Recombinational cloning using nucleic acids having recombination sites

• US 20040253631A1
• Filed: 07/19/2004
• Published: 12/16/2004
• Est. Priority Date: 10/24/1997
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Fox, Donna K., Temple, Gary F., Hartley, James L., Brasch, Michael A.
• Granted Patent: US 7,408,049 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

Abstract

Recombinational cloning is provided by the use of nucleic acids, vectors and methods, in vitro and in vivo, for moving or exchanging segments of DNA molecules using engineered recombination sites and recombination proteins to provide chimeric DNA molecules that have the desired characteristic(s) and/or DNA segment(s).

Claims

1. A composition comprising:

(a) an isolated nucleic acid molecule comprising one or more site-specific recombination sites; and

(b) one or more topoisomerases bound to said isolated nucleic acid molecule.

2. The composition of claim 1, wherein said site-specific recombination sites are att sites.

3. The composition of claim 1, wherein said site-specific recombination sites are lox sites.

4. The composition of claim 3, wherein said lox sites are loxP sites.

5. The composition of claim 4, wherein said loxP sites are selected from the group consisting of loxP and loxP511.

6. The composition of claim 2, wherein said att sites are selected from the group consisting of attB, attP, attL and attR.

7. The composition of claim 6, wherein said attB sites are selected from the group consisting of attB1 (SEQ ID NO:

6), attB2 (SEQ ID NO;

7) and attB3 (SEQ ID NO;

8).

8. The composition of claim 6, wherein said attP sites are selected from the group consisting of attP1 (SEQ ID NO:

15) and attP2 (SEQ ID NO;

16).

9. The composition of claim 6, wherein said attL sites are selected from the group consisting of attL1 (SEQ ID NO:

12), attL2 (SEQ ID NO;

13) and attL3 (SEQ ID NO;

14).

10. The composition of claim 6, wherein said attR sites are selected from the group consisting of attR1 (SEQ ID NO:

9), attR2 (SEQ ID NO;

10) and attR3 (SEQ ID NO;

11).

11. A composition comprising:

(a) an isolated nucleic acid molecule comprising two or more site-specific recombination sites; and

(b) one or more topoisomerases bound to said isolated nucleic acid molecule.

12. The composition of claim 11, wherein said two or more site-specific recombination sites do not recombine with each other.

13. An isolated nucleic acid molecule comprising:

(a) one or more site-specific recombination sites; and

(b) one or more topoisomerase recognition sites.

14. The isolated nucleic acid molecule of claim 13, wherein said site-specific recombination sites are att sites are selected from the group consisting of attB, attP, attL and attR.

15. The isolated nucleic acid molecule of claim 13, wherein said site-specific recombination sites are lox sites selected from the group consisting of loxP and loxP511.

16. The isolated nucleic acid molecule of claim 14, wherein said attB sites are selected from the group consisting of attB1 (SEQ ID NO:

6), attB2 (SEQ ID NO;

7) and attB3 (SEQ ID NO;

8).

17. The isolated nucleic acid molecule of claim 14, wherein said attP sites are selected from the group consisting of attP1 (SEQ ID NO:

15) and attP2 (SEQ ID NO;

16).

18. The isolated nucleic acid molecule of claim 14, wherein said attL sites are selected from the group consisting of attL1 (SEQ ID NO:

12), attL2 (SEQ ID NO;

13) and attL3 (SEQ ID NO;

14).

19. The isolated nucleic acid molecule of claim 14, wherein said attR sites are selected from the group consisting of attR1 (SEQ ID NO:

9), attR2 (SEQ ID NO;

10) and attR3 (SEQ ID NO;

11).

20. An isolated nucleic acid molecule comprising:

(a) two or more recombination sites that do not recombine with each other; and

(b) one or more topisomerase recognition sites.