Generation and selection of novel DNA-binding proteins

Abstract

Novel DNA-binding proteins, especially repressors of gene expression, are obtained by variegation of genes encoding known binding proteins and selection for proteins binding the desired target DNA sequence. A novel selection vector may be used to reduce artifacts. Heterooligomeric proteins which bind to a target DNA sequence which need not be palindromic are obtained by a variety of methods, e.g., variegation to obtain proteins binding symmetrized forms of the half-targets and heterodimerization to obtain a protein binding the entire asymmetric target.

Patent number: 5198346
Filing date: Jul 26, 1990
Issue date: Mar 30, 1993
Inventors: Robert C. Ladner, Sonia K. Guterman, Rachel B. Kent, Arthur C. Ley
Assignee: Protein Engineering Corp.
Primary Examiner: John D. Ulm


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What is claimed is:

1. A method of obtaining first and second genes encoding first and second homooligomeric DNA binding proteins which hybridize to form a hybrid heteroologomeric DNA binding protein which binds to a predetermined ultimate target double stranded DNA sequence, said sequence being nonpalindromic, said sequence comprising a left target-sequence and right target subsequence each of at least 4 base pairs length, said method comprising:

producing a first gene encoding a first DNA-binding oligomeric protein binding to a first target sequence and a second gene encoding a second DNA-binding oligomeric protein binding to a second target sequence, wherein said first and second DNA-binding proteins each have at least two essentially dyad-symmetric DNA-binding domains, where said first target sequence is a palindrome or gapped palindrome and comprises said left target subsequence and a palindrome-completing subsequence, and where said second target sequence is a palindrome or gapped palindrome and comprises said right target subsequence and a palindrome-completing subsequence, whereby one of the DNA-binding domains of the second DNA-binding protein binds to the right target subsequence, said genes being produced by a process of at least partially random mutation followed by selection for the binding of the corresponding protein to the corresponding target sequence,

wherein said first and second proteins can hybridize so as to obtain a heterooligomeric DNA-binding protein comprising a DNA-binding domain recognizing the left target subsequence and a DNA-binding domain recognizing the right target subsequence

whereby said heterooligomeric protein has an affinity for the ultimate target DNA.

2. The method of claim 1 wherein at least one of said first and second genes is obtained by

(a) providing a cell culture, said cell culture comprising a plurality of cells, each cell bearing a selection vector, said selection vector comprising a first and a second operon, each comprising at least one expressible gene, the genes of said first and second operons being different, a copy of the target DNA sequence being included in each operon and positioned therein so that under forward selection conditions the transformed cells enjoy a selective advantage if they express a protein or polypeptide which binds to said copies of the target DNA sequence, said cell culture being transformed with a variegated gene encoding potential DNA-binding proteins or polypeptides, where said cells collectively can express a plurality of different but sequence-related potential DNA-binding proteins or polypeptides,

(b) causing the cells of such culture to express said potential DNA-binding proteins or polypeptides;

(c) exposing the cells to forward selection conditions to select for cells which express a protein or polypeptide which preferentially binds to said target DNA sequence; and

(d) recovering the selected cells bearing a gene coding for such protein or polypeptide.

3. The method of claim 2 wherein the level of variegation is such that from 10.sup.6 to 10.sup.9 different potential DNA-binding proteins can be expressed.

4. The method of claim 2 wherein a gene coding for a known DNA binding protein having a helix-turn-helix DNA binding motif is variegated.

5. The method of claim 2 wherein a gene encoding a known DNA binding protein picked from the group consisting of Cro from phage .lambda., cI repressor from phage .lambda., Cro from phage 434, cI repressor from phage 434, P22 repressor, E. coli tryptophan repressor, E. coli CAP, P22 Arc, P22 Mnt, E. coli lactose repressor, MAT-a1-alpha2 from yeast, Polyoma Large T antigen, SV40 Large T antigen, Adenovirus E1A, and TFIIIA from Xenopus laevis is variegated to obtain genes coding on expression for a plurality of potential target DNA-binding proteins.

6. The method of claim 2 wherein said variegated gene comprises at least one variegated codon, said codon having three base positions, each variegated codon being characterized by a mixture of bases at at least one base position wherein the mixture of bases for at least one base position is non-equimolar.

7. The method of claim 2 wherein the ultimate target double stranded DNA sequence is an HIV sequence.

8. The method of claim 7 wherein the ultimate target doublet stranded DNA sequence is HIV 353-369 or a subsequence thereof comprising at least eight base paris.

9. The method of claim 2 wherein at least one of said operons comprises a selectable beneficial gene, an occludible promoter operably linked to said beneficial gene and directing its transcription, an occluding promoter occluding transcription of said beneficial gene, and a copy of the target DNA sequence positioned so that the binding of said protein or polypeptide to said copy represses said occluding promoter and thereby facilitates transcription of said beneficial gene.

10. The method of claim 9 wherein the beneficial gene is aadA.

11. The method of claim 10 wherein the occludible promoter is the aadA promoter and the occluding promoter is Pcon.

12. The method of claim 2 wherein said selection vector comprises:

a) a first operon, which operon comprises:

i) a first binding marker gene(s),

ii) a first promoter directing expression of said binding marker gene(s), and

iii) a first copy of the target DNA sequence, where said target DNA sequence interferes substantially with expression of the first gene(s) if and only if a protein expressed by the transformed cell binds to the target DNA sequence,

(b) a second operon, which operon comprises:

i) a second binding marker gene(s),

ii) a second promoter directing expression of said binding marker gene(s); and

iii) a second copy of the target DNA sequence, where said target DNA sequence interferes substantially with expression of said gene(s) if and only if a protein expressed by the transformed cell binds to the target DNA sequence,

where the binding marker genes of said first and second operons are different, and where, when said cells are exposed to forward selection conditions the gene products of said first and second binding marker genes are deleterious to the cell.

13. The method of claim 12 wherein the binding marker genes are functionally unrelated.

14. The method of claim 12 wherein the promoters of said first and second operons are different.

15. The method of claim 12 wherein a plurality of genetic elements essential to the maintenance of the vector or the survival of the transformed cells under conditions that select for presence of said vector, said operons and said genetic elements being positioned on said vector so no single deletion even can render nonfunctional more than one of said operons without also rendering nonfunctional one of said essential genetic elements.

16. The method of claim 12, said vector further comprising a gene (pdbp) coding for a potential DNA-binding protein or polypeptide, said gene comprising:

a) a coding region that codes for a polypeptide, each domain of said polypeptide having at least 50% sequence identity to a known DNA-binding domain, and

b) a promoter operably linked to said coding region for controlling its expression.

17. The method of claim 12 wherein at least one of said genetic elements comprises a beneficial gene, and a control promoter operably linked to said beneficial gene, but where no instance of said target DNA sequence is associated with said genetic element.

18. The method of claim 17 wherein the control promoter is essentially identical to the promoter of one of said selectable binding marker operons, so that proteins binding to the latter promoter will also bind to the control promoter and thereby inhibit expression of said beneficial gene.

19. The method of claim 12 wherein under reverse selection conditions the gene products of said binding marker genes are beneficial to the transformed cells.

