Method for determining polynucleotide sequence variations

• US 20030138834A1
• Filed: 01/15/2003
• Published: 07/24/2003
• Est. Priority Date: 08/19/1998
• Status: Abandoned Application

• Current Assignee: BioVentures, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: BioVentures, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Dawson, Elliott P.
• Application Number:
  US10/346,156
• Publication Number:
  US 20030138834A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• 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: C12Q 2563/107 fluorescence

Abstract

A method for determining the presence, location or identity, or a combination of these, of the nucleotides in a polynucleotide. A method for determining the presence, location or identity, or a combination of these, of one or more than one nucleotide difference between a first polynucleotide and a second polynucleotide, or between more than two polynucleotides.

Claims

1. A method for determining the presence, location or identity, or a combination of these, of the nucleotides in a first polynucleotide, or for determining the presence, location or identity, or a combination of these, of one or more than one nucleotide difference between a first polynucleotides and a second polynucleotide, comprising:

a) providing a sample of the first polynucleotide;

b) selecting a region of the first polynucleotide potentially containing the variation;

c) subjecting the selected region to a template producing amplification reaction to produce a first plurality of double stranded polynucleotide templates which includes the selected region;

d) selecting a region of the templates potentially containing the variation;

e) producing a first family of labeled, linear polynucleotide fragments from both strands of the templates simultaneously by a fragment producing reaction including,

i) a set of at least two primers comprising a first primer and a second primer,

ii) at least four types of non-sequence-terminating nucleotides, comprising at least two different sets of two Watson-Crick-pairing nucleotides or nucleotide analogs, and

iii) two types of sequence-terminating non-Watson-Crick-pairing nucleotides or nucleotide analogs, comprising a first terminator and a second terminator;

where one or more than one of the non-sequence-terminating nucleotides or the sequence-terminating non-Watson-Crick-pairing nucleotides is a nucleotide analog;

where the first primer and the second primer flank the selected region of the template strands;

where the first primer has a first primer label and the second primer has a second primer label;

where at least a portion of one of the types of non-sequence-terminating nucleotides is labeled with a first nucleotide label;

where the first terminator is labeled with a first terminator label and the second terminator is labeled with a second terminator label;

where each of the first primer label, the second primer label, the first nucleotide label, the first terminator label and the second terminator label are all distinguishable from each other;

where each of the first family of fragments are terminated by either the first terminator or the second terminator at the 3′

end of the fragment; and

where the first family of fragments includes one or more than one fragment terminating at each possible base, represented by the either the first terminator or the second terminator, of that portion of the selected region of both template strands flanked by a primer; and

f) determining the location and identity of the bases in the selected region of the first polynucleotide by detecting the first primer label, the second primer label, the first nucleotide label, the first terminator label and the second terminator label present in the fragments.

2. The method of claim 1, additionally comprising comparing the location and identity of the bases determined with the location and identity of bases from a second polynucleotide, thereby identifying the presence and identity of a variation in a nucleotide sequence between the selected region of the first polynucleotide and a corresponding region of the second polynucleotide, after determining the location and identity of the bases in the selected region of the first polynucleotide.

3. The method of claim 1, where the selected region of the first polynucleotide comprises a plurality of discontinuous sequences on the first polynucleotide.

4. The method of claim 1, where the template producing amplification reaction comprises subjecting the selected region to PCR.

5. The method of claim 1, where the template producing amplification reaction comprises subjecting the selected region to RT-PCR.

6. The method of claim 1, where the first plurality of double stranded polynucleotide templates comprises double stranded nucleic acid strands of between about 50 and 50,000 nucleotides per strand.

7. The method of claim 1, further comprising purifying the temples to remove other amplification reaction components after subjecting the selected region to a template producing amplification reaction.

8. The method of claim 1, where the fragment producing amplification reaction comprises subjecting the selected