Recombinational cloning using engineered recombination sites

• US 6,171,861 B1
• Filed: 01/12/1998
• Issued: 01/09/2001
• Est. Priority Date: 06/07/1995
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Hartley, James L., Brasch, Michael A.
• Primary Examiner(s): Elliot, George C.
• Assistant Examiner(s): SANDALS, WILLIAM O
• Application Number:
  US09/005,476
• Time in Patent Office: 1,093 Days
• Field of Search: 435/172.3, 435/455, 435/462, 435/465, 435/320.1, 530/350, 536/23.1, 536/24.1
• US Class Current: 435/455
• 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 method for in vitro cloning of a nucleic acid of interest, comprising:

(a) mixing in vitro a first vector comprising at least a first recombination site and a second vector comprising at least a second recombination site, wherein said first and/or second vector further comprises a nucleic acid of interest;

(b) incubating said mixture in the presence of at least one recombination protein under conditions sufficient to cause recombination of at least said first and second recombination sites, thereby producing a chimeric nucleic acid molecule comprising said nucleic acid of interest;

(c) contacting one or more hosts with said mixture; and

(d) selecting for a host comprising said chimeric nucleic acid molecule, and selecting against a host comprising said first vector and against a host comprising said second vector, thereby cloning said nucleic acid of interest.

2. The method of claim 1, wherein said at least one recombination protein is encoded by a bacteriophage selected from the group consisting of bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1.

3. The method of claim 1, wherein said at least one recombination protein is encoded by bacteriophage lambda.

4. The method of claim 1, wherein said at least one recombination protein is selected from the group consisting of Int, IHF, Xis and Cre.

5. The method of claim 1, wherein said at least one recombination protein is selected from the group consisting of Int, IHF and Xis.

6. The method of claim 1, wherein said at least one recombination protein is selected from the group consisting of α

δ

, Tn3 resolvase, Hin, Gin, Cin and Flp.

7. The method of claim 1, wherein said at least one recombination protein is Cre.

8. The method of claim 1, wherein said recombination sites are selected from the group consisting of alt and lox P sites.

9. The method of claim 1, wherein said recombination sites are alt sites.

10. The method of claim 1, wherein said recombination sites are lox P sites.

11. The method of claim 1, wherein said method produces a chimeric vector comprising said first and second vectors.

12. The method of claim 1, wherein said first and/or second recombination site contains at least one mutation that removes one or more stop codons.

13. The method of claim 1, wherein said first and/or second recombination site contains at least one mutation that avoids hairpin formation.

14. The method of claim 1, wherein said first and/or second recombination site comprises at least a first nucleic acid sequence selected from the group consisting of SEQ ID NOs:

1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

15. The method of claim 1, wherein said host is E. coli.

16. The method of claim 1, wherein said first and/or second recombination site comprises at least a first nucleic acid sequence selected from the group consisting of a mutated att recombination site containing at least one mutation that enhances recombinational specificity, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

17. The method of claim 16, wherein said mutated att site comprises at least one nucleic acid sequence as set forth in SEQ ID NOs.:

1-16.

18. The method of claim 1, wherein said selection is accomplished using at least one Selectable marker.

19. The method of claim 18, wherein said Selectable marker is selected from the group consisting of an antibiotic resistance gene and a toxic gene.

20. A composition comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first nucleic acid sequence selected from the group consisting of SEQ ID NOs:

1-6, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

21. A kit for in vitro cloning of DNA segments comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first nucleic acid sequence selected from the group consisting of SEQ ID NOs:

1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

22. A kit comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule, said nucleic acid molecule comprising at least a first att recombination site which comprises a core region having at least one mutation that enhances recombination efficiency or specificity in vitro in the formation of a Cointegrate DNA or a Product DNA molecule.

23. The kit of claim 22, wherein said recombination site confers at least one enhancement selected from the group consisting of (i) enhancing excisive recombination;

(ii) enhancing integrative recombination;

(iii) decreasing the requirement for host factors;

(iv) increasing the efficiency of the formation reaction by recombination of said Cointegrate DNA or of said Product DNA;

(v) increasing the specificity of the formation reaction by recombination of said Cointegrate DNA or of said Product DNA; and

(vi) increasing the specificity or yield of a subsequent recombination reaction of, or subsequent isolation of, the Product DNA.

24. The kit of claim 22, wherein said core region comprises a nucleic acid sequence selected from the group consisting of:

a) RKYCWGCTTTYKTRTACNAASTSGB (m-att) (SEQ ID NO;

1);

b) AGCCWGCTTTYKTRTACNAACTSGB (m-attB) (SEQ ID NO;

2);

c) GTTCAGCTTTCKTRTACNAACTSGB (m-attR) (SEQ ID NO;

3);

d) AGCCWGCTTTCKTRTACNAAGTSGB (m-attL) (SEQ ID NO;

4);

c) GTTCAGCTTTYKTRTACNAAGTSGB (m-attP1) (SEQ ID NO;

5);

and a corresponding or complementary DNA or RNA sequence, wherein R=A or G;

K=G or T/U;

Y=C or T/U;

W=A or T/U;

N=A or C or G or T/U;

S=C or G; and

B=C or G or T/U.

25. The kit of claim 22, wherein said core region comprises a nucleic acid sequence selected from the group consisting of:

a) AGCCTGCTTTTTTGTACAAACTTGT (attB1) (SEQ ID NO;

6);

b) AGCCTGCTTTCTTGTACAAACTTGT (attB2) (SEQ ID NO;

7);

c) ACCCAGCTTTCTTGTACAAACTTGT (attB3) (SEQ ID NO;

8);

d) GTTCAGCTTTTTTGTACAAACTTGT (attR1) (SEQ ID NO;

9);

e) GTTCAGCTTTCTTGTACAAACTTGT (attR2) (SEQ ID NO;

10);

f) GTTCAGCTTTCTTGTACAAAGTTGG (attR3) (SEQ ID NO;

11);

g) AGCCTGCTTTTTTGTACAAAGTTGG (attL1) (SEQ ID NO;

12);

h) AGCCTGCTTTCTTGTACAAAGTTGG (attL2) (SEQ ID NO;

13);

i) ACCCAGCTTTCTTGTACAAAGTTGG (attL3) (SEQ ID NO;

14);

j) GTTCAGCTTTTTTGTACAAAGTTGG (attP1) (SEQ ID NO;

15);

k) GTTCAGCTTTCTTGTACAAAGTTGG (attP2, P3) (SEQ ID NO;

16);

and a corresponding or complementary DNA or RNA sequence.

26. A method of making a reaction mixture, comprising mixing at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first nucleic acid sequence selected from the group consisting of SEQ ID NOs:

1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

27. A reaction mixture made by the method of claim 26.