Collections of uniquely tagged molecules

• US 6,607,878 B2
• Filed: 10/06/1997
• Issued: 08/19/2003
• Est. Priority Date: 10/06/1997
• Status: Expired due to Fees

• Current Assignee: Stratagene California
• Original Assignee: Stratagene California
• Inventors: Sorge, Joseph A.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/944,410
• Publication Number:
  US 20030050453A1
• Time in Patent Office: 2,143 Days
• Field of Search: 435/6, 435/7.94, 530/360, 436/501, 536/23.1, 935/78
• US Class Current: 506/4
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence

Abstract

This invention is directed to methods and kits for creating and analyzing molecules using uniquely identifiable tags. The invention is also directed to methods and kits that use uniquely identifiable tags for sequencing DNA, for determining mutations, including substitutions, deletions, and additions, in sample genes, and monitoring mRNA populations.

Claims

1. A composition comprising a mixture of different species of molecules, wherein at least some of said molecules are derived from a combinatorial synthesis process, wherein at least some of the species are linked to a tag comprising linked information encoding elements wherein the combination of elements imparts a physical property that is not characteristic of any one element alone and wherein at least some of said tags have a unique physical property when compared to other tags, wherein the physical property of the combination of elements identifies the species of molecule without determining the physical property of each element of said tag, and wherein said tags do not consist solely of nucleotides.

2. The composition in claim 1 wherein the mixture contains at least 10 species of molecules.

3. The composition in claim 1 wherein each tag has a uniquely identifiable molecular weight.

4. The composition in claim 1 wherein each tag comprises a reporter subunit and a base subunit that associate with one another, such that the reporter subunit dissociates from the base subunit under uniquely identifiable dissociation conditions.

5. The composition in claim 4 wherein a reporter subunit comprises a protein, nucleic acid, or other affinity reagent that recognizes the base subunit.

6. The composition in claim 5 wherein the reporter subunit comprises an oligonucleotide.

7. The composition in claim 6 wherein the reporter subunit is immobilized.

8. The composition in claim 4 wherein the base subunit comprises an oligonucleotide.

9. The composition in claim 8 wherein the base subunit is immobilized.

10. The composition in claim 8 wherein the reporter subunit comprises an oligonucleotide and wherein the reporter and base subunits are perfectly complementary.

11. The composition in claim 8 wherein the reporter subunit comprises an oligonucleotide and wherein the reporter and base subunits are not perfectly complementary.

12. The composition in claim 4 wherein the reporter subunit is a hydrocarbon.

13. The composition in claim 12 wherein the reporter subunits are hydrocarbons of varying chain length and/or hydrophobicity.

14. The composition in claim 4 wherein the reporter subunit is an amino acid residue.

15. The composition in claim 4 wherein the reporter subunits are amino acid residues of varying numbers of residues and/or isoelectric points.

16. The composition in claim 4 wherein temperature is used to vary the dissociation conditions.

17. The composition in claim 4 wherein the mixture is in a disassociating agent such that the concentration of the disassociating agent is varied directly or indirectly to vary the dissociation conditions.

18. The composition in claim 17 wherein a change in pH is used to vary the dissociation conditions.

19. The composition in claim 17 wherein formamide is used to vary the dissociation conditions.

20. The composition in claim 17 wherein urea is used to vary the dissociation conditions.

21. The composition in claim 4 wherein a field is used to vary the dissociation conditions.

22. The composition in claim 21 wherein the field is an electrical field.

23. The composition in claim 21 wherein the field is a magnetic field.

24. The composition in claim 1 wherein each tag comprises uniquely identifiable information encoding elements, wherein the order of the elements within the tag provides information regarding the identity of the tag.

25. The composition in claim 24 wherein the information encoding elements have uniquely identifiable isotopic emissions.

26. The composition in claim 25 wherein the information encoding elements include radioisotopes.

27. The composition in claim 24 wherein the information encoding elements have uniquely identifiable molecular weights.

28. The composition in claim 24 wherein the information encoding elements have uniquely identifiable physical sizes.

29. The composition in claim 24 wherein the information encoding elements have uniquely identifiable light absorbing or emitting properties.