20. The method of claim 19 wherein each of the first and second operons confers a phenotype selected independently but not-identically from the group consisting of: galT,K.sup.+, tetA.sup.+, lacZ.sup.+, pheS.sup.+, argP.sup.+, thyA.sup.+, crp.sup.+, pyrF.sup.+, ptsM.sup.+, secA.sup.+ /malE.sup.+ /lacZ.sup.+, ompA.sup.+, btuB.sup.+, lamB.sup.+, tonA.sup.+, cir.sup.+, tsx.sup.+, aroP.sup.+, cysK.sup.+, and dctA.sup.+.

21. The method of claim 12 wherein the vector comprises a plurality of codons, each variegated codon has a root mean square deviation from a flat distribution over the allowed amino acids of less than 0.08.

22. The method of claim 21 wherein the variation at each variegated codon allows all twenty possible amino acids.

23. The method of claim 22 wherein at any variegated codon the expected ratio of occurrence of (Lys+Arg) codons to (Asp+Glu) codon is 0.8 to 1.25.

24. A method of obtaining genes encoding a heterooligomeric protein which binds to a predetermined ultimate target double stranded DNA sequence, said sequence being nonpalindromic, said sequence comprising a left target subsequence and a right target subsequence each of at least 4 base pairs lengths, said method comprising:

(a) providing a first gene encoding a first DNA-binding oligomeric protein binding to a first target sequence and a second gene encoding a second DNA-binding oligomeric protein binding to a second target sequence, wherein said first and second DNA-binding proteins each have at least two dyad-symmetric DNA-binding domains, wherein said first and second DNA-binding proteins each have a dimerization interface, where said first target sequence is a palindrome or gaped palindrome and comprises said left target subsequence and a palindrome-completing subsequence, whereby one of the dyad-symmetric DNA-binding domains of the first DNA-binding protein binds to said left target subsequence, and where said second target sequence is a palindrome or gaped palindrome and comprises said right target subsequence and a palindrome completing subsequence, whereby one of the dyad-symmetric DNA-binding domains of the second DNA-binding protein binds to the right target subsequence,

(b) variegating the dimerization interface of the protein encoded by one of said first or second genes to obtain variegants thereof and reverse selecting for expression from said variegant of a first oligomerization mutant protein, encoded by a variegant of said variegated gene, which is no longer capable of forming a homooligomer that can bind to said first or second target sequence, respectively, and verifying that said oligomerization mutant protein maintains a tertiary structure similar to the protein form which is descended,

(c) variegating the dimerization interface of the protein encoded by the other of said first or second genes to obtain variegants thereof,

(d) providing host cells carrying the gene encoding said first oligomerization mutant protein and a variegant gene of step (c), each operably linked to a promoter functional in the host cell, and

(e) forward selecting for expression from a step (c) variegant gene of a second oligomerization mutant protein which is capable of forming a heterooligomer with said first oligomerization mutant protein, said heterooligomer binding said ultimate target DNA sequence, and

(f) isolating the genes encoding said heterooligomer.

25. The method of claim 24 wherein both of said first and second genes are provided by a process comprising (i) mutation of one or more preselected codons to encode a plurality of predetermined expected amino acids at each preselected codon, in predetermined expected proportions, and thereby obtain a plurality of different potential DNA binding proteins, and (ii) selection for genes encoding proteins which bind the corresponding target sequence.

26. The method of claim 25 in which the first and second DNA binding proteins either are unable to hybridize at all and still bind DNA, or, if they do hybridize, have a substantially diminished affinity or specificity for the corresponding subsequence of the target DNA sequence.

27. The method of claim 25 wherein at least one of said first and second genes is obtained by

(a) providing a cell culture, said cell culture comprising a plurality of cells, each cell bearing a selection vector, said selection vector comprising a first and a second operon, each comprising at least one expressible gene, the genes of said first and second operons being different, a copy of the target DNA sequence being included in each operon and positioned therein so that under forward selection conditions the transformed cells enjoy a selective advantage if they express a protein or polypeptide which binds to said copies of the target DNA sequence, said cell culture being transformed with a variegated gene encoding potential DNA-binding proteins or polypeptides, where said cells collectively can express a plurality of different but sequence-related potential DNA-binding proteins or polypeptides,

(b) causing the cells of such culture to express said potential DNA-binding proteins or polypeptides;

(c) exposing the cells to forward selection conditions to select for cells which express a protein or polypeptide which preferentially binds to said target DNA sequence; and

(d) recovering the selected cells bearing said first or second gene coding for such protein or polypeptide.

28. The method of claim 27 wherein the level of variegation is such that from 10.sup.6 to 10.sup.9 different potential DNA-binding proteins can be expressed.

29. The method of claim 27 wherein a gene coding for a known DNA binding protein having a helix-turn-helix DNA binding motif is variegated.

30. The method of claim 27 wherein a gene encoding a known DNA binding protein picked from the group consisting of Cro from phage .lambda., cI repressor from phage .lambda., Cro from phage 434, cI repressor from phage 434, P22 repressor, E. coli tryptophan repressor, E. coli CAP, P22 Arc, P22 Mnt, E. coli lactose repressor, MAT-a1-alpha2 from yeast, Polyoma Large T antigen, SV40 Large T antigen, Adenovirus E1A, and TFIIIA from Xenopus laevis is variegated to obtain genes coding on expression for a plurality of potential target DNA-binding proteins.

31. The method of claim 27 wherein said variegated gene comprises at least one variegated codon, said codon having three base positions, each variegated codon being characterized by a mixture of bases at at least one base position wherein the mixture of bases for at least one base position is non-equimolar.

32. The method of claim 27 wherein the ultimate target double stranded DNA sequence is an HIV sequence.

33. The method of claim 32 wherein the ultimate target double stranded DNA sequence is HIV 353-369 or a subsequence thereof comprising at least eight base paris.

34. The method of claim 27 wherein at least one of said operons comprises a selectable beneficial gene, an occludible promoter operably linked to said beneficial gene and directing its transcription, an occluding promoter occluding transcription of said beneficial gene, and a copy of the target DNA sequence positioned so that the binding of said protein or polypeptide to said copy represses said occluding promoter and thereby facilitates transcription of said beneficial gene.

35. The method of claim 34 wherein the beneficial gene is aadA.

36. The method of claim 35 wherein the occludible promoter is the aadA promoter and the occluding promoter is Pcon.

37. The method of claim 27 wherein said selection vector comprises:

a) a first operon, which operon comprises:

i) a first binding marker gene(s),

ii) a first promoter directing expression of said binding marker gene(s), and

iii) a first copy of the target DNA sequence, where said target DNA sequence interferes substantially with expression of the first gene(s) if and only if a protein expressed by the transformed cell binds to the target DNA sequence,

(b) a second operon, which operon comprises:

i) a second binding marker gene(s),

ii) a second promoter directing expression of said binding marker gene(s); and

iii) a second copy of the target DNA sequence, where said target DNA sequence interferes substantially with expression of said gene(s) if and only if a protein expressed by the transformed cell binds to the target DNA sequence,

where the binding marker genes of said first and second operons are different, and where, when said cells are exposed to forward selection conditions the gene products of said first and second binding marker genes are deleterious to the cell.

