Method for analyzing polynucleotides

• US 6,566,059 B1
• Filed: 09/10/1999
• Issued: 05/20/2003
• Est. Priority Date: 10/01/1998
• Status: Expired due to Term

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Variagenics, Inc. (Arca Biopharma, Inc.)
• Inventors: Verdine, Gregory L., Stanton, Vincent P. Jr., Wolfe, Jia Liu
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/394,467
• Time in Patent Office: 1,348 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.3, 536/25.3
• US Class Current: 435/6.12
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

The present invention relates to methods for the analysis of polynucleotides including detection of variance in nucleotide sequence without the need for full sequence determination, full sequence determination of a polynucleotide, genotyping of DNA and labeling a polynucleotide fragment during the process of cleaving it into fragments.

Claims

1. A method for detecting a variance in a nucleotide sequence in a polynucleotide or for genotyping a polynucleotide suspected to contain a known polymorphism or mutation, comprising:

a. replacing a natural nucleotide at greater than 90% of its points of occurrence in a polynucleotide, provided the points of occurrence are not in a primer sequence, with a modified-base nucleotide to form a modified polynucleotide;

b. contacting the modified polynucleotide with a non-enzymatic reagent or reagents that cleave(s) it at greater than 90% of the points of occurrence of the modified-base nucleotide to give a set of fragments; and

,

c. analyzing the set of fragments to detect a variance in or to genotype the polynucleotide.

2. The method of claim 1, wherein the modified-base nucleotide comprises modified adenine.

3. The method of claim 2, wherein the modified adenine is 7-deaza-7-nitroadenine.

4. The method of claim 3, wherein cleaving the modified polynucleotide comprises contacting it with chemical base.

5. The method of claim 3, wherein cleaving the modified polynucleotide comprises contacting it with a phosphine.

6. The method of claim 5, wherein contacting the modified polynucleotide with a phosphine comprises contacting it with tris(2-carboxyethyl)phosphine.

7. The method of claim 1, wherein the modified-base nucleotide comprises modified cytosine.

8. The method of claim 7, wherein the modified cytosine comprises azacytosine.

9. The method of claim 7, wherein the modified cytosine is a cytosine substituted at the 5-position with an electron withdrawing group.

10. The method of claim 9, wherein the electron withdrawing group is nitro or halo.

11. The method of claim 8, wherein cleaving the modified polynucleotide comprises contacting it with chemical base.

12. The method of claim 11, wherein cleaving the modified polynucleotide comprises contacting it with tris(2-carboxyethyl)phosphine.

13. The method of claim 1, wherein the modified-base nucleotide comprises modified guanine.

14. The method of claim 13, wherein the modified guanine is 7-nitrodeaza-guanine.

15. The method of claim 14, wherein cleaving the modified polynucleotide comprises contacting it with chemical base.

16. The method of claim 13, wherein the modified guanine is N²-allylguanine.

17. The method of claim 16, wherein cleaving the modified polynucleotide comprises contacting it with an electrophile.

18. The method of claim 17, wherein the electrophile is iodine.

19. The method of claim 1, wherein the modified-base nucleotide is modified thymine or modified uracil.

20. The method of claim 19, wherein the modified thymine or modified uracil is 5-hydroxyuracil.

21. The method of claim 20, wherein cleaving the modified polynucleotide comprises:

a. contacting the modified polynucleotide with a chemical oxidant; and

,

b. then contacting it with chemical base.

68. The method of any of claims 1, 22, 56 and 57, wherein the percentage replacement of a natural nucleotide with a modified nucleotide, the percentage cleavage at the modified nucleotide or both the percentage replacement and the percentage cleavage is greater than 95%.

69. The method of any of claims 1, 22, 56 and 57, wherein the percentage replacement of a natural nucleotide with a modified nucleotide, the percentage cleavage at the modified nucleotide or both the percentage replacement and the percentage cleavage is greater than 99%.

22. A method for detecting a variance in a nucleotide sequence in a polynucleotide or for genotyping a polynucleotide suspected to contain a known polymorphism or mutation, comprising:

a. replacing a natural nucleotide at greater than 90% of its points of occurrence in a polynucleotide, provided the points of occurrence are not in a primer sequence, with a modified-sugar nucleotide to form a modified polynucleotide;

b. contacting the modified polynucleotide with a non-enzymatic reagent or reagents that cleave(s) the modified polynucleotide at greater than 90% of the points of occurrence of the modified-sugar nucleotide to give a set of fragments; and

,

c. analyzing the set of fragments to detect a variance in or to genotype the polynucleotide.

23. The method of claim 22, wherein the modified-sugar nucleotide comprises a 2-ketosugar.

24. The method of claim 23, wherein cleaving the modified polynucleotide comprises contacting it with chemical base.

25. The method of claim 22, wherein the modified-sugar nucleotide comprises arabinose.

26. The method of claim 25, wherein cleaving the modified polynucleotide comprises contacting it with chemical base.

27. The method of claim 22, wherein the modified-sugar nucleotide comprises a 4-hydroxymethyl group.

28. The method of claim 27, wherein cleaving the modified polynucleotide comprises contacting it with chemical base.

29. The method of claim 22, wherein the modified-sugar nucleotide comprises hydroxycyclopentane.

30. The method of claim 29, wherein the hydroxycyclopentane comprises 1-hydroxy- or 2-hydroxycyclopentane.

31. The method of claim 29, wherein cleaving the modified polynucleotide comprises contacting it with chemical base.

32. The method of claim 22, wherein the modified-sugar nucleotide comprises an azidosugar.

33. The method of claim 32, wherein the azidosugar comprises a 2′

-azido, a 4′

-azido or a 4′

-azidomethyl sugar.