30. The composition in claim 24 wherein the information encoding elements have uniquely identifiable magnetic properties.

31. The composition of claim 24 wherein the information encoding elements have uniquely identifiable electrical properties.

32. The composition of claim 24 wherein the information encoding elements have uniquely identifiable molecular compositions.

33. The composition of claim 32 wherein the uniquely identifiable molecular compositions can be identified by NMR.

34. The composition of claim 32 wherein the uniquely identifiable molecular compositions can be identified by atomic force microscopy.

35. The composition of claim 24 wherein the information encoding elements have uniquely identifiable molecular structures.

36. The composition of claim 35 wherein the uniquely identifiable molecular structures can be identified by their three dimensional structure.

37. The composition of claim 35 wherein the uniquely identifiable molecular structures can be identified by their affinity for an antibody or an antigen.

38. The composition of claim 35 wherein the uniquely identifiable molecular structures can be identified by their affinity for a ligand.

67. The composition of claim 1 wherein the combined physical property that is unique to each tag can be determined in a system wherein the different molecular species can diffuse in a liquid phase.

69. The composition of claim 1 wherein the physical property is molecular mass.

70. The composition of claim 1 wherein said tag can be identified without the need for first isolating each of the tags prior to performing a decoding procedure.

74. A method comprising identifying tagged species present in the composition of claims 1, 68, or 72 by detecting one or more physical properties of the tags in said compositon.

75. The method of claim 74 wherein the physical property comprises at least one of:

electrophoretic mobility, light absorption, nuclear magnetic resonance, radioisotopic emission, light frequency emission, light intensity emission, radiofrequency emission, mobility in an electric field, mobility in a pH gradient, isoelectric focussing pH, mobility in a magnetic field, gel filtration mobility, molecular size, melting temperature, annealing temperature, binding to a nucleic acid probe, binding energy, dissociation energy, dielectric constant, electrical charge, molecular mass, fluorescent emission, rate of diffusion, color, harmonic frequency, electrical conductivity, electrical resistance, molecular shape, efficiency as a substrate in a ligation reaction, efficiency as a substrate in a polymerization reaction, or efficiency as a substrate in a restriction enzyme digestion reaction.

76. The method in claim 74 wherein the tag is a single stranded nucleic acid and the physical property is the binding to a second nucleic acid.

77. The method in claim 74 wherein the physical property detected is the annealing of the tags to two or more target nucleic acid molecules.

78. The method of claim 74 wherein the tag comprises duplexed nucleic acids.

79. The method of claim 78 wherein the physical property detected is the denaturation of the duplexed nucleic acids.

80. The method of claim 74 wherein each tagged species comprises a uniquely identifiable tag comprising information encoding elements created by a method comprising:

(i) performing parallel first synthesis reactions in separate reaction vessels, wherein a first information encoding element is coupled to a first synthesis product produced in each vessel, each of the first encoding elements being unique for the first synthesis product in each separate reaction vessel;

(ii) mixing the first synthesis products;

(iii) placing portions of the mixture from (ii) into new separate reaction vessels and performing parallel second synthesis reactions to modify the first synthesis products to create a second synthesis product in the new separate reaction vessels, wherein a second information encoding element is coupled to the second synthesis product in each vessel, each of the second information encoding elements being unique for each of the second synthesis reactions in each separate reaction vessel such that the combination of elements imparts a physical property that is unique to each tag in said mixture, and wherein the physical property of the combination of elements identifies the species of molecule without the need to determine a physical property of each element of said tag independently.

81. The method of claim 80, wherein said tagged species creation method further comprises:

(iv) mixing the second synthesis products;

(v) placing portions of the mixture from (iv) into new separate reaction vessels and performing parallel third synthesis reactions to modify the second synthesis products to create third synthesis products in the new separate reaction vessels, wherein a third information encoding element is coupled to the third synthesis product in each vessel, each of the third information encoding elements being unique for each of the third synthesis reactions in each separate reaction vessel such that the combination of elements imparts a physical property that is unique to each tag in said mixture, and wherein the physical property of the combination of elements identifies the species of molecule without the need to determine a physical property of each element of said tag independently.