38. The method of claim 37 wherein the binding marker genes are functionally unrelated.

39. The method of claim 37 wherein the promoters of said first and second operons are different.

40. The method of claim 37 wherein a plurality of genetic elements essential to the maintenance of the vector or the survival of the transformed cells under conditions that select for presence of said vector, said operons and said genetic elements being positioned on said vector so no single deletion event can render nonfunctional more than one of said operons without also rendering nonfunctional one of said essential genetic elements.

41. The method of claim 37, said vector further comprising a gene (pdbp) coding for a potential DNA-binding protein or polypeptide, said gene comprising:

a) a coding region that codes for a polypeptide, each domain of said polypeptide having at least 50% sequence identity to a known DNA-binding domain, and

b) a promoter operably linked to said coding region for controlling its expression.

42. The method of claim 37 wherein at least one of said genetic elements comprises a beneficial gene, and a control promoter operably linked to said beneficial gene, but where no instance of said target DNA sequence is associated with said genetic element.

43. The method of claim 42 wherein the control promoter is essentially identical to the promoter of one of said selectable binding marker operons, so that proteins binding to the latter promoter will also bind to the control promoter and thereby inhibit expression of said beneficial gene.

44. The method of claim 37 wherein under reverse selection conditions the gene products of said binding marker genes are beneficial to the transformed cells.

45. The method of claim 44 wherein each of the first and second operons confers a phenotype selected independently but not-identically from the group consisting of: galT,K.sup.+, tetA.sup.+, lacZ.sup.+, pheS.sup.+, argP.sup.+, thyA.sup.+, crp.sup.+, pyrF.sup.+, ptsM.sup.+, secA.sup.+ /malE.sup.+ /lacZ.sup.+, ompA.sup.+, btuB.sup.+, lamB.sup.+, tonA.sup.+, cir.sup.+, tsx.sup.+, aroP.sup.+, cysK.sup.+, and dctA.sup.+.

46. The method of claim 37 wherein the vector comprises a plurality of codons, each variegated codon has a root mean square deviation from a flat distribution over the allowed amino acids of less than 0.08.

47. The method of claim 46 wherein the variation at each variegated codon allows all twenty possible amino acids.

48. The method of claim 47 wherein at any variegated codon the expected ratio of occurrence of (Lys+Arg) codons to (Asp+Glu) codon is 0.8 to 1.25.

Determining DNA sequences by mass spectrometry

Abstract

This invention relates to the methods, apparatus, reagents and mixtures of reagents for sequencing natural or recombinant DNA and other polynucleotides. In particular, this invention relates to a method for sequencing polynucleotides based on mass spectrometry to determine which of the four bases (adenine, guanine, cytosine or thymine) is a component of the terminal nucleotide. In particular, the present invention relates to identifying the individual nucleotides by the mass of stable nuclide markers contained within either the dideoxynucleotides, the DNA primer, or the deoxynucleotide added to the primer. This invention is particularly useful in identifying specific DNA sequences in very small quantities in biological products produced by fermentation or other genetic engineering techniques. The invention is therefore useful in evaluating safety and other health concerns related to the presence of DNA in products resulting from genetic engineering techniques.

Patent number: 5003059
Filing date: Jun 20, 1988
Issue date: Mar 26, 1991
Inventor: Thomas M. Brennan
Assignee: Genomyx, Inc.
Primary Examiner: Gary L. Kunz


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What is claimed is:

1. In a process for determining DNA sequence by the dideoxynucleotide chain termination method, the improvement comprising incorporating .sup.32 S, .sup.33 S, .sup.34 S and .sup.36 S in the formation of the chain terminated sequence so that a unique sulfur isotope is associated with the terminal nucleotide in the chain terminated sequence, separating the chain terminated sequences by capillary gel electrophoresis, combusting the separated chain terminated sequences to convert the incorporated sulfur to SO.sub.2, and determining the terminal nucleotide by measuring .sup.64 SO.sub.2, .sup.65 SO.sub.2, .sup.67 SO.sub.2, and .sup.68 SO.sub.2 in a mass spectrometer thereby determining the sequence of the DNA.

2. A process according to claim 1 wherein the isotope is incorporated only into the 2', 3'-dideoxyribonucleotides.

3. A process according to claim 1 wherein the isotope is incorporated only into the 2'-deoxyribonucleotides.

4. A process according to claim 1 wherein the isotope is incorporated into a DNA primer.

Processes for inserting DNA into eucaryotic cells

Abstract

The present invention relates to processes for inserting DNA into eucaryotic cells, particularly DNA which includes a gene or genes coding for desired proteinaceous materials for which no selective criteria exist. The insertion of such DNA molecules is accomplished by cotransforming eucaryotic cells with such DNA together with a second DNA which corresponds to a gene coding for a selectable marker.

Patent number: 4399216
Filing date: Feb 25, 1980
Issue date: Aug 16, 1983
Inventors: Richard Axel, Michael H. Wigler, Saul J. Silverstein
Assignee: The Trustees of Columbia University

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What is claimed is:

1. A process for inserting foreign DNA I into a suitable eucaryotic cell which comprises cotransforming said eucaryotic cell with said foreign DNA I and with unlinked foreign DNA II which codes for a selectable phenotype not expressed by said ducaryotic cell, said cotransformation being carried out under suitable conditions permitting survival or identification of eucaryotic cells which have acquired said selectable phenotype, said foreign DNA I being incorporated into the chromosomal DNA of said eucaryotic cell.

2. A process in accordance with claim 1 wherein said foreign DNA I codes for proteinaceous material which is not associated with a selectable phenotype.

3. A process in accordance with claim 2 wherein said foreign DNA I codes for interferon protein.

4. A process in accordance with claim 2 wherein said foreign DNA I codes for insulin.

5. A process in accordance with claim 2 wherein said foreign DNA I codes for growth hormone.

6. A process in accordance with claim 2 wherein said foreign DNA I codes for a clotting factor.

7. A process in accordance with claim 2 wherein said foreign DNA I codes for a viral antigen or an antibody.

8. A process in accordance with claim 2 wherein said foreign DNA I codes for an enzyme.

9. A process in accordance with claim 1 wherein said foreign DNA I is substantially purified.

10. A process in accordance with claim 1 wherein said foreign DNA I has been obtained from restriction endonuclease cleavage of eucaryotic chromosomal DNA.

11. A process in accordance with claim 1 wherein said foreign DNA I and DNA II have been treated with calcium phosphate.

12. A process in accordance with claim 1 wherein said eucaryotic cell is a mammalian cell.

13. A process in accordance with claim 12 wherein said mammalian cell is an erythroblast.

14. A process in accordance with claim 12 wherein said mammalian cell is a fibroblast.

15. A process in accordance with claim 1 wherein said foreign DNA I is present in an amount relative to said DNA II which codes for a selectable phenotype in the range from about 1:1 to about 100,000:1.

16. A process in accordance with claim 1 wherein said DNA II which codes for a selectable phenotype comprises the gene for thymidine kinase from herpes simplex virus.

17. A process in accordance with claim 1 wherein said DNA II which codes for proteinaceous material which is associated with a selectable phenotype comprises the gene for adenine phosphoribosyltransferase.

