Recombinational cloning using nucleic acids having recombination sites

• US 6,277,608 B1
• Filed: 04/22/1999
• Issued: 08/21/2001
• Est. Priority Date: 10/24/1997
• Status: Expired due to Term

• 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): Guzo, David
• Assistant Examiner(s): Leffers, Jr., Gerald G
• Application Number:
  US09/296,280
• Time in Patent Office: 852 Days
• Field of Search: 435/6, 435/91.42, 435/320.1, 435/69.1, 435/91.1, 536/23.1, 536/24.2
• US Class Current: 435/91.4
• 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 cloning or subcloning desired nucleic acid molecules comprising

a) combining in vitro or in vivo

i) one or more Insert Donor molecules comprising one or more nucleic acid segments flanked by two or more recombination sites, wherein said recombination sites do not substantially recombine with each other;

ii) two or more different Vector Donor molecules, each comprising two or more recombination sites, wherein said recombination sites do not substantially recombine with each other; and

iii) one or more site specific recombination proteins; and

b) incubating the combination under conditions sufficient to transfer one or more of said nucleic acid segments into said different Vector Donor molecules, thereby producing two or more different Product molecules.

2. The method of claim 1, wherein said Insert Donor molecules are obtained from genomic DNA.

3. The method of claim 1, wherein said Insert Donor molecules are obtained from cDNA.

4. The method of claim 1, wherein said Insert Donor molecules are produced by chemical synthesis.

5. The method of claim 1, wherein said Vector Donor molecules comprise at least one Selectable marker.

6. The method of claim 5, wherein the Selectable marker comprises at least one DNA segment selected from the group consisting of:

(a) a DNA segment that encodes a product that provides resistance in a recipient cell against otherwise toxic compounds;

(b) a DNA segment that encodes a product that is otherwise lacking in a recipient cell;

(c) a DNA segment that encodes a product that suppresses the activity of a gene product in a recipient cell;

(d) a DNA segment that encodes a product that can be identified;

(e) a DNA segment that encodes a product that inhibits a cell function in a recipient cell;

(f) a DNA segment that inhibits the activity of any of the DNA segments of (a)-(e) above;

(g) a DNA segment that binds a product that modifies a substrate;

(h) a DNA segment that encodes a specific nucleotide recognition sequence which can be recognized by a protein, an RNA, DNA or chemical,

(i) a DNA segment that, when deleted, directly or indirectly confers sensitivity to cell killing by particular compounds within a recipient cell;

(j) a DNA segment that encodes a product that is toxic in a recipient cell; and

(k) a DNA segment that can be used to isolate or identify a desired molecule.

7. The method of claim 6, wherein said Selectable marker comprises at least one marker selected from the group consisting of an antibiotic resistance gene, a tRNA gene, an auxotrophic marker, a toxic gene, a phenotypic marker, an antisense oligonucleotide, a restriction endonuclease, a restriction endonuclease cleavage site, an enzyme cleavage site, a protein binding site, and a sequence complementary to a PCR primer sequence.

8. The method of claim 1, wherein said Vector Donor molecules comprise prokaryotic and/or eukaryotic vectors.

9. The method of claim 8, wherein said eukaryotic vectors comprise vectors which replicate in yeast cells, plant cells, fish cells, eukaryotic cells, mammalian cells, or insect cells.

10. The method of claim 8, wherein said prokaryotic vectors comprise vectors which replicate in gram negative or gram positive bacteria.

11. The method of claim 10, wherein said prokaryotic vectors comprise vectors which replicate in bacteria of the genus Escherichia, Salmonella, Bacillus, Streptomyces or Pseudemonas.

12. The method of claim 11, wherein said prokaryotic vector comprises a vector which replicates in E. coli.

13. The method of claim 8, wherein said Vector Donor molecules are selected from the group consisting of cloning vectors, sequencing vectors, expression vectors, fusion vectors, 2-hybrid vectors, and reverse 2-hybrid vectors.

14. The method of claim 8, wherein said eukaryotic vectors are selected from the group consisting of pFastBac, pFastBac HT, pFastBac DUAL, pSFV, pTet-Splice, pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, YACneo, pSVK3, pSVL, pMSG, pCH110, pKK232-8, p3′

SS, pXT1, pSG5, pPbac, pMbac, pMC1neo, and pOG44, pYES2, pAC360, pBlueBacHis, pVL1392, pBlueBacIII, pCDM8, pcDNA1, pZeoSV, pcDNA3 pREP4, pCEP4, and pEBVHis.

15. The method of claim 8, wherein said prokaryotic vectors are selected from the group consisting of pcDNA II, pSL301, pSE280, pSE380, pSE420, pTrcHis, pRSET, pGEMEX-1, pGEMEX-2, pET, pTrc99A, pKK223-3, pGEX, pEZZ18, pRIT2T, pMC1871, pKK233-2, pKK388-1, and pProEx-HT.

16. The method of claim 13, wherein said 2-hybrid and reverse 2-hybrid vectors are selected from the group consisting of pPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGAD1-3, pGAD10, pACt, pACT2, pGADGL, pGADGH, pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA, pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5, pNLexA, and pYESTrp.

17. The method of claim 1, wherein said Insert Donor molecules comprise a vector.

18. The method of claim 1, wherein said Insert Donor molecules comprise a DNA segment produced by amplification.

19. The method of claim 18, wherein said amplification is accomplished by PCR.

20. The method of claim 19, wherein said Insert Donor is linear.

21. The method of claim 20, wherein said Insert Donor comprises at least one recombination site at one or both termini of said linear molecule.

22. The method of claim 1, wherein said recombination sites are selected from the group consisting of loxP, attB, attP, attL, and attR.

23. (Once amended) The method of claim 1, wherein said recombination proteins are selected from the group consisting of Int, Cre, Flp, and Res.