82. The method of claim 74 wherein each tagged species comprises a uniquely identifiable tag comprising information encoding elements created by a method comprising:

(i) performing parallel first enzymatic reactions in separate reaction vessels, wherein a first information encoding element is coupled to a first enzymatic product produced in each vessel, each of the first encoding elements being unique for the first enzymatic product in each separate reaction vessel;

(ii) mixing the first enzymatic products;

(iii) placing portions of the mixture from (ii) into new separate reaction vessels and performing parallel second enzymatic reactions to modify the first enzymatic products to create second enzymatic products in the new separate reaction vessels, wherein a second information encoding element is coupled to the second enzymatic product in each vessel, each of the second information encoding elements being unique for each of the second enzymatic reactions in each separate reaction vessel such that the combination of elements imparts a physical property that is unique to each tag in said mixture, and wherein the physical property of the combination of elements identifies the species of molecule without the need to determine a physical property of each element of said tag independently.

83. The method of claim 82 wherein said tagged species creation method further comprises:

(iv) mixing the second enzymatic products; and

(v) placing portions of the mixture from (iv) into new separate reaction vessels and performing parallel third enzymatic reactions to modify the second enzymatic products to create third enzymatic products in the new separate reaction vessels, wherein a third information encoding element is coupled to the third enzymatic product in each vessel, each of the third information encoding elements being unique for each of the third enzymatic reactions in each separate reaction vessel such that the combination of elements imparts a physical property that is unique to each tag in said mixture, and wherein the physical property of the combination of elements identifies the species of molecule without the need to determine a physical property of each element of said tag independently.

84. The composition of claims 1, 68, or 72 wherein the physical property comprises at least one of:

electrophoretic mobility, light absorption, nuclear magnetic resonance, radioisotopic emission, light frequency emission, light intensity emission, radiofrequency emission, mobility in an electric field, mobility in a pH gradient, isoelectric focussing pH, mobility in a magnetic field, gel filtration mobility, molecular size, melting temperature, annealing temperature, binding to a nucleic acid probe, binding energy, dissociation energy, dielectric constant, electrical charge, molecular mass, fluorescent emission, rate of diffusion, color, electrical conductivity, electrical resistance, molecular shape, efficiency as a substrate in a ligation reaction, efficiency as a substrate in a polymerization reaction, or efficiency as a substrate in a restriction enzyme digestion reaction.

85. The composition of claim 1, wherein the mixture of different species comprises at least one hundred species.

88. The method of claim 74, wherein said detection is simultaneous.

39. A method for creating a mixture of at least one hundred species of molecules wherein at least some of said molecules are linked to a uniquely identifiable tag comprising information encoding elements comprising:

(I) performing parallel first synthesis reactions in separate reaction vessels, wherein a first information encoding element is linked to a first synthesis product produced in each vessel, wherein at least some of the first encoding elements are unique for the first synthesis product in each separate reaction vessel;

(ii) mixing the first synthesis products;

(iii) placing portions of the mixture from (ii) into new separate reaction vessels and performing parallel second synthesis reactions to modify the first synthesis products to create a second synthesis product in the new separate reaction vessels, wherein a second information encoding element is linked to the first information encoding element that is linked to the second synthesis product in each vessel, wherein at least some of the second information encoding elements are unique for each of the second synthesis reactions in each separate reaction vessel such that the combination of elements imparts a physical property that is unique to at least some of the tags in said mixture, wherein the physical property of the combination of elements identifies the species of molecule without the need to determine a physical property of each element of said tag independently, and wherein the tags do not consist solely of nucleic acid encoding elements.

40. A method of claim 39, further comprising:

(iv) mixing the second synthesis products;

(v) placing portions of the mixture from (iv) into new separate reaction vessels and performing parallel third chemical synthesis reactions to modify the second synthesis products to create third chemical synthesis products in the new separate reaction vessels, wherein a third information encoding element is coupled to the third synthesis product in each vessel, each of the third information encoding elements being unique for each of the third chemical synthesis reactions in each separate reaction vessel such that the order of coupling of such first, second, and third information encoding elements can be determined without first isolating each of the tags.

