Recombinational cloning using engineered recombination sites

• US 7,304,130 B2
• Filed: 11/02/1999
• Issued: 12/04/2007
• Est. Priority Date: 06/07/1995
• Status: Expired due to Fees

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Hartley, James L., Brasch, Michael A.
• Primary Examiner(s): Vogel; Nancy
• Application Number:
  US09/432,085
• Publication Number:
  US 20030100110A1
• Time in Patent Office: 2,954 Days
• Field of Search: 530/350, 530/300, 530/402, 536/23.1, 536/23.4, 536/24.1, 536/23, 435/69.1, 435/69.7, 435/91.1, 435/91.4
• US Class Current: 530/350
• 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

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 fusion polypeptide encoded by a coding region of a nucleic acid molecule, wherein said coding region compnses:

(a) a first nucleic acid comprising a full length sequence selected from the group consisting of SEQ ID NOs;

1-16, a loxP sequence, a DNA sequence complementary to any of the full length sequences of SEQ ID NOs;

1-16 or a loxP sequence, and an RNA sequence corresponding to any of the full length sequences of SEQ ID NOs;

1-16 or a loxP sequence; and

(b) a second nucleic acid sequence encoding a tag sequence, wherein said first nucleic acid sequence and said second nucleic acid sequence are co-transcribed, wherein said fusion polypeptide comprises amino acids encoded by said first nucleic acid sequence.

2. The polypeptide of claim 1, wherein said tag sequence is a GST tag or a His tag.

3. The polypeptide of claim 1, wherein said nucleic acid molecule further comprises a third nucleic acid sequence encoding a selectable marker, a cloning site, a restriction site, a promoter, an operon or an origin of replication.

4. The polypeptide of claim 1, where said nucleic acid molecule further comprises a third nucleic acid sequence encoding a selectable marker.

5. The polypeptide of claim 4, wherein said selectable marker is selected from the group consisting of a reporter gene, a toxic gene and a phenotypic marker.

6. The polypeptide of claim 4, wherein said selectable marker confers resistance to an antibiotic.

7. The polypeptide of claim 6, wherein said antibiotic is selected from the group consisting of chloramphenicol, ampicillin, tetracycline, and kanamycin.

8. The polypeptide of claim 5, wherein said reporter gene is phoA or CAT.

9. A fusion polypeptide encoded by a coding region of a nucleic acid molecule, wherein said coding region comprises:

(a) a first nucleic acid comprising a full length sequence selected from the group consisting of SEQ ID NOs;

1-16, a loxP sequence, a DNA sequence complementary to any of the full length sequences of SEQ ID NOs;

1-16 or a loxP sequence, and an RNA sequence corresponding to any of the full length sequences of SEQ ID NOs;

1-16 or a loxP sequence; and

(b) a second nucleic acid sequence encoding a GST or a His tag sequence, wherein said first nucleic acid sequence and said second nucleic acid sequence are co-transcribed,

wherein said fusion polypeptide comprises amino acids encoded by said first nucleic acid sequence.

10. The polypeptide of claim 9, wherein said nucleic acid molecule further comprises a third nucleic acid sequence encoding a selectable marker, a cloning site, a restriction site, a promoter, an operon or an origin of replication.

11. The polypeptide of claim 9, wherein said nucleic acid molecule further comprises a third nucleic acid sequence encoding a selectable marker.

12. The polypeptide of claim 11, wherein said selectable marker is selected from the group consisting of a reporter gene, a toxic gene and a phenotypic marker.

13. The polypeptide of claim 11, wherein said selectable marker confers resistance to an antibiotic.

14. The polypeptide of claim 13, wherein said antibiotic is selected from the group consisting of chloramphenicol, ampicillin, tetracycline, and kanamycin.

15. The polypeptide of claim 12, wherein said reporter gene is phoA or CAT.

16. A fusion polypeptide encoded by a coding region of a nucleic acid molecule, wherein said coding region comprises:

(a) a first nucleic acid comprising a full length sequence selected from the group consisting of a lambda or lambdoid phage attB sequence, a DNA sequence complementary to the full length lambda or lambdoid phage attB sequence, and an RNA sequence corresponding to the full length lambda or lambdoid phage attB sequence; and

(b) a second nucleic acid sequence encoding a GST or a His tag sequence, wherein said first nucleic acid sequence and said second nucleic acid sequence are co-transcribed, wherein said fusion polypeptide comprises amino acids encoded by said lambda or lambdoid attB sequence, amino acids encoded by said DNA sequence complementary to said lambda or lambdoid phage attB sequence, or amino acids encoded by said RNA sequence corresponding to said lambda or lambdoid phage attB sequence.

17. The polypeptide of claim 16, wherein said nucleic acid molecule further comprises a third nucleic acid sequence encoding a selectable marker, a cloning site, a restriction site, a promoter, an operon or an origin of replication.

18. The polypeptide of claim 16, wherein said nucleic acid molecule further comprises a third nucleic acid sequence encoding a selectable marker.

19. The polypeptide of claim 18, wherein said selectable marker is selected from the group consisting of a reporter gene, a toxic gene and a phenotypic marker.

20. The polypeptide of claim 18, wherein said selectable marker confers resistance to an antibiotic.

21. The polypeptide of claim 20, wherein said antibiotic is selected from the group consisting of chloramphenicol, ampicillin, tetracycline, and kanamycin.

22. The polypeptide of claim 19, wherein said reporter gene is phoA or CAT.