18. A process in accordance with claim 1 wherein said DNA II which codes for a selectable phenotype comprises a gene associated with drug resistance.

19. A process in accordance with claim 18 wherein said gene associated with drug resistance is the gene coding for a mutant dihydrofolate reductase which renders cells resistant to methotrexate.

20. A eucaryotic cell into which foreign DNA I has been inserted in accordance with the process of claim 1.

21. A mammalian cell into which foreign DNA I has been inserted in accordance with the process of claim 1.

22. A process for producing a foreign proteinaceous material which comprises cotransforming a eucaryotic cell in accordance with the process of claim 1, culturing or cloning said cotransformed eucaryotic cell under suitable conditions to yield a multiplicity of eucaryotic cells producing said foreign proteninaceous material and recovering said proteinaceous material from said eucaryotic cells.

23. A process in accordance with claim 22 wherein said proteinaceous material comprises interferon protein, insulin, growth hormone, clotting factor, viral antigen or antibody.

24. A process in accordance with claim 22 wherein said eucaryotic cell is a mammalian cell.

25. A method of detecting eucaryotic cells which have been transformed with foreign DNA I which is not associated with a selectable phenotype which comprises cotransforming said eucaryotic cell with said DNA I and with DNA II which is associated with a selectable phenotype in accordance with the process of claim 1, and screening for eucaryotic cells so cotransformed.

26. A process for inserting foreign DNA I into a eucaryotic cell which comprises cotransforming said eucaryotic cell with said foreign DNA I and with unlinked foreign DNA II which codes for a selectable phenotype not expressed by said eucaryotic cell, said cotransformation being carried out in a suitable medium and in the presence of conditions permitting identification and recovery of eucaryotic cells which have acquired said selectable phenotype.

27. A process for cotransforming a suitable eucaryotic cell which comprises transforming under suitable conditions said eucaryotic cell with foreign DNA I and with foreign DNA II, said DNA I and DNA II being unlinked and said DNA II coding for a selectable phenotype not expressed by said eucaryotic cell prior to cotransformation.

28. A process for inserting purified foreign DNA I coding for proteinaceous material which is not associated with a selectable phenotype into a suitable eucaryotic cell which comprises cotransforming said eucaryotic cell with said foreign DNA I and with unlinked foreign DNA II coding for proteinaceous material which is associated with a selectable phenotype, said cotransformation being carried out under suitable conditions permitting survival or identification of eucaryotic cells which have acquired said selectable phenotype, said foreign DNA I being incorporated into the chromosomal DNA of said eucaryotic cell.

29. A process in accordance with claim 28 wherein said proteinaceous material which is not associated with a selectable phenotype comprises interferon protein, insulin, growth hormone, clotting factor, viral antigen or antibody.

30. A eucaryotic cell into which foreign DNA I has been inserted in accordance with the process of claim 28.

31. A process for inserting a multiplicity of foreign DNA I molecules corresponding to multiple copies of a gene coding for a proteinaceous material into a suitable eucaryotic cell which comprises cotransforming said eucaryotic cell with said multiplicity of foreign DNA I molecules and with a multiplicity of unlinked foreign DNA II molecules coding for a selectable phenotype not expressed by said eucaryotic cell, said cotransformation being carried out under suitable conditions permitting survival or identification of eucaryotic cells which have acquired said multiplicity of genes coding for said selectable phenotype.

32. A process in accordance with claim 31 wherein said foreign DNA I codes for proteinaceous material which is not associated with a selectable phenotype.

33. A process in accordance with claim 32 wherein said foreign DNA I codes for interferon protein.

34. A process in accordance with claim 32 wherein said foreign DNA I codes for insulin.

35. A process in accordance with claim 32 wherein said foreign DNA I codes for growth hormone.

36. A process in accordance with claim 32 wherein said foreign DNA I codes for a clotting factor.

37. A process in accordance with claim 32 wherein said foreign DNA I codes for a viral antigen or an antibody.

38. A process in accordance with claim 32 wherein said foreign DNA I codes for an enzyme.

39. A process in accordance with claim 31 wherein said foreign DNA I is substantially purified.

40. A process in accordance with claim 31 wherein said foreign DNA I has been obtained from restriction endonuclease cleavage of eucaryotic chromosomal DNA.

41. A process in accordance with claim 31 wherein said foreign DNA I and DNA II have been treated with calcium phosphate.

42. A process in accordance with claim 31 wherein said eucaryotic cell is a mammalian cell.

43. A process in accordance with claim 42 wherein said mammalian cell is an erythroblast.

44. A process in accordance with claim 42 wherein said mammalian cell is a fibroblast.

45. A process in accordance with claim 31 wherein said foreign DNA I is present in an amount relative to said DNA II which codes for proteinaceous material associated with a selectable phenotype in the range from about 1:1 to about 100,000:1.

46. A process in accordance with claim 31 wherein said foreign DNA II which does for proteinaceous material which is associated with a selectable phenotype comprises a gene associated with drug resistance.

47. A process in accordance with claim 46 wherein said gene associated with drug resistance is a gene coding for a mutant dihydrofolate reductase which renders cells resistant to methotrexate.

48. A process in accordance with claim 31 wherein said foreign DNA I is incorporated into the chromosomal DNA of said eucaryotic cell.

49. A eucaryotic cell into which foreign DNA I has been inserted in accordance with the process of claim 31.

50. A mammalian cell into which foreign DNA I has been inserted in accordance with the process of claim 31.

51. A process for producing a foregin proteinaceous material which comprises cotransforming a eucaryotic cell in accordance with the process of claim 31, maintaining said cotransformed eucaryotic cell under suitable conditions to produce said foreign proteinaceous material, and recovering said proteinaceous material so produced.

52. A process in accordance with claim 51 wherein said proteinaceous material comprises interferon protein, insulin, growth hormone, clotting factor, viral antigen or antibody.

53. A process in accordance with claim 51 wherein said eucaryotic cell is mammalian cell.

54. A process for generating a multiplicity of foregin DNA I molecules corresponding to multiple copies of a gene in a eucaryotic cell which comprises tranforming said eucaryotic cell with a molecule which is formed by linking one of said foreign DNA I molecules to a DNA II molecule corresponding to an amplifiable gene for a dominant selectable phenotype not expressed by said eucaryotic cell, and culturing the transformed eucaryotic cells in the presence of successively elevated concentrations of an agent permitting survival or identification of eucaryotic cells which have acquired multiple copies of said amplifiable gene, said transformation and culturing being carried out under suitable conditions.

55. A process in accordance with claim 54 wherein said foreign DNA I codes for proteinaceous material which is not associated with a selectable phenotype.

56. A process in accordance with claim 55 wherein said foreign DNA I codes for interferon protein.

57. A process in accordance with claim 55 wherein said foreign DNA I codes for insulin.

58. A process in accordance with claim 55 wherein said foreign DNA I codes for growth hormone.

59. A process in accordance with claim 55 wherein said foreign DNA I codes for a clotting factor.

60. A process in accordance with claim 55 wherein said foreign DNA I codes for a viral antigen or antibody.

61. A process in accordance with claim 55 wherein said foregin DNA I codes for an enzyme.

62. A process in accordance with claim 54 wherein said foreign DNA I is substantially purified.

63. A process in accordance with claim 54 wherein said foreign DNA I has been obtained from restriction endonuclease cleavage of eucaryotic chromosomal DNA.