41. A method for creating a mixture of at least one hundred species of molecules wherein at least some of said molecules are linked to a uniquely identifiable tag comprising information encoding elements comprising:

(i) performing parallel first enzymatic reactions in separate reaction vessels, wherein a first information encoding element is linked to a first enzymatic product produced in each vessel, wherein at least some of the first encoding elements are unique for the first enzymatic product in each separate reaction vessel;

(ii) mixing the first enzymatic products;

(iii) placing portions of the mixture from (ii) into new separate reaction vessels and performing parallel second enzymatic reactions to modify the first enzymatic products to create second enzymatic products in the new separate reaction vessels, wherein a second information encoding element is linked to the first information encoding element that is linked to the second enzymatic product in each vessel, wherein at least some of the second information encoding elements are unique for at least some of the second enzymatic reactions in at least some of the separate reaction vessels such that the order of coupling of the first and second information encoding elements of at least some of the tags can be determined without first isolating each of the tags, and wherein the tags do not consist solely of nucleic acid encoding elements.

42. A method of claim 41, further comprising:

(iv) mixing the second enzymatic products;

(v) placing portions of the mixture from (iv) into new separate reaction vessels and performing parallel third enzymatic reactions to modify the second enzymatic products to create third enzymatic products in the new separate reaction vessels, wherein a third information encoding element is coupled to the third enzymatic product in each vessel, each of the third information encoding elements being unique for each of the third enzymatic reactions in each separate reaction vessel such that the order of coupling of such first, second, and third information encoding elements can be determined without first isolating each of the tags.

43. The method in claim 42 wherein the information encoding elements have uniquely identifiable isotopic emissions.

44. The method in claim 43 wherein the information encoding elements include radioisotopes.

45. The method in claim 42 wherein the information encoding elements have uniquely identifiable molecular weights.

46. The method in claim 42 wherein the information encoding elements have uniquely identifiable physical sizes.

47. The method in claim 42 wherein the information encoding elements have uniquely identifiable light absorbing or emitting properties.

48. The method in claim 42 wherein the information encoding elements have uniquely identifiable magnetic properties.

49. The method of claim 42 wherein the information encoding elements have uniquely identifiable electrical properties.

50. The method of claim 42 wherein the information encoding elements have uniquely identifiable molecular methods.

51. The method of claim 42 wherein the information encoding elements have uniquely identifiable molecular structures.

52. The method in claim 42 wherein the enzymatic reaction is an amplification reaction.

53. The method in claim 52 wherein the enzymatic reactions utilize the polymerase chain reaction.

54. The method in claim 52 wherein the enzymatic reactions utilize the ligase chain reaction.

55. The method in claim 52 wherein the enzymatic reactions amplify certain reaction substrates in preference to other reaction substrates based on the nucleotide sequence of such substrates.

56. The method in claim 42 wherein the tags comprise nucleic acids.

57. A composition comprising a mixture of different species of molecules, wherein at least some of said molecules are derived from a combinatorial synthesis process, wherein at least some of the species are linked to a tag, said tag comprising at least two linked uniquely identifiable information encoding elements that are unique to at least two variable positions on the species wherein the combination of elements imparts a physical property that is not characteristic of any element alone and wherein at least some of the tags have a unique physical property when compared to other tags, wherein the physical property of the combination of elements identifies the species of molecule without determining the physical property of each element of said tag, and wherein the tags do not consist solely of nucleic acid encoding elements.

58. A composition comprising a mixture of different species of molecules, wherein at least some of said molecules are derived from a combinatorial synthesis process, wherein each species is linked to a tag, said tag comprising operatively linked information encoding elements wherein the combination of elements imparts a physical property that is unique to each tag in said mixture, wherein the physical property of the combination of elements identifies the species of molecule without the need to determine a physical property of each element of said tag independently, and wherein each tag comprises a reporter subunit and a base subunit that associate with one another, such that the reporter subunit dissociates from the base subunit under uniquely identifiable dissociation conditions.

