Method and mixture reagents for analyzing the nucleotide sequence of nucleic acids by mass spectrometry

• US 6,218,118 B1
• Filed: 07/09/1998
• Issued: 04/17/2001
• Est. Priority Date: 07/09/1998
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Sampson, Jeffrey R., Yakhini, Zohar H., Tsalenko, Anna M., Sampas, Nicholas M., Myerson, Joel, Webb, Peter G.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Siew, Jeffrey
• Application Number:
  US09/112,437
• Time in Patent Office: 1,013 Days
• Field of Search: 435/6, 435/91.2, 435/91.5, 436/173.1, 536/23.1, 536/25.32, 536/24.33, 536/24.31
• US Class Current: 506/4
• CPC Class Codes: B01J 2219/00454 by chemical cleavage from the solid support
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

Methods and reagents are disclosed which satisfy the need for more sensitive, more accurate and higher through-put analyses of target nucleic acid sequences. The methods and reagents may be generically applied to generally any target nucleic acid sequence and do not require a priori information about the presence, location or identity of mutations in the target nucleic acid sequence. The reagents of the invention are mixtures of natural and mass-modified oligonucleotide precursors having a high level of coverage and mass number complexity. A method is also disclosed for analyzing a target nucleic acid sequence employing the mixtures of natural and mass-modified oligonucleotide precursors and chemical or enzymatic assays to alter the mass of the oligonucleotide precursors prior to mass spectral analysis, generally via MALDI-TOF. The enzymatic assay may be a polymerase extension assay or a ligase assay. The kits for carrying out the methods of the invention are also disclosed.

Claims

1. A mixture or set of sub-mixtures comprising natural and mass-modified X-mer precursors,

wherein said X-mer precursors have a minimum length of 3 nucleotides,

wherein said mixture has a minimum mixture coverage complexity of at least 56/N or wherein said set of sub-mixtures has a composite mixture coverage complexity of at least 56/N, wherein N represents the number of distinct X-mer precursors in the mixture,

wherein mass number complexity of said mixture is greater than the mass number complexity of any natural equivalent of said mixture or wherein mass number complexity of said set of sub-mixtures is greater than the mass number complexity of any natural equivalent of said set of sub-mixtures,

wherein each sub-mixture in said set has a reduced mixture coverage complexity as compared with the composite mixture coverage complexity and wherein each sub-mixture further comprises a plurality of X-mer precursors,

wherein said length is selected independently for each X-mer precursor, and

wherein each of said X-mer precursors in said mixture is represented by a single chemical species.

3. The mixture or set of sub-mixtures of claim 1 or 2 wherein said mixture has a mixture coverage complexity of at least about ½

when said mixture contains at least 128 discrete X-mers, or wherein said set of sub-mixtures has a composite mixture coverage complexity of at least about ½

when said set of sub-mixtures contains at least 128 discrete X-mers.

4. The mixture or set of sub-mixtures of claim 1 or 2, wherein said mixture has a mixture coverage complexity of at least about ¼

when said mixture contains at least 256 discrete X-mers, or wherein in said set of sub-mixtures has a composite mixture coverge complexity of at least about ¼

when said set of sub-mixtures contains at least 256 discrete X-mers.

5. The mixture or set of sub-mixtures of claim 1 or 2, wherein said mixture has a mixture coverage complexity of at least about ⅛

when said mixture contains at least 512 discrete X-mers, or wherein said set of sub-mixtures has a composite mixture coverage complexity of at least about ⅛

when said set of sub-mixtures contains at least 512 discrete X-mers.

6. The mixture or set of sub-mixtures of claim 1 or 2 wherein said X-mer precursors have an ionization tag.

7. The mixture or set of sub-mixtures of claim 1 or 2 wherein nucleotide sequences of the precursors of said mixture or set of sub-mixtures are known.