64. A process in accordance with claim 54 wherein said foreign DNA I and DNA II have been treated with calcium phosphate.

65. A process in accordance with claim 54 wherein said eucaryotic cell is mammalian cell.

66. A process in accordance with claim 65 wherein said mammalian cell is an erythroblast.

67. A process in accordance with claim 65 wherein said mammalian cell is a fibroblast.

68. A process in accordance with claim 54 wherein said foreign DNA I is present in an amount relative to said DNA II which codes for proteinaceous material associated with a selectable phenotype in the range from about 1:1 to about 100,000:1.

69. A process in accordance with claim 54 wherein said DNA II which codes for proteinaceous material which is associated with a selectable phenotype comprises a gene associated with resistance to a drug or chemical antagonist.

70. A process in accordance with claim 69 wherein said gene associated with resistance to a drug or chemical anatgonist is a gene coding for a mutant dihydrofolate reductase which renders cells resistant to methotrexate.

71. A process in accordance with claim 54 wherein said foreign DNA I is incorporated into the chromosomal DNA of said eucaryotic cell.

72. A eucaryotic cell into which foreign DNA I has been inserted in accordance with the process of claim 54.

73. A mammalian cell into which foreign DNA I has been inserted in accordance with the process of claim 54.

Specific DNA probes in diagnostic microbiology

Abstract

Method and compositions for infectious disease diagnosis and epidemiology involving labeled nucleotide probes complementary to nucleic acid coding for a characteristic pathogen product. Clinical isolates are cultivated, expanding the number of microorganisms, the resulting colonies lysed, the genome normally denatured and then fixed. Alternatively, clinical samples (stool, sputum, pus, etc.) are spotted onto an inert support. The sample is treated in such a way that the DNA is liberated from microbes present in the sample and complexed onto the support. The DNA is normally denatured and fixed in this process. Subsequently, a labelled polynucleotide probe specific for a DNA sequence characteristic of a pathogenic product suspected of being present in the clinical sample is contacted with the fixed genomic single stranded nucleic acid under hybridizing conditions. Hybridization of probes to the single stranded nucleic acid is diagnostic of the presence of the pathogen.

Patent number: 4358535
Filing date: Dec 8, 1980
Issue date: Nov 9, 1982
Inventors: Stanley Falkow, Stephen L. Moseley
Assignee: Board of Regents of the University of Washington


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What is claimed is:

1. A method for detecting the presence of a pathogen in a clinical sample suspected of containing said pathogen, said method comprising:

depositing said sample on an inert support;

treating said sample to affix genetic material of any of said pathogen present in said sample to said support in substantially single stranded form at substantially the same site on said support where said sample was deposited;

contacting said fixed single stranded genetic material with a labeled probe having a nucleotide sequence of at least about 25 bases at least substantially complementary to a nucleotide sequence of a structural gene characteristic of said pathogen, said contacting being under hybridizing conditions at a predetermined stringency; and

detecting duplex formation on said support by means of said label.

2. A method according to claim 1, including the additional step of cultivating said deposited sample to produce at least one individual colony.

3. A method according to claims 1 or 2, wherein said depositing is performed by streaking or spotting.

4. A method according to claim 2, wherein said support is an inert porous filter and said cultivating comprises:

maintaining said filter in contact with a nutrient gel; and said contacting comprises

placing said filter on a bibulous material wetted with reagent solution capable of removing other than genetic material to leave single stranded genetic material.

5. A method according to claims 1, 2 or 4, wherein said pathogen is a unicellular organism.

6. A method according to claim 5, wherein said unicellular organism is a bacterium.

7. A method according to claims 1, 2 or 4, wherein said pathogen is a virus.

8. A method according to claims 1, 2 or 4, wherein said pathogen is a multicellular organism.

9. A method according to claims 1, 2 or 4, wherein said structural gene codes for an excreted product.

10. A method according to claims 1, 2 or 4, wherein said structural gene codes for a cytoplasmic product.

11. A method for detecting the presence of a unicellular pathogen in a sample suspected of containing said pathogen, said method comprising:

depositing said sample on an inert porous filter as a plurality of individual portions;

transferring said filter to a bibulous material wetted with a reagent solution capable of lysing said pathogen and denaturing the genetic material of said pathogen to provide single stranded DNA;

heating said filter to fix said single stranded DNA at substantially the same site as the individual portion from which said genetic material is derived;

contacting said fixed single stranded DNA with a labeled probe having a nucleotide sequence of at least about 25 bases at least substantially complementary to a nucleotide sequence of a structural gene characteristic of said pathogen under hybridizing conditions of a predetermined stringency,; and

detecting duplex formation on said support by means of said label.

12. A method according to claim 11, including the step of:

cultivating said individual portions on said inert porous filter by contacting said filter with a nutrient gel to produce individual colonies of said pathogen, and wherein said reagent solution is a dilute aqueous alkaline solution.

13. A method according to claims 11 or 12, wherein said label is a radionuclide.

14. A method according to claims 11 or 12, wherein said label is a fluorescent molecule.

15. A method according to claims 11 or 12, wherein said unicellular pathogen is a bacterium.

16. A method according to claim 15, wherein said label is a radionuclide.

17. A method for detecting the presence of a gram negative bacillus in a clinical isolate suspected of containing said bacillus, said method comprising:

spotting said clinical isolate onto an inert porous filter;

contacting said spotted inert porous filter with a nutrient gel, whereby nutrients diffuse to said bacillus in said spot, whereby a colony forms;

transferring said filter supporting said colony onto a bibulous material containing a reagent solution for lysing said bacillus and denaturing the genome of said bacillus to provide single stranded DNA at substantially the same site as said colony;

heating said filter to fix said single stranded DNA to said filter;

contacting said filter with said fixed single stranded DNA, with a radioactively labeled probe having a nucleotide sequence of at least about 25 bases and at least substantially complementary to a nucleotide sequence of a structural gene characteristic of said bacillus under hybridizing conditions of a predetermined stringency; and

detecting duplex formation on said support by means of said radioactive isotope.

18. A method according to claim 17, wherein said structural gene codes for a released product.

19. A method according to claim 18, wherein said released product is a toxin.

20. A method according to claims 17, 18 or 19, wherein said bacillus is enterotoxigenic Escherichia coli.

Monoclonal antibody specific for DNA.RNA hybrids

Abstract

A monoclonal antibody specific for DNA.multidot.RNA duplexes, particularly DNA.multidot.RNA heteropolymer duplexes, characterized by having cross-reactivity for binding to single- or double-stranded DNA or RNA as measured by competitive immunoassay of less than about 1:1000, and preferably less than 1:10,000, and an affinity for DNA.multidot.RNA heteropolymer duplexes greater than 10.sup.9 L/mole. The monoclonal antibody is prepared by conventional somatic cell hybridization techniques wherein the host animal is preferably immunized with an immunogen comprising a random DNA.multidot.RNA heteropolymer. The antibody, particularly in a labeled form, is useful in the specific detection of DNA.multidot.RNA duplexes in a test medium such as a nucleic acid hybridization assay mixture.