59. A method for determining the sequence of a nucleic acid comprising:

shearing the nucleic acid into random fragments;

specifically amplifying the random fragments based on all possible combinations of nucleotides at positions 1 to X at one end of each of the random fragments to create first stage amplification products, wherein X is an integer from 2 to 6;

encoding at least one first stage tag element that specifically corresponds to each possible combination of nucleotides at positions 1 to X at the end of each fragment;

specifically amplifying the first stage amplification products based on all possible combinations of specific nucleotides at positions X+1 or X+1 to Y of each of the first stage amplification products to create second stage products, wherein Y is an integer from 4 to 12;

encoding at least one second stage tag element that specifically corresponds to each possible combination of nucleotides at positions X+1 or X+1 to Y of each first stage product thereby creating a tag that uniquely encodes nucleotides at positions 1 to X+1 or 1 to Y of each fragment;

simultaneously decoding the tags without prior separation of the tags to determine the nucleotides of each fragment at positions 1 to X+1 or 1 to Y; and

determining the nucleic acid sequence by the overlap of the nucleotides identified at positions 1 to X+1 or 1 to Y of the fragments.

60. A method for detecting mutations in a sample nucleic acid sequence comprising:

randomly shearing a wild type nucleic acid sequence into wild type fragments;

creating probes that encode nucleotides at positions 1 to X at one end of each of the wild type fragments, wherein X is an integer from 2 to 6, and each of said probes being complementary to each of said wild type fragments but lacking a complementary sequence for nucleotides at positions 1 to X+1 of each wild type fragment;

randomly shearing the sample nucleic acid sequence into sample fragments;

separately exposing the sample fragments and the wild type fragments to the probes;

performing primer extension with the probes to encode each nucleotide at position X+1 of each sample fragment and each wild type fragment;

simultaneously decoding the probes to determine each nucleotide at position X+1 of each sample fragment and each wild type fragment; and

comparing the decoded results of the sample fragments to the wild type fragments to detect differences between specific positions of the wild type nucleic acid sequence and the sample nucleic acid sequence.

61. A method for detecting mutations in a sample nucleic acid sequence comprising:

randomly shearing the sample nucleic acid sequence into sample fragments;

separately exposing the sample fragments and wild type fragments, obtained from randomly sheared wild type nucleic acid sequence, to the probes, said probes encoding nucleotides at positions 1 to X at one end of each of the wild type fragments, wherein X is an integer from 2 to 6, and each of said probes being complementary to each of said wild type fragments but lacking a complementary sequence for nucleotides at positions 1 to X+1 of each wild type fragment;

performing primer extension with the probes to encode each nucleotide at position X+1 of each sample fragment and each wild type fragment;

simultaneously decoding the probes to determine each nucleotide at position X+1 of each sample fragment and each wild type fragment; and

comparing the decoded results of the sample fragments to the wild type fragments to detect differences between specific positions of the wild type nucleic acid sequence and the sample nucleic acid sequence.

62. A kit for detecting mutations in a sample nucleic acid sequence comprising:

wild type fragments obtained from a randomly sheared wild type nucleic acid sequence; and

probes encoding nucleotides at positions 1 to X at one end of each of the wild type fragments, wherein X is an integer from 2 to 6, and each of said probes being complementary to each of said wild type fragments but lacking a complementary sequence for nucleotides at positions 1 to X+1 of each wild type fragment.

63. A method for detecting the presence or absence of or quantity of particular mRNA in a sample comprising:

creating reference cDNA from a reference mRNA population;

cleaving the reference cDNA with a restriction enzyme that is specific for a specific X consecutive nucleotides, wherein X is an integer from 2 to 6, to create reference cDNA fragments each having the same nucleotides at positions 1 to X at one end;

creating probes that encode nucleotides at positions X+1 to Y of each of the cDNA fragments, wherein Y is an integer from 4 to 12, and each of said probes being complementary to each of said cDNA fragments but lacking a complementary sequence for nucleotides at positions 1 to Y+1 of each cDNA fragment;

creating target cDNA from target mRNA in a sample;

cleaving the target cDNA with the restriction enzyme that is specific for X consecutive nucleotides to create target cDNA fragments;

exposing the target cDNA fragments to the probes;

performing primer extension with the probes to encode each nucleotide at position Y+1 of each target cDNA fragment that is present that corresponds to a reference cDNA fragment;

detecting the presence or absence of or quantity of target cDNA fragments corresponding to reference cDNA fragments to detect the presence or absence of or quantity of target mRNA in the sample.