8. The mixture or set of sub-mixtures of claim 1 or 2 wherein at least some of said mass-modified X-mer precursors comprises at least one mass tag or at least one chemical modification of an internucleoside linkage, a sugar backbone, or a nucleoside base.

56. A kit for carrying out a method of analyzing a target nucleic acid sequence, comprising:

a. the mixture or the set of sub-mixtures of claim 1;

b. an enzyme having a nuclcotide polymerase activity; and

c. a multiplicity of nucleotides selected from the group consisting of natural chain-terninating triphosphates and mass-modified chain-terminating triphosphates.

57. A kit for carrying out a method of analyzing a target nucleic acid sequence, comprising:

a. the mixture or the set of sub-mixtures of claim 1;

b. an enzyme having DNA polymerase activity;

c. a multiplicity of nucleotides selected from the group consisting of natural chain-terminating triphosphates and mass-modified chain-terminating triphosphates; and

d. a multiplicity of extension nucleotide triphosphates.

58. A kit for carrying out a method of analyzing a target nucleic acid sequence, comprising:

a. the mixture or the set of sub-mixtures of claim 1;

b. an enzyme having DNA polymerase activity;

c. a multiplicity of nucleotides selected from the group consisting of natural chain-terminating triphosphates and mass-modified chain-terminating triphosphates; and

d. a nuclease.

59. A kit for carrying out a method of analyzing a target nucleic acid sequence, comprising:

a. the mixture or the set of sub-mixtures of claim 1;

b. an enzyme having DNA polymerase activity;

c. a multiplicity of nucleotides selected from the group consisting of natural chain-terminating triphosphates and mass-modified chain-terminating triphosphates;

d. a nuclease; and

e. a multiplicity of extension nucleotide triphosphates.

60. A kit for carrying out a method of analyzing a target nucleic acid sequence, comprising:

a. the mixture or the set of sub-mixtures of claim 1;

b. a DNA polymerase;

c. a multiplicity of nucleotides selected from the group consisting of natural chain-terminating triphosphates and mass-modified chain-terminating triphosphates;

d. a 5′

-exonuclease; and

e. a multiplicity of extension nucleotide triphosphates and 5′

-(α

)-phosphothioate analogues.

61. A kit for carrying out a method of analyzing a target nucleic acid sequence, comprising:

a. the mixture or the set of sub-mixtures of claim 1; and

b. a DNA ligase.

62. A kit for carrying out a method of analyzing a target nucleic acid sequence, comprising:

a. the mixture or the set of sub-mixtures of claim 1;

b. a DNA ligase; and

c. a nuclease.

63. A kit for carrying a method of analyzing a target nucleic acid sequence, comprising:

a the mixture or the set of sub-mixtures of claim 1; and

b. a condensing agent.

64. A kit for carrying out a method of analyzing a target nucleic acid sequence having a 3′

-end and a 5′

-end, comprising;

a. the mixture or the set of sub-mixtures of claim 1;

b. a DNA ligase; and

c. a n array comprising;

(a) a surface; and

(b) a multiplicity of nucleic acid sequence probes comprising;

(i) a cleavable linker attached to said surface; and

(ii) a nucleic acid sequence having a 3′

-end and a terminal 5′

-phosphate wherein said 3′

-end of said nucleic acid sequence is attached to said cleavable linker.

65. A kit for carrying out a method of analyzing a target nucleic acid sequence having a 3′

-end and a 5′

-end, comprising;

a. the mixture or the set of sub-mixtures of claim 1;

b. a condensing agent; and

c. an array comprising;

(a) a surface; and

(b) a multiplicity of nucleic acid sequence probes comprising;

(i) a cleavable linker attached to said surface; and

(ii) a nucleic acid sequence having a 3′

-end and a terminal 5′

-phosphate wherein said 3′

-end of said nucleic acid sequence is attached to said cleavable linker.

