Use of multiple recombination sites with unique specificity in recombinational cloning

• US 7,393,632 B2
• Filed: 12/11/2000
• Issued: 07/01/2008
• Est. Priority Date: 12/10/1999
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Byrd, Devon R. N., Temple, Gary F., Hartley, James L., Brasch, Michael A., Cheo, David
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/732,914
• Publication Number:
  US 20020007051A1
• Time in Patent Office: 2,759 Days
• Field of Search: 435/91.41, 435/6, 435/462
• US Class Current: 435/6.16
• CPC Class Codes: C07K 14/47 from mammals
• 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 15/102 Mutagenizing nucleic acids
• 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

The present invention provides compositions and methods for recombinational cloning. The compositions include vectors having multiple recombination sites with unique specificity. The methods permit the simultaneous cloning of two or more different nucleic acid molecules. In some embodiments the molecules are fused together while in other embodiments the molecules are inserted into distinct sites in a vector. The invention also generally provides for linking or joining through recombination a number of molecules and/or compounds (e.g., chemical compounds, drugs, proteins or peptides, lipids, nucleic acids, carbohydrates, etc.) which may be the same or different. Such molecules and/or compounds or combinations of such molecules and/or compounds can also be bound through recombination to various structures or supports according to the invention.

Claims

1. A method of cloning a nucleic acid molecule comprising:

(a) providing a first nucleic acid segment flanked by a first and a second recombination site and a second nucleic acid segment flanked by a third and a fourth recombination site, wherein none of the recombination sites flanking the first and second nucleic acid segment is capable of recombining with any of the other sites flanking the first and second nucleic acid segment;

(b) providing a vector comprising a fifth, sixth, seventh and eighth recombination site, wherein each of the fifth, sixth, seventh and eighth recombination sites is capable of recombining with one of the first, second, third or fourth recombination site; and

(c) conducting a recombination reaction such that the two nucleic acid segments are recombined into the vector thereby cloning the first and the second nucleic acid segments.

2. A method of cloning two or more nucleic acid segments, comprising:

(a) providing two or more nucleic acid segments, each segment flanked by two recombination sites which do not recombine with each other;

(b) providing a vector comprising a number of recombination sites equal to twice the number of nucleic acid segments, wherein each of the recombination sites is capable of recombining with one of the recombination sites flanking one of the nucleic acid segments; and

(c) conducting a recombination reaction such that the nucleic acid segments are recombined into the vector thereby cloning the nucleic acid segments,

wherein transcription of at least two of the nucleic acid segments results in the production of a single RNA.

3. The method of claim 2, wherein at least one of the nucleic acid segments produces a sense RNA strand upon transcription and at least one of the nucleic acid segments produces an antisense RNA strand upon transcription.

4. The method of claim 3, wherein the sense RNA and antisense RNA have at least one complementary region and are capable of hybridizing to each other.

5. The method of claim 2, wherein the nucleic acid segments comprise one or more libraries of nucleic acid molecules which encode variable domains of antibody molecules.

6. The method of claim 5, wherein the one or more libraries comprise nucleic acid molecules which encode variable domains of antibody light and heavy chains.

7. The method of claim 2, further comprising screening to identify nucleic acid molecules which encode proteins having binding specificity for one or more antigens.

8. A method of cloning at least one nucleic acid molecule comprising:

(a) providing a first, a second and a third nucleic acid segment, wherein the first nucleic acid segment is flanked by a first and a second recombination site, the second nucleic acid segment is flanked by a third and a fourth recombination site and the third nucleic acid segment is flanked by a fifth and a sixth recombination site, wherein the second recombination site is capable of recombining with the third recombination site and none of the first, fourth, fifth or sixth recombination sites is capable of recombining with any of the first through sixth recombination sites;

(b) providing a vector comprising a seventh and an eighth recombination site flanking a first negative selectable marker and comprising a ninth and a tenth recombination site flanking a second negative selectable marker, wherein none of the seventh through tenth recombination sites can recombine with any of the seventh through tenth recombination sites;

(c) conducting a first recombination reaction such that the second and the third recombination sites recombine; and

(d) conducting a second recombination reaction such that the first and the fourth recombination sites recombine with the seventh and the eighth recombination sites and the fifth and the sixth recombination sites recombine with the ninth and the tenth recombination sites thereby cloning the first, second and third nucleic acid segments.