34. The method of claim 32, wherein cleaving the modified polynucleotide comprises contacting it with tris(2-carboxyethyl)phosphine (TCEP).

35. The method of claim 22, wherein the modified-sugar nucleotide comprises a group capable of photolyzing to form a free radical.

36. The method of claim 35, wherein the group capable of photolyzing to form a free radical is phenylselenyl or t-butylcarboxy.

37. The method of claim 35, wherein cleaving the modified polynucleotide comprises exposing it to ultraviolet light.

38. The method of claim 22, wherein the modified-sugar nucleotide comprises a cyanosugar.

39. The method of claim 38, wherein the cyanosugar comprises a 2′

-cyanosugar or a 2″

-cyanosugar.

40. The method of claim 38, wherein cleaving the modified polynucleotide comprises contacting it with chemical base.

41. The method of claim 22, wherein the modified-sugar nucleotide comprises an electron withdrawing group.

42. The method of claim 41, wherein the electron withdrawing group is selected from the group consisting of fluorine, azido, methoxy and nitro.

43. The method of claim 42, wherein the electron withdrawing group is located at the 2′

, 2″

or 4′

position of the modified-sugar nucleotide.

44. The method of claim 41, wherein cleaving the modified polynucleotide comprises contacting it with chemical base.

45. The method of claim 22, wherein the modified-sugar nucleotide comprises an electron-withdrawing element in the sugar ring.

46. The method of claim 45, wherein the electron-withdrawing element is nitrogen.

47. The method of claim 46, wherein the nitrogen replaces the ring oxygen of the modified sugar.

48. The method of claim 46, wherein the nitrogen replaces a ring carbon of the modified sugar.

49. The method of claim 47, wherein cleaving the modified polynucleotide comprises contacting it with chemical base.

50. The method of claim 48, wherein cleaving the modified polynucleotide comprises contacting it with chemical base.

51. The method of claim 22, wherein the modified-sugar nucleotide comprises a mercapto group.

52. The method of claim 51, wherein the mercapto group is located in the 2′

position of the sugar.

53. The method of claim 51, wherein cleaving the modified polynucleotide comprises contacting it with chemical base.

54. The method of claim 22, wherein the modified-sugar nucleotide comprises a 5′

-methylenyl-sugar, a 5′

-keto-sugar or a 5′

,5′

-difluoro-sugar.

55. The method of claim 54, wherein cleaving the modified polynucleotide comprises contacting it with chemical base.

56. A method for detecting a variance in a nucleotide sequence in a polynucleotide or for genotyping a polynucleotide suspected to contain a known polymorphism or mutation, comprising:

a. replacing a first natural nucleotide and a second, different natural nucleotide in the polynucleotide with a first modified nucleotide and a second, different modified nucleotide, respectively, at greater than 90% of their points of occurrence in the polynucleotide to give a modified polynucleotide;

b. contacting the modified polynucleotide with a non-enzymatic reagent or reagents that cleave(s) the modified polynucleotide at greater than 90% of the sites in the polynucleotide where the first modified nucleotide is followed immediately in sequence by the second modified nucleotide to give a set of fragments; and

,

c. analyzing the set of fragments to detect a variance in or to genotype the polynucleotide.

57. The method of claim 56, wherein:

the first modified nucleotide is covalently bonded at its 5′

position to a nitrogen atom of a phosphoramidate group; and

,

the second modified nucleotide comprises a 2′

-hydroxy group and is contiguous to, and 5′

of, the first modified nucleotide.

58. The method of claim 57, wherein cleaving the modified polynucleotide comprises contacting it with acid.

59. The method of claim 56, wherein:

the first modified nucleotide is covalently bonded at its 3′

position to a nitrogen atom of a phosphoramidate group; and

,

the second modified nucleotide comprises a 2′

-hydroxy group and is contiguous to, and 3′

of, the first modified nucleotide.

60. The method of claim 59, wherein cleaving the modified polynucleotide comprises contacting it with acid.

61. The method of claim 56, wherein:

the first modified nucleotide is covalently bonded at its 5′

position to an oxygen atom of an alkylphosphonate or an alkylphosphorotriester group; and

,

the second modified nucleotide comprises a 2′

-hydroxy group and is contiguous to, and 3′

of, the first modified nucleotide.

62. The method of claim 61, wherein cleaving the modified polynucleotide comprises contacting it with acid.

63. The method of claim 56, wherein:

the first modified nucleotide has an electron-withdrawing group at its 4′

position; and

,

the second modified nucleotide comprises a 2′

-hydroxy group and is contiguous to, and 5′

of, the first modified nucleotide.

64. The method of claim 63, wherein cleaving the modified polynucleotide comprises contacting it with acid.

65. A method for detecting a variance in a nucleotide sequence in a polynucleotide or for genotyping a polynucleotide suspected to contain a known polymorphism, comprising:

a. replacing a natural nucleotide at greater than 90% of its points of occurrence in a polynucleotide with a modified-phosphodiester nucleotide to form a modified polynucleotide;

b. contacting the modified polynucleotide with a non-enzymatic reagent or reagents that cleave(s) the modified polynucleotide at greater than 90% of the points of occurrence of the modified-phosphodiester nucleotide to give a set of fragments; and

,

c. analyzing the set of fragments to detect a variance in or to genotype the polynucleotide.

66. The method of claim 65, wherein the modified-phosphodiester nucleotide comprises phosphorothioate.

67. The method of claim 65, wherein the modified-phosphorothioate nucleotide comprises phosphoroamidate.