2. A mixture or set of sub-mixtures comprising natural and mass-modified X-mer precursors,

wherein said X-mer precursors have a minimum length of 3 nucleotides,

wherein said mixture has a minimum mixture coverage complexity of at least 56/N or wherein said set of sub-mixtures has a composite mixture coverage complexity of at least 56/N, wherein N represents the number of distinct X-mer precursors in the mixture,

wherein mass number complexity of said mixture is greater than the mass number complexity of any natural equivalent of said mixture or wherein mass number complexity of said set of sub-mixtures is greater than the mass number complexity of any natural equivalent of said set of sub-mixtures,

wherein each sub-mixture in said set has a reduced mixture coverage complexity as compared with the composite mixture coverage complexity and wherein each sub-mixture further comprises a plurality of X-mer precursors,

wherein said length is selected independently for each X-mer precursor,

wherein each of said X-mer precursors in said mixture is represented by a single chemical species, and

wherein said X-mer precursors have a determined isotopic composition.

9. A method of analyzing a target nucleic acid sequence, comprising the steps of:

(1) hybridizing a mixture or set of sub-mixtures comprising natural and mass-modified X-mer precursors to the target nucleic acid sequence,

wherein said mixture has a minimum mixture coverage complexity of at least 56/N or wherein said set of sub-mixtures has a composite mixture coverage complexity of at least 56/N, wherein N represents the number of distinct X-mer precursors in the mixture,

wherein each sub-mixture in said set has a reduced mixture coverage complexity as compared with the composite mixture coverage complexity and further comprises a plurality of X-mer precursors,

wherein said length is selected independently for each X-mer precursor,

wherein each of said X-mer precursors in said mixture is represented by a single chemical species, and

wherein said X-mer precursors comprise a 3′

-end and a 5′

-end,

(2) processing said hybrids to alter the mass of said X-mer precursor portions of said hybrids in a target sequence-mediated reaction; and

(3) analyzing the product of step (2) via mass spectrometry.

10. The method of claim 9 wherein said X-mer precursors have a determined isotopic composition.

11. The method of claim 9 wherein said mass number complexity (MNC) of said mixture is greater than the mass number complexity of any natural equivalent of said mixture or wherein said mass number complexity (MNC) of said set of sub-mixtures is greater than the mass number complexity of any natural equivalent of said set of sub-mixtures.

12. The method of claim 9 wherein said mixture has a mixture coverage complexity of at least about ½

when said mixture contains at least 128 discrete X-mers, and wherein said set of sub-mixtures has a composite mixture coverage complexity of at least about ½

when said set of sub-mixtures contains at least 128 discrete X-mers.

13. The method of claim 9 wherein said mixture has a mixture coverage complexity of at least about ¼

when said mixture contains at least 256 discrete X-mers, or wherein said set of sub-mixtures has a composite mixture coverage complexity of at least about ¼

when said set of sub-mixtures contains at least 256 discrete X-mers.

14. The method of claim 9 wherein said mixture has a mixture coverage complexity of at least about ⅛

when said mixture contains at least 512 discrete X-mers, or wherein said set of sub-mixtures has a composite mixture coverage complexity of at least about ⅛

when said set of sub-mixtures contains at least 512 discrete X-mers.

15. The method of claim 9 wherein said mixture is provided in at least two reaction mixtures.

16. The method of claim 9 wherein at least some of said mass-modified X-mer precursors comprise at least one mass tag or at least one chemical modification of a internucleoside linkage, a sugar backbone, or a nucleoside base.

17. The method of claim 9 wherein said processing step comprises altering the mass of said X-mer precursor portions of said hybrids by an amount greater than that defined by the mass difference between the lightest and heaviest X-mer in the mixture.

18. The method of claim 9 further comprising the step of:

purifying the products of step (2) prior to analysis via mass spectrometry.

19. The method of claim 9 further comprising the step of:

separating the products of step (2) prior to analysis via mass spectrometry.