9. A method of cloning at least one nucleic acid molecule comprising:

(a) providing a first, a second and a third nucleic acid segment, wherein the first nucleic acid segment is flanked by a first and a second recombination site, the second nucleic acid segment is flanked by a third and a fourth recombination site and the third nucleic acid segment is flanked by a fifth and a sixth recombination site, wherein the second recombination site is capable of recombining with the third recombination site and the fourth recombination site is capable of recombining with the fifth recombination site;

(b) providing a vector comprising a seventh and an eighth recombination site; and

(c) conducting at least one recombination reaction such that the second and the third recombination sites recombine and the fourth and the fifth recombination sites recombine and the first and the sixth recombination sites recombine with the seventh and the eighth recombination sites respectively, thereby cloning the first, second and third nucleic acid segments.

10. The method of claim 9, wherein the recombination reaction is conducted in the presence of one or more recombination proteins under conditions which favor the recombination.

11. A method of cloning n nucleic acid fragments, wherein n is an integer greater than 2, comprising:

(a) providing a 1^st through an n^th nucleic acid segment, each segment flanked by two recombination sites, wherein the recombination sites are selected such that one of the two recombination sites flanking the i^th segment, n_i, reacts with one of the recombination sites flanking the n<sub>i−

1</sub>th segment and the other recombination site flanking the i^th segment reacts with one of the recombination sites flanking the n_i+1th segment;

(b) providing a vector comprising at least two recombination sites, wherein one of the two recombination sites on the vector reacts with one of the sites on the 1^st nucleic acid segment and another site on the vector reacts with a recombination site on the n^th nucleic acid segment; and

(c) conducting at least one recombination reaction such that all of the nucleic acid fragments are recombined into the vector.

12. The method of claim 11, wherein the recombination reaction is conducted in the presence of one or more recombination proteins under conditions which favor the recombination.

13. The method of claim 12, wherein the recombination proteins comprise one or more proteins selected from the group consisting of:

(a) Cre;

(b) Int;

(c) IHF;

(d) Xis;

(e) Fis;

(f) Hin;

(g) Gin;

(h) Cin;

(i) Tn3 resolvase;

(j) TndX;

(k) XerC; and

(l) XerD.

14. The method of claim 11, wherein the recombination sites of the nucleic acid segments and the vector comprise one or more recombination sites selected from the group consisting of:

(a) lox sites;

(b) psi sites;

(c) dif sites;

(d) cer sites;

(e) frt sites;

(f) att sites; and

(g) mutants, variants, and derivatives of the recombination sites of (a), (b), (c), (d), (e), or (f) which retain the ability to undergo recombination.

15. The method of claim 14, wherein the recombination sites which recombine with each other comprise att sites having identical seven base pair overlap regions.

16. A method of joining two or more segments of nucleic acid, comprising:

(a) providing two or more segments of nucleic acid, each segment comprising at least one recombination site capable of recombining with a recombination site present on the other segment; and

(b) contacting the segments with one or more recombination proteins under conditions causing recombination between the recombination sites, thereby joining the segments,

wherein the expression product is a ribozyme or an inhibitory RNA molecule.

17. A method of joining n nucleic acid segments, wherein n is an integer greater than 2, comprising:

(a) providing a 1^st through an n^th nucleic acid segment, each segment flanked by two recombination sites, wherein the recombination sites are selected such that one of the two recombination sites flanking the i^th segment, n_i, reacts with one of the recombination sites flanking the n<sub>i−

1</sub>th segment and the other recombination site flanking the i^th segment reacts with one of the recombination sites flanking the n_i+1th segment; and

(b) contacting the segments with one or more recombination proteins under conditions causing the segments to join.

18. The method of claim 17, wherein the recombination proteins comprise one or more proteins selected from the group consisting of:

(a) Cre;

(b) Int;

(c) IHF;

(d) Xis;

(e) Fis;

f) Hin;

(g) Gin;

(h) Cin;

(i) Tn3 resolvase;

(j) TndX;

(k) XerC; and

(l) XerD.

19. The method of claim 17, wherein the recombination sites which recombine with each other comprise att sites having identical seven base pair overlap regions.

20. The method of claim 19, wherein the first three nucleotides of the seven base pair overlap regions of the recombination sites which recombine with each other comprise nucleotide sequences selected from the group consisting of:

(a) AAA;

(b) AAC;

(c) AAG;

(d) AAT;

(e) ACA;

(f) ACC;

(g) ACG;

(h) ACT;

(i) AGA;

(j) AGC;

(k) AGG;

(l) AGT;

(m) ATA;

(n) ATC;

(o) ATG; and

(p) ATT.