Patent number: 4833084
Filing date: Aug 26, 1985
Issue date: May 23, 1989
Inventor: Robert J. Carrico
Assignee: Miles Inc.
Primary Examiner: Jeremy M. Jay

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What is claimed is:

1. The monoclonal antibody secreted by hybridoma cell line ATCC HB 8730.

2. Hybridoma cell line ATCC HB 8730.

Arabinonucleic acid probes for DNA/RNA assays

Abstract

A novel nucleic acid, arabinonucleic acid, is provided as a probe in nucleic acid assays. The arabinose moiety of the probe can be detected with anti-arabinose antibody-label conjugates.

Patent number: 4760017
Filing date: Dec 23, 1985
Issue date: Jul 26, 1988
Inventor: Randy M. McCormick
Assignee: E. I. Du Pont de Nemours and Company
Primary Examiner: Robert Benson

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What is claimed is:

1. A probe for the detection of a preselected nucleic acid sequence comprising single stranded arabinonucleic acid consisting essentially of arabinonucleotides having 3' and 5' internucleotide linkages with a base linked at the 1' position, said base being selected from the group consisting essentially of adenine, guanine, cytosine, thymine and uracil, said bases having a sequence complementary to said preselected nucleic acid sequence.

2. A method for identifying a preselected nucleic acid sequence in a sample comprising the steps of:

(a) rendering the nucleic acid in said sample single-stranded;

(b) immobilizing the single-stranded nucleic acids onto a support;

(c) contacting said immobilized single-stranded nucleic acids with a single-stranded arabinonucleic acid consisting essentially of arabinonucleotides having 3' and 5' internucleotide linkages with a base linked at the 1' position, said base being selected from the group consisting essentially of adenine, guanine, cytosine, thymine and uracil, said bases having a sequence complementary to said preselected nucleic acid sequence, under conditions that allow a hybridization reaction to occur;

(d) washing said support to remove arabinonucleic acid not incorporated into the hybrid formed on the support; and

(e) determining the presence of arabinonucleic acid in the hybrid formed on the support by contacting it with an anti-arabinose antibody-label conjugate and detecting said label.

3. The method of claim 2 wherein the label is an enzyme.

Product and process for isolating DNA, RNA and proteins

Abstract

Solutions and methods are disclosed for the effective, simple isolation/extraction of DNA, RNA and proteins from a single biological material sample, such as cells, tissues and biological fluids. The preferred solutions include effective amounts of a chaotropic agent(s), buffer, reducing agent, and may or may not include an organic solvent. Genomic DNA and total RNA can be isolated utilizing the solutions and methods of the invention in as little as 20 minutes, and proteins in as little as 30 minutes.

Patent number: 5945515
Filing date: Jul 31, 1995
Issue date: Aug 31, 1999
Inventor: Piotr Chomczynski
Primary Examiner: David S. Romeo

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What is claimed is:

1. A solution for isolating substantially pure and undegraded RNA, DNA and proteins from biological material, said solution comprising:

at least one chaotropic agent,

a buffer present in an amount sufficient to maintain the pH of said solution in the range of about 6 to about 7.5.

an organic solvent present at a concentration in the range of about 13 to about 23% (v/v) of said solution, and

at least one chelating agent.

2. The solution of claim 1 wherein said chelating agent is selected from the group consisting of ethylenediamine tetraacetic acid and citric acid.

3. The solution of claim 1 further comprising a detergent.

4. The solution of claim 3 wherein said detergent is selected from the group consisting of sarcosine and polyoxyethylenesorbitan.

5. A method of isolating substantially pure and undegraded RNA, DNA and proteins from biological material, comprising the steps of:

a) homogenizing a biological material sample in the solution of claim 2 to form an homogenate;

b) recovering substantially pure, undegraded RNA from the homogenate by sedimentation;

c) thereafter precipitating DNA in the remaining homogenate by adding an additional amount of organic solvent thereto, and recovering the precipitated DNA by one of sedimentation or spooling; and

d) thereafter precipitating proteins from the remaining homogenate by adding an additional amount of organic solvent thereto, and recovering the precipitated proteins by sedimentation.

6. The method of claim 5 wherein said additional amount of organic solvent added to precipitate DNA is added to achieve a concentration in the range of about 28 to about 38% (v/v).

7. The method of claim 6 wherein said organic solvent is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethylsulfoxide.

8. The method of claim 5 wherein said additional amount of organic solvent added to precipitate proteins is added to achieve a concentration in the range of about 78 to about 82% (v/v).

9. The method of claim 8 wherein said organic solvent is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethylsulfoxide.

10. A method of isolating substantially pure and undegraded RNA from biological material, comprising the steps of:

a) homogenizing a biological material sample in a solution of claim 1 to form an homogenate; and

b) recovering substantially pure RNA from the homogenate by sedimentation.

11. A method of isolating substantially pure and undegraded DNA from biological material, comprising the steps of:

a) homogenizing a biological material sample in a solution of claim 1 to form an homogenate; and

b) precipitating DNA in the homogenate by adding an additional amount of an organic solvent thereto for a concentration of organic solvent in the range of about 28% to about 38% (v/v), and recovering the precipitated DNA by one of spooling and sedimentation.

12. The method of claim 11 wherein said organic solvent is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethylsulfoxide.

13. A method of isolating substantially pure and undegraded RNA, DNA and proteins from biological material, comprising the steps of:

a) homogenizing a biological material sample in a solution comprising:

at least one chaotropic agent, and

a buffer present in an amount sufficient to maintain the pH of said solution in the range of about 6 to about 7.5, to form an homogenate;

b) removing unhomogenized material from the homogenate by sedimentation;

c) precipitating RNA in the remaining homogenate by adding thereto an organic solvent to achieve a concentration in the range of about 13 to about 23% (v/v), and recovering the precipitated RNA by sedimentation;

d) thereafter precipitating DNA in the remaining homogenate by adding an additional amount of organic solvent thereto, and recovering the precipitated DNA by one of spooling or sedimentation; and

e) thereafter precipitating proteins from the remaining homogenate by adding an additional amount of an organic solvent thereto, and recovering the precipitated proteins by sedimentation.

14. The method of claim 13 wherein said organic solvent used for precipitating RNA is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethysulfoxide.

15. The method of claim 13 wherein said additional amount of organic solvent added to precipitate DNA is added to achieve a concentration in the range of about 28 to about 38% (v/v).

16. The method of claim 15 wherein said organic solvent is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethylsulfoxide.

17. The method of claim 13 wherein said additional amount of organic solvent added to precipitate proteins is added to achieve a concentration in the range of about 78 to about 82% (v/v).

18. The method of claim 17 wherein said organic solvent is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethylsulfoxide.

19. A method of isolating substantially pure and undegraded RNA from biological material, consisting essentially of the following steps:

a) homogenizing a biological material sample in a solution consisting essentially of:

at least one chaotropic agent, and

a buffer present in an amount sufficient to maintain the pH of said solution in the range of about 6 to about 7.5, to form an homogenate;

b) removing unhomogenized material from the homogenate by sedimentation; and

c) precipitating RNA in the homogenate by adding thereto an organic solvent to achieve a concentration in the range of about 13 to about 23% (v/v), and recovering the precipitated RNA by sedimentation.