20. The method of claim 9 wherein steps (1)-(2) are conducted in solution.

21. The method of claim 9 wherein steps (1)-(2) are conducted with a surface-bound mixture.

22. The method of claim 9 wherein said products are analyzed via MALDI-TOF mass spectrometry.

23. The method of claim 9 wherein said processing step comprises a target sequence mediated enzymatic assay.

24. The method of claim 23 wherein said enzymatic assay is an assay selected from a polymerase extension assay and a ligase assay.

25. The method of claim 9 wherein said processing step comprises extending said hybridized X-mer precursors by polymerizing at least one nucleotide at said 3′

-end of said hybridized X-mer precursors.

26. The method of claim 9 wherein said processing step comprises extending said hybridized X-mer precursors by polymerizing a single nucleotide at said 3′

-end of said hybridized X-mer precursors.

27. The method of claim 25 wherein hybridized X-mer precursors are extended using an enzyme having a nucleotide polymerase activity.

28. The method of claim 25 wherein said nucleotide is a chain-terminating nucleotide triphosphate.

29. The method of claim 28 wherein said chain-terminating nucleotide triphosphate is a nucleotide selected from the group consisting of natural dideoxynucleotide triphosphates and mass-modified dideoxynucleotide triphosphates.

30. The method of claim 29 wherein the mass of said mass-modified dideoxynucleotide triphosphate is greater than that defined by the mass difference between the lightest and heaviest X-mer in the mixture.

31. The method of claim 25 further comprising the step of:

digesting the products of step (2) prior to analysis via mass spectrometry.

32. The method of claim 31, wherein said nucleotide is a nucleotide selected from the group consisting of:

extension nucleotide triphosphates, natural dideoxynucleotide triphosphates, and mass-modified dideoxynucleotide triphosphates.

33. The method of claim 32, wherein said extension nucleotide triphosphates are nucleotides selected from the group consisting of:

deoxynucleotides, 5′

-(α

)-phosphothioate analogues, 5′

-N-(α

)-phosphoramidate analogues and ribonucleotides.

34. The method of claim 31 wherein said digestion step is carried out with a nuclease.

35. The method of claim 34 wherein said nuclease is 5′

-exonuclease.

36. The method of claim 35 wherein said 5′

-exonuclease is an enzyme selected from the group consisting of DNA polymerase and T7 Gene 6.

37. The method of claim 31 wherein said digestion step is carried out via a chemical reaction.

38. The method of claim 9 wherein said processing step comprises ligating adjacent X-mer precursors using a DNA ligase.

39. The method of claim 9 wherein said processing step comprises ligating adjacent X-mer precursors using a condensing agent.

40. The method of claim 39 wherein said condensing agent is selected from the group consisting of carbodiimides and cyanogen bromide derivatives.

41. The method of claim 9 wherein said processing step comprises a chemical assay.

66. The method of claim 9, 10 or 11, wherein said X-mer precursors have an ionization tag.

67. The method of claim 28 wherein said chain-terminating nucleotides have an ionization tag.

69. The method of claim 9, wherein the step (1) of hybridizing, the mixture or set of sub-mixtures comprises mass-modified X-mer precursors.

42. A method of analyzing a target nucleic acid sequence having a 3′

-end and a 5′

-end, comprising the steps of;

(1) hybridizing said target nucleic acid sequence to a multiplicity of nucleic acid probes in an array comprising;

(a) a surface; and

(b) said multiplicity of nucleic acid sequence probes comprising;

(i) a cleavable linker attached to said surface; and

(ii) a nucleic acid sequence having a 3′

-end and a terminal 5 ′

-phosphate wherein said 3′

-end of said nucleic acid sequence is attached to said cleavable linker;

(2) hybridizing a mixture or sat of sub-mixtures comprising natural and mass-modified X-mer precursors to the target nucleic acid sequence,

wherein said mixture has a minimum mixture coverage complexity of at least 56/N or wherein said set of sub-mixtures has a composite mixture coverage complexity of at least 56/N, wherein N represents the number of distinct X-mer precursors in the mixture,

wherein each sub-mixture in said set has a reduced mixture coverage complexity as compared with the composite mixture coverage complexity and wherein each sub-mixture further comprises a plurality of X-mer precursors,

wherein said length is selected independently for each X-mer precursor,

wherein each of said X-mer precursors in said mixture or said sub-mixture is represented by a single chemical species, and

wherein said X-mer precursors comprise a 3′

-end and a 5′

-end,

(3) ligating said hybridized X-mer precursors located adjacent to said terminal 5′

-phosphate with said surface-bound probe to form a hybridized precursor/probe complex with said target nucleic acid sequence attached thereto; and

(4) cleaving said complex at said cleavable linker; and

(5) analyzing said complex via mass spectrometry.

43. The method of claim 42, wherein said mass number complexity (MNC) of said mixture is greater than the mass number complexity of any natural equivalent of said mixture or wherein said mass number complexity (MNC) of said set of sub-mixtures is greater than the mass number complexity of any natural equivalent of said set of sub-mixtures.

44. The method of claim 42, wherein said mixture has a mixture coverage complexity of at least about ½

when said mixture contains at least 128 discrete X-mers, and wherein said set of sub-mixtures has a composite mixture coverage complexity of at least about ½

when said set of sub-mixtures contains at least 128 discrete X-mers.

45. The method of claim 42, wherein said mixture has a mixture coverage complexity of at least about ¼

when said mixture contains at least 256 discrete X-mers, or wherein said set of sub-mixtures has a composite mixture coverage complexity of at least about ¼

when said set of sub-mixtures contains at least 256 discrete X-mers.

46. The method of claim 42, wherein said mixture has a mixture coverage complexity of at least about ⅛

when said mixture contains at least 512 discrete X-mers, or wherein said set of sub-mixtures has a composite mixture coverage complexity of at least about ⅛

when said set of sub-mixtures contains at least 512 discrete X-mers.

47. The method of claim 42, wherein nucleotide sequences of the X-mer precursors of said mixture or said set of sub-mixtures are known.

48. The method of claim 42, wherein said mixture is provided in at least two reaction mixtures.

49. The method of claim 42, wherein at least some of said mass-modified X-mer precursors comprise at least one mass tag or at least one chemical modification of a internucleoside linkage, a sugar backbone, or a nucleoside base.

50. The method of claim 42 wherein said hybridized X-mer precursor ligated with said probe using a DNA ligase.

51. The method of claim 42, wherein said hybridized X-mer precursor ligated with said probe using a condensing agent.

52. The method of claim 51, wherein condensing agent is selected from the group consisting of carbodiimides and cyanogen bromide derivatives.

53. The method of claim 42 wherein said cleavable linker is a photocleavable linker.

54. The method of claim 42 wherein said cleavable linker is a chemical cleavable linker.

55. The method of claim 42 wherein said complexes are analyzed via MALDI-TOF mass spectrometry.

70. The method of claim 42, wherein the step (2) of hybridizing a mixture or set of sub-mixtures to the target nucleic acid sequence, the mixture or set of sub-mixtures comprises mass-modified X-mer precursors.

68. A mixture or set of sub-mixtures comprising mass-modified X-mer precursors,

wherein said X-mer precursors have a minimum length of 3 nucleotides,

wherein said mixture has a minimum mixture coverage complexity of at least 56/N or wherein said set of sub-mixtures has a composite mixture coverage complexity of at least 56/N, wherein N represents the number of distinct X-mer precursors in the mixture,

wherein mass number complexity of said mixture or said set of sub-mixtures is greater than the mass number complexity of any natural equivalent of said mixture or said set of sub-mixtures,

wherein each sub-mixture in said set has a reduced mixture coverage complexity as compared with the composite mixture coverage complexity and wherein each sub-mixture further comprises a plurality of X-mer precursors,

wherein said length is selected independently for each X-mer precursor, and

wherein each of said X-mer precursors in said mixture is represented by a single chemical species.