21. The method of claim 19, wherein the first three nucleotides of the seven base pair overlap regions of the recombination sites which recombine with each other comprise nucleotide sequences selected from the group consisting of:

(a) CAA;

(b) CAC;

(c) CAG;

(d) CAT;

(e) CCA;

(f) CCC;

(g) CCG;

(h) CCT;

(i) CGA;

(j) CGC;

(k) CGG;

(l) CGT;

(m) CTA;

(n) CTC;

(o) CTG; and

(p) CTT.

22. The method of claim 19, wherein the first three nucleotides of the seven base pair overlap regions of the recombination sites which recombine with each other comprise nucleotide sequences selected from the group consisting of:

(a) GAA;

(b) GAC;

(c) GAG;

(d) GAT;

(e) GCA;

(f) GCC;

(g) GCG;

(h) GCT;

(i) GGA;

(j) GGC;

(k) GGG;

(l) GGT;

(m) GTA;

(n) GTC;

(o) GTG; and

(p) GTT.

23. The method of claim 19, wherein the first three nucleotides of the seven base pair overlap regions of the recombination sites which recombine with each other comprise nucleotide sequences selected from the group consisting of:

(a) TAA;

(b) TAC;

(c) TAG;

(d) TAT;

(e) TCA;

(f) TCC;

(g) TCG;

(h) TCT;

(i) TGA;

(j) TGC;

(k) TGG;

(l) TGT;

(m) TTA;

(n) TTC;

(o) TTG; and

(p) TTT.

24. The method of claim 17, further comprising inserting the nucleic acid segments joined in step (b) into a vector.

25. The method of claim 17, wherein the joined nucleic acid segments undergo intramolecular recombination to form a circular molecule.

26. The method of claim 25, wherein the recombination sites which undergo recombination to form the circular molecule are located at the 5′

and 3′

termini of the one or more of the nucleic acid segments.

27. The method of claim 17, wherein one or more of the nucleic acid segments encodes a selectable marker.

28. The method of claim 17, wherein one or more of the nucleic acid segments contains an origin of replication.

29. The method of claim 17, wherein some or all of the nucleic acid segments comprise nucleic acid molecules of one or more libraries.

30. The method of claim 17, wherein the one or more libraries comprise polynucleotides which encode variable domains of antibody molecules.

31. The method of claim 30, wherein at least one of the nucleic acid segments encodes a polypeptide linker for connecting variable domains of antibody molecules.

32. The method of claim 31, wherein the one or more libraries comprise polynucleotides which encode variable domains of antibody light and heavy chains.

33. The method of claim 17, further comprising screening to identify nucleic acid molecules which encode proteins having one or more identifiable activities.

34. The method of claim 33, wherein the one or more identifiable activities comprise binding specificity for one or more antigens.

35. The method of claim 33, wherein the one or more identifiable activities comprise an enzymatic activity.

36. The method of claim 33, wherein the one or more identifiable activities comprise an activity associated with a selectable marker.

37. The method of claim 17, wherein at least two of the nucleic acid segments encode expression products involved in the same biochemical pathway or biological process.

38. The method of claim 37, wherein the nucleic acid segments encode at least two different subunits of a multimeric enzyme complex.

39. The method of claim 17, wherein the nucleic acid segments encode at least two different enzymes which participate in reactions in the same biochemical pathway.

40. The method of claim 17, wherein the biochemical pathway leads to the production of an antibiotic or a carbohydrate.

41. A method of making a population of recombinant host cells comprising introducing nucleic acid segments joined by the method of claim 17 into a host cell.

42. A method of altering properties of a cell comprising introducing into the cell nucleic acid segments joined by the method of claim 17.

43. The method of claim 39, wherein the biochemical pathway leads to the post-translational modification of proteins.

44. The method of claim 43, wherein the post-translational modification is glycosylation or sialation.

45. The method of claim 44, wherein the cell is a bacterial cell.

46. A method of synthesizing a protein comprising:

(a) providing a nucleic acid molecule comprising at least one recombination site and comprising a coding sequence containing at least one suppressible stop codon;

(b) providing a vector comprising at least one recombination site and a coding sequence;

(c) causing recombination in vitro such that the nucleic acid molecule is inserted into the vector to produce a modified vector with the two coding sequences connected in frame and separated by said stop codon;

(d) transforming a host cell which expresses a suppressor tRNA with the modified vector; and

(e) causing expression of the two coding sequences such that a fusion protein encoded by at least a portion of both of the coding sequences is produced.

47. The method according to claim 46, wherein the stop codon is selected from the group consisting of amber, opal and ochre codons.

