Recombinational cloning using nucleic acids having recombination sites

• US 7,408,049 B2
• Filed: 07/19/2004
• Issued: 08/05/2008
• Est. Priority Date: 10/24/1997
• Status: Expired due to Fees

• 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.
• Primary Examiner(s): Qian; Celine X
• Application Number:
  US10/893,235
• Publication Number:
  US 20040253631A1
• Time in Patent Office: 1,478 Days
• Field of Search: None
• US Class Current: 536/23.1
• 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, outside of a cell, comprising:

(a) a first isolated nucleic acid molecule having one or more lox recombination sites and one or more topoisomerase recognition sites;

(b) a second isolated nucleic acid molecule having two or more lox recombination sites and two or more topoisomerase recognition sites; and

(c) a topoisomerase.

2. The composition of claim 1, wherein the lox sites are selected from the group consisting of loxP and loxP511.

3. The composition of claim 1, wherein at least one of the one or more lox recombination sites of the first isolated nucleic acid molecule may recombine with at least two of the two or more lox recombination sites of the second isolated nucleic acid molecule.

4. The composition of claim 1, wherein at least two of the two or more lox recombination sites of the second isolated nucleic acid molecule flank at least two of the two or more topoisomerase recognition sites.

5. The composition of claim 1, wherein the topoisomerase is a type I topoisomerase.

6. The composition of claim 1, wherein the two or more topoisomerase recognition sites are recognized by a type I topoisomerase.

7. The composition of claim 1, wherein the topoisomerase is bound to the first isolated nucleic acid molecule.

8. The composition of claim 1, wherein the second isolated nucleic acid molecule further comprises an origin of replication.

9. The composition of claim 1, wherein the second isolated nucleic acid molecule further comprises a selectable marker.

10. The composition of claim 9, wherein the selectable marker is an antibiotic resistance gene.

11. A composition, outside of a cell, comprising:

(a) an isolated nucleic acid molecule having one or more lox recombination sites; and

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

12. The composition of claim 11 further comprising a second isolated nucleic acid molecule having two or more lox recombination sites and two or more topoisomerase recognition sites.

13. The composition of claim 12, wherein the lox sites are selected from the group consisting of loxP and loxP511.

14. The composition of claim 12, wherein at least one of the one or more lox recombination sites of the first isolated nucleic acid molecule may recombine with at least two of the two or more lox recombination sites of the second isolated nucleic acid molecule.

15. The composition of claim 12, wherein at least two of the two or more lox recombination sites of the second isolated nucleic acid molecule flank at least two of the two or more topoisomerase recognition sites.

16. The composition of claim 12, wherein the topoisomerase is a type I topoisomerase.

17. The composition of claim 12, wherein the two or more topoisomerase recognition sites are recognized by a type I topoisomerase.

18. The composition of claim 12, wherein the second isolated nucleic acid molecule further comprises an origin of replication.

19. The composition of claim 12, wherein the second isolated nucleic acid molecule further comprises a selectable marker.

20. The composition of claim 19, wherein the selectable marker is an antibiotic resistance gene.