claim 1, which inducibly expresses an inhibitor of RecBCD, selected from the group consisting of lambda Gam, T3 gene 5.9 protein, and P22 Abc2 protein.

6. The cell of claim 1, which inducibly expresses said Cre recombinase protein.

7. The cell of claim 1, which is competent.

9. The cell of claim 1, wherein said integrated Cre recombinase gene is operably linked to the arabinose promoter

10. A method of circularizing a linear DNA molecule comprising at least two loxP sites, comprising introducing said DNA into said cells of claim 1, wherein said linear DNA molecule is joined at said loxP sites.

11. The method of claim 10, wherein said cell lacks a functional recB gene.

12. The method of claim 10, wherein said cell lacks a functional recC gene.

13. The method of claim 10, wherein said RecBCD activity of said cell is transiently repressed.

14. The method of claim 10, wherein said cell inducibly expresses an inhibitor of RecBCD selected from the group consisting of lambda Gam, T3 gene 5.9 protein, and P22 Abc2 protein.

15. The method of claim 10, wherein said cell inducibly expresses said Cre recombinase protein.

16. The method of claim 10, wherein said cell is competent.

17. The cell of claim 16, wherein said Cre recombinase is transiently expressed by a process comprising:

treating said cell to induce expression of said Cre recombinase prior to making said cells competent.

18. The cell of claim 1, wherein said integrated Cre recombinase gene is operably linked to the arabinose promoter.

8. The cell of claim 8, wherein said Cre recombinase is transiently expressed by a process comprising:

treating said cell to induce expression of said Cre recombinase prior to making said cells competent.