64. A method for detecting the presence of absence of or quantity of particular mRNA in a sample comprising:

creating target cDNA from target mRNA in the sample;

cleaving the target cDNA with a restriction enzyme that is specific for X consecutive nucleotides to create target cDNA fragments each having the same nucleotides at positions 1 to X, wherein X is an integer from 2 to 6;

exposing the target cDNA fragments to probes, said probes each encoding the same nucleotides at positions 1 to X as the target cDNA fragments, each of said probes also encoding nucleotides at positions X+1 to Y of reference cDNA fragments created from a reference mRNA population and cleaved by the restriction enzyme, wherein Y is an integer from 4 to 12, and each of said probes being complementary to each of said reference cDNA fragments but lacking a complementary sequence for nucleotides at positions 1 to Y+1 of each reference cDNA fragment;

performing primer extension with the probes to encode each nucleotide at position Y+1 of each target cDNA fragment that is present that corresponds to a reference cDNA fragment;

detecting the presence or absence of or quantity of target cDNA fragments corresponding to reference cDNA fragments to detect the presence or absence of or quantity of target mRNA in the sample.

65. A kit for detecting the presence or absence of or quantity of particular mRNA in a sample comprising:

multiple probes each encoding the same nucleotides at positions 1 to X, said positions 1 to X being associated with a given restriction enzyme, wherein X is an integer from 2 to 6, each of said probes also encoding nucleotides at positions X+1 to Y of reference cDNA fragments created from a reference mRNA population and cleaved by the given restriction enzyme, wherein Y is an integer from 4 to 12, and each of said probes being complementary to each of said reference cDNA fragments but lacking a complementary sequence for nucleotides at positions 1 to Y+1 of each reference cDNA fragment.

66. A method for identifying molecular species from a mixture of at least one hundred different molecular species comprising:

performing multiple consecutive sets of parallel different syntheses;

creating a tag for each molecular species generated from the consecutive sets of syntheses using information encoding elements that each specifically correspond to a particular synthesis and that identify the order of syntheses, wherein said tags comprise linked information encoding elements wherein the combination of elements imparts a physical property that is unique for at least some of said tags in said mixture and that can be determined without the need to determine a physical property of each element of said tag; and

decoding the tags without determining the physical property of each element of said tags to determine at least some of the molecular species created by the consecutive sets of parallel different syntheses.

68. A composition comprising a mixture of at least one hundred different species of molecules, wherein at least some of said molecules are derived from a combinatorial synthesis process and wherein each species is linked to a tag, said tag comprising linked elements wherein the order of elements imparts a physical property that is unique to each tag within the mixture.

86. The composition of claim 68, wherein the tags can be identified without the need for first isolating each of the tags prior to performing a decoding step.

71. A method for identifying tags in a composition comprising a mixture of at least one hundred different species of molecules, wherein at least some of the species are linked to a tag, said tag comprising linked information encoding elements wherein the combination of at least two encoding elements imparts a physical property that is not characteristic of any one element alone and wherein at least some of the tags have a unique physical property when compared to other tags and wherein the physical property of the combination of elements identifies the species of molecule, comprising:

identifying said tags based on said physical property that is unique to said tag and not based on the mere order of said encoding elements.

72. A composition comprising a mixture of at least one hundred different species of molecules, wherein at least some of said molecules are derived from a combinatorial synthesis process, wherein each species is linked to a tag, said tag comprising linked information encoding elements wherein the combination of elements imparts a physical property that is unique to each tag in said mixture and that can be determined without the need to determine the sequence of elements in said tag.

73. The composition of claim 72 wherein said tag can be identified without the need for first isolating each of the tags prior to performing a decoding procedure.

87. A composition comprising a mixture of at least one hundred different species of molecules, wherein at least some of said molecules are derived from a combinatorial synthesis process, wherein each species is linked to a tag, said tag comprising linked information encoding elements wherein the combination of elements imparts a physical property that is unique to each tag in said mixture and wherein said tag does not consist solely of nucleotides.