20. The method of claim 19 wherein said organic solvent is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethylsulfoxide.

21. A method of isolating substantially pure and undegraded DNA from biological material, consisting essentially of the following steps:

a) homogenizing a biological material in a solution comprising:

at least one chaotropic agent, and

a buffer present in an amount sufficient to maintain the pH of said solution in the range of about 8 to about 12, to form an homogenate;

b) removing unhomogenized material from the homogenate by sedimentation, and

c) precipitating DNA in the homogenate by adding thereto an organic solvent to achieve a concentration in the range of about 28 to about 38% (v/v), and recovering the precipitated DNA by one of spooling or sedimentation.

22. The method of claim 21 wherein said organic solvent is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethylsulfoxide.

Shelf-stable product and process for isolating RNA, DNA and proteins

Abstract

Shelf-stable solvent solutions and methods for simultaneously isolating RNA, DNA and proteins from biological samples are disclosed. The solvent solutions include phenol and a guanidinium compound, preferably at a concentration below about 2M, which is effective in isolating substantially pure and undegraded RNA, substantially pure and undegraded DNA, and proteins from the same biological sample.

Patent number: 5346994
Filing date: Jan 28, 1992
Issue date: Sep 13, 1994
Inventor: Piotr Chomczynski

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What is claimed is:

1. A solvent solution comprising: effective amounts of phenol, a guanidinium compound and a thiocyanate compound selected from the group consisting of ammonium thiocyanate and sodium thiocyanate for extracting substantially pure and undegraded RNA, substantially pure and undegraded DNA, and proteins from biological tissue.

2. The solvent solution of claim 1, said thiocyanate compound being present at a concentration in the range of about 0.1-0.6M, based on the total volume of said solvent solution.

3. A solvent solution comprising: effective amounts of phenol, a guanidinium compound and sodium acetate present at a concentration of about 0.1M, based on the total volume of said solvent solution, said solvent solution having a pH of about 5.0 for extracting substantially pure and undegraded RNA, substantially pure and undegraded DNA, and proteins from biological tissue.

4. A solvent solution comprising: effective amounts of phenol, a guanidium compound and a phenol solubilizer for extracting substantially pure and undegraded RNA, substantially pure and undegraded DNA, and proteins from biological tissue.

5. The solvent solution of claim 4, said phenol solubilizer being glycerol.

6. The solvent solution of claim 5, said glycerol being present in the range of about 3%-10% by volume of said solvent solution, based on the total volume of said solvent solution.

7. The solvent solution of claim 6, said phenol being present in the range of about 30%-50% by volume of said solvent solution, based on the total volume of said solvent solution.

8. A solvent solution for extracting substantially pure RNA, DNA and proteins from biological tissue, said solvent solution comprising:

(a) guanidinium thiocyanate at a concentration in the range of about 0.5-2M, based on the total volume of said solvent solution;

(b) a buffer in an amount sufficient to maintain the pH of said solvent solution in the range of about 4-6;

(c) phenol in the amount of about 30%-50% by volume based on the total volume of said solvent solution; and

(d) a phenol solubilizer in the amount of about 3%-10% by volume based on the total volume of said solvent solution.

9. The solvent solution of claim 8 further comprising ammonium thiocyanate at a concentration in the range of about 0.1-0.6M, based on the total volume of said solvent solution.

10. The solvent solution of claim 8, said phenol solubilizer being glycerol.

11. The solvent solution of claim 8, said guanidinium thiocyanate concentration being about 0.8M.

12. The solvent solution of claim 9, said ammonium thiocyanate concentration being about 0.4M.

13. The solvent solution of claim 8, said buffer being sodium acetate.

14. The solvent solution of claim 13, said sodium acetate being present at a concentration of about 0.1M, based on the total volume of said solvent solution, said solvent solution having a pH of about 5.0.

15. The solvent solution of claim 10, said glycerol comprising about 5% by volume of said solvent solution.

16. The solvent solution of claim 8, said phenol comprising about 38% by volume of said solvent solution.

17. A method of isolating substantially pure RNA, DNA and proteins from biological tissue, comprising the steps of:

(a) homogenizing a tissue sample in the solvent solution of claim 1 to form a homogenate;

(b) adding a water-insoluble organic solvent to said homogenate and sedimenting to form a mixture consisting of an aqueous phase containing substantially pure, undegraded RNA, an organic phase containing proteins, and an interphase containing substantially pure, undegraded DNA;

(c) precipitating RNA from the aqueous phase by the addition of a lower alcohol thereto and recovering the precipitated RNA by sedimentation;

(d) precipitating proteins from the organic phase by the addition of a lower alcohol thereto and recovering the precipitated proteins by sedimentation; and

(e) recovering DNA from the interphase by washing the interphase with a predetermined amount of said solvent solution, sedimentation of the DNA and removal of any phenol and salt contamination from the DNA.

18. The method of claim 17 wherein said water-insoluble organic solvent added to said homogenate is chloroform.

19. The method of claim 17 wherein the lower alcohol added to the aqueous phase is isopropanol.

20. The method of claim 17 wherein the lower alcohol added to the organic phase is isopropanol.

21. A method of isolating substantially pure RNA, DNA and proteins from biological tissue, comprising the steps of:

(a) homogenizing a tissue sample in the solvent solution of claim 1 to form a homogenate;

(b) adding a water-insoluble organic solvent to said homogenate and sedimenting to form a mixture consisting of an aqueous phase containing substantially pure, undegraded RNA, an organic phase containing proteins, and an interphase containing substantially pure, undegraded DNA;

(c) precipitating RNA from the aqueous phase by the addition of a lower alcohol thereto and recovering the precipitated RNA by sedimentation;

(d) extracting the organic phase and interphase with water;

(e) precipitating proteins from the organic phase by the addition of a lower alcohol thereto and recovering the precipitated proteins by sedimentation; and

(f) precipitating DNA from the interphase by the addition of CsCl, sodium citrate solution and a lower alcohol thereto and recovering the precipitated DNA by sedimentation.

22. The method of claim 21 wherein said water-insoluble organic solvent added to said homogenate is chloroform.

23. The method of claim 21 wherein the lower alcohol added to the aqueous phase is isopropanol.

24. The method of claim 21 wherein the lower alcohol added to the organic phase is isopropanol.

25. The method of claim 21 wherein the lower alcohol added to the interphase is ethanol.

26. A method of isolating substantially pure RNA, DNA and proteins from biological tissue, comprising the steps of:

(a) homogenizing a tissue sample in the solvent solution of claim 1 to form a homogenate;

(b) sedimenting substantially pure, undegraded DNA from said homogenate, washing the sedimented DNA with an amount of said solvent solution, and removing any phenol and salt contamination from the DNA;

(c) adding a water-insoluble organic solvent to the residual homogenate subsequent to said DNA sedimenting step, and thereafter sedimenting to form a mixture having an aqueous phase containing substantially pure, undegraded RNA and an organic phase containing proteins;

(d) precipitating RNA from the aqueous phase by the addition of a lower alcohol thereto and recovering the precipitated RNA by sedimentation;

(e) precipitating proteins from the organic phase by the addition of a lower alcohol thereto and recovering the precipitated proteins by sedimentation.