48. The method according to claim 46, wherein the vector comprises a gene which encodes at least one suppressor tRNA molecule.

49. The method according to claim 46, wherein the chromosome of the host cell comprises a gene which encodes at least one suppressor tRNA molecule.

50. The method according to claim 46, further comprising the steps of transforming the host cell with a nucleic acid molecule comprising a gene which encodes at least one suppressor tRNA molecule.

51. The method according to claim 46, wherein the fusion protein comprises an N- or C-terminal tag encoded by at least a portion of the vector.

52. The method according to claim 51, wherein the tag is selected from the group consisting of:

(a) glutathione S-transferase;

(b) β

-glucuronidase;

(c) green fluorescent protein;

(d) yellow fluorescent protein;

(e) red fluorescent protein;

cyan fluorescent protein;

(g) maltose binding protein;

(h) a six histidine tag; and

(i) an epitope tag.

53. A method for determining the gene expression profile in a cell or tissue comprising:

(a) generating at least one population of cDNA molecules from RNA obtained from the cell or tissue, wherein cDNA molecules of the population comprise at least one recombination site capable of recombining with at least one recombination site present on cDNA molecules of the same or a different population;

(b) contacting the nucleic acid molecules of (a) with one or more recombination proteins under conditions which cause the nucleic acid molecules to join;

(c) determining the sequence of the joined nucleic acid molecules; and

(d) comparing the sequence of said joined nucleic acid molecules to nucleic acid sequences cataloged in public databases to identify the gene expression profile.

54. The method of claim 53, wherein the joined cDNA molecules are inserted into a vector which contains sequencing primer binding sites flanking the insertion site.

55. The method of claim 53, wherein the joined cDNA molecules are separated by attB recombination sites.

56. The method of claim 55, wherein the attB recombination sites which recombine with each other have identical seven base pair overlap regions.

57. The method of claim 53, wherein the joined cDNA molecules contain between about 10 and about 30 nucleotides which corresponds to the RNA obtained from the cell or tissue.

58. A method of producing a population of hybrid nucleic acid molecules comprising:

(a) mixing at least a first population of nucleic acid and molecules, wherein;

one or more nucleic acid molecules of said population comprises one or more recombination sites, with at least one target nucleic acid molecule comprising one or more recombination sites;

(b) causing some or all of the nucleic acid molecules of the at least first population to recombine in vitro with all or some of the target nucleic molecules, thereby forming the population of hybrid nucleic acid molecules; and

(c) selecting for the population of hybrid nucleic acid molecules and against the first population of nucleic acid molecules and against the target nucleic acid molecules.

59. A method of cloning at least one nucleic acid molecule comprising:

(a) providing a first population of nucleic acid molecules wherein all or a portion of such molecules are flanked by a first and a second recombination site;

(b) providing at least one nucleic acid segment encoding a polypeptide selected from the group consisting of;

(i) the Fc portion of an immunoglobin;

(ii) β

-glucuronidase;

(iii) a fluorescent protein;

(iv) a purification tag;

(v) an epitope tag;

(vi) maltose binding protein;

(vii) a six histidine tag; and

(viii) glutathione S-transferase, flanked by a third and a fourth recombination site, wherein either the first or the second recombination site is capable of recombining with either the third or the fourth recombination site;

(c) conducting a recombination reaction in vitro such that all or a portion of the nucleic acid molecules in the population is recombined with the segment to form a second population of nucleic acid molecules; and

(d) cloning the second population of nucleic acid molecules.

60. A method of producing a nucleic acid molecule comprising:

(a) providing a first nucleic acid molecule comprising at least one recombination site and comprising a coding sequence containing at least one suppressible stop codon;

(b) providing a second nucleic acid molecule comprising at least one recombination site and a coding sequence; and

(c) causing recombination in vitro such that the first nucleic acid molecule is joined to the second nucleic acid molecule to produce a third nucleic acid molecule in which the two coding sequences are connected in frame and are separated by said stop codon.

61. The method of claim 60, wherein said first nucleic acid molecule and/or said second nucleic acid molecule and/or third nucleic acid molecule is a vector.

62. The method of claim 60, further comprising:

(d) transforming a host cell which expresses a suppressor tRNA with the third nucleic acid molecule.

63. The method of claim 60, wherein the stop codon is selected from the group consisting of amber, opal and ochre codons.

64. The method of claim 60, wherein said first and/or second nucleic acid molecule comprises a gene which encodes at least one suppressor tRNA molecule.

65. The method of claim 62 wherein the chromosome of the host cell comprises a gene which encodes at least one suppressor tRNA molecule.