RECOMBINATIONAL CLONING USING NUCLEIC ACIDS HAVING RECOMBINATION SITES

• US 20100267120A1
• Filed: 07/06/2009
• Published: 10/21/2010
• Est. Priority Date: 06/07/1995
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Fox, Donna K., Temple, Gary F., Hartley, James L., Brasch, Michael A.
• Application Number:
  US12/497,915
• Publication Number:
  US 20100267120A1
• Time in Patent Office: Days
• US Class Current: 435/252.310
• 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. An in vitro method of cloning an amplification product comprising:

(a) obtaining an amplification product comprising a first recombination site and a second recombination site which do not recombine with each other; and

(b) combining said amplification product in vitro with a vector comprising a third recombination site and a fourth recombination site which do not recombine with each other, under conditions such that recombination occurs between said first and third and said second and fourth recombination sites, thereby producing a product vector.

2. The method of claim 1, further comprising inserting said product vector into a host cell.

3. The method of claim 1, wherein said vector is an expression vector.

4. The method of claim 1, wherein said vector comprises at least one additional nucleic acid sequence selected from the group consisting of a selectable marker, a cloning site, a restriction site, a promoter, an operon, an origin of replication, and a gene or partial gene.

5. The method of claim 1, wherein said vector comprises at least one origin of replication.

6. The method of claim 1, wherein said vector comprises at least one promoter.

7. The method of claim 1, wherein said vector comprises at least one selectable marker.

8. The method of claim 1, wherein said amplification product is linear.

9. The method of claim 1, wherein said first, second, third or fourth recombination sites are lox sites or functional mutants thereof.

10. The method of claim 9, wherein said lox sites are selected from the group consisting of loxP sites and loxP511 sites.

11. The method of claim 1, wherein said first, second, third or fourth recombination sites are att sties or functional mutants thereof.

12. The method of claim 11, wherein said att sites are selected from the group consisting of attB sites, attP sites, attL sites and attR sites.

13. The method of claim 1, wherein said first, second, third or fourth recombination sites are selected from the group consisting of a lox site, an att site, an FRT site, and functional mutants thereof.

14. The method of claim 1, wherein said amplification product and said vector are combined in the presence of at least one recombination protein.

15. The method of claim 14, wherein said recombination protein is Cre.

16. The method of claim 14, wherein said recombination protein is selected from the group consisting of Int, Xis and IHF.

17. The method of claim 1, wherein said amplification product is a polymerase chain reaction product.

18. The method of claim 17, wherein said polymerase chain reaction product is linear.