27. The method of claim 26 wherein said water-insoluble organic solvent added to said residual homogenate is chloroform.

28. The method of claim 26 wherein said lower alcohol added to the aqueous phase is isopropanol.

29. The method of claim 26 wherein said lower alcohol added to the organic phase is isopropanol.

30. A method of isolating substantially pure RNA, DNA and proteins from biological tissue, comprising the steps of:

(a) precipitating RNA from an aqueous phase obtained from a mixture consisting of an aqueous phase containing substantially pure, undegraded RNA, an organic phase containing proteins, and an interphase containing substantially pure, undegraded DNA, said mixture formed by adding a water-insoluble organic solvent to a homogenate and sedimenting, said homogenate formed by homogenizing a tissue sample in a solvent solution comprising effective amounts of phenol and a guanidinium compound for extracting substantially pure and undegraded RNA, substantially pure and undegraded DNA, and proteins from biological tissue, by the addition of a lower alcohol thereto and recovering the precipitated RNA by sedimentation;

(b) precipitating proteins from an organic phase obtained from a mixture consisting of an aqueous phase containing substantially pure, undegraded RNA, an organic phase containing proteins, and an interphase containing substantially pure, undegraded DNA, said mixture formed by adding a water-insoluble organic solvent to a homogenate and sedimenting, said homogenate formed by homogenizing a tissue sample in a solvent solution comprising effective amounts of phenol and a guanidinium compound for extracting substantially pure and undegraded RNA, substantially pure and undegraded DNA, and proteins from biological tissue, by the addition of a lower alcohol thereto and recovering the precipitated proteins by sedimentation; and

(c) recovering DNA from an interphase obtained from a mixture consisting of an aqueous phase containing substantially pure, undegraded RNA, an organic phase containing proteins, and an interphase containing substantially pure, undegraded DNA, said mixture formed by adding a water-insoluble organic solvent to a homogenate and sedimenting, said homogenate formed by homogenizing a tissue sample in a solvent solution comprising effective amounts of phenol and a guanidinium compound for extracting substantially pure and undegraded RNA, substantially pure and undegraded DNA, and proteins from biological tissue, by washing the interphase with a predetermined amount of said solvent solution, sedimentation of the DNA and removal of any phenol and salt contamination from the DNA.

Chimeric DNA-RNA catalytic sequences

Abstract

This invention provides chimeric DNA/RNA catalytic molecules useful to cleave RNA sequences.

Patent number: 5149796
Filing date: Apr 30, 1991
Issue date: Sep 22, 1992
Inventors: John J. Rossi, Pairoj Chang, Bruce E. Kaplan
Assignee: City of Hope

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What is claimed is:

1. A catalytic molecule capable of cleaving an RNA sequence at a known ribozyme cleavage site said molecule having the formula

3' X - AAAG - Y - AGUAAGUC - Z 5'

or

3' X - CAAAG - Y - AGUAAGUC - Z 5'

in which X and Z are DNA sequences that base pair with an RNA substrate at positions juxtaposed to said known cleavage site,

AAAG, CAAAG and AGUAGUC are RNA sequences,

Y is a DNA sequence that base pairs inter se in a manner required to permit said RNA sequences to cleave said substrate at said cleavage site.

2. A catalytic molecule capable of cleaving an RNA sequence, said molecule having catalytic RNA moieties linked to first and second DNA moieties which base pair with the substrate RNA sequences flanking the cleavage site and interconnected by a third DNA sequence which base pairs inter se to facilitate said cleavage.

3. A molecule including the construct shown by FIG. 1 or FIG. 2.

Process for obtaining DNA, RNA, peptides, polypeptides, or protein

Abstract

The present invention is directed to a process for the production of a peptide, polypeptide, or protein having a predetermined property. In accordance with one embodiment, the process begins by producing by way of synthetic polynucleotide coupling, stochastically generated polynucleotide sequences. A library of expression vectors containing such stochastically generated polynucleotide sequences is formed. Next, host cells containing the vectors are cultured so as to produce peptides, polypeptides, or proteins encoded by the stochastically generated polynucleotide sequences. Screening or selection is carried out on such host cells to identify a peptide, polypeptide, or protein produced by the host cells which has the predetermined property. The stochastically generated polynucleotide sequence which encodes the identified peptide, polypeptide, or protein is then isolated and used to produce the peptide, polypeptide, or protein having the predetermined...

Patent number: 5763192
Filing date: Jun 5, 1995
Issue date: Jun 9, 1998
Inventors: Stuart Alan Kauffman, Marc Ballivet
Assignee: IXSYS, Incorporated
Primary Examiner: Hankyel T. Park

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What is claimed is:

1. A process for the production of a peptide, polypeptide, or protein having a predetermined property, comprising the steps of:

producing by enzymatic or chemical coupling, stochastically generated polynucleotide sequences;

forming a library of expression vectors containing such stochastically generated polynucleotide sequences;

culturing host cells containing the vectors to produce peptides, polypeptides, or proteins encoded by the stochastically generated polynucleotide sequences;

carrying out screening or selection on such host cells, to identify a peptide, polypeptide, or protein produced by the host cells having the predetermined property;

isolating a stochastically generated polynucleotide sequence which encodes the identified peptide, polypeptide, or protein;

using the isolated sequence to produce the peptide, polypeptide, or protein having the predetermined property.

2. The process of claim 1, wherein said peptide, polypeptide or protein having said binding property comprises at least one epitope similar to an amino acid sequence of one of the epitopes of a given antigen.

3. The process of claim 1, wherein said peptide, polypeptide or protein having said binding property comprises stimulating or modifying the effects of a biologically active molecule, and screening and/or selection of the clones of transformed host cells producing at least one peptide or polypeptide having this property is carried out by preparing antibodies against that molecule, and utilizing these antibodies so obtained to identify those clones containing those peptides or polypeptides, then by growing the clones thus identified and separating and purifying the peptide or polypeptide produced by these clones, and finally by submitting these peptide(s) or polypeptide(s) to an assay in vitro to verify that it has in fact the capacity to simulate or modify the effects of the said molecule.

4. The process of claims 2 or 3 for the preparation of a vaccine, said process comprising antibodies against a pathogenic agent that are obtained and used to identify those clones producing at least one protein having at least one epitope similar to an amino acid sequence of one of the epitopes of the pathogenic agent, that the corresponding clones of transformed host cells are grown in such a manner as to produce this protein, that the protein is isolated and purified from the cultures of clones of cells and that this protein is used for the production of a vaccine against the pathogenic agent.

5. The process of claim 4 for the preparation of an anti-hepatitis B virus vaccine, said process comprising that at least one capsid protein of the hepatitis B virus is extracted and purified, that this protein is injected into the body of an animal capable of forming antibodies against this protein, that these antibodies are recovered and purified, that these antibodies are used to identify those clones producing at least one protein having at least one epitope similar to one of the epitopes of the hepatitis B virus, that the clones of transformed host cells corresponding to these clones are grown in a manner to produce this protein, that this protein is isolated and purified from these cultures of host cells, and that this protein is used for the production of an anti-hepatitis B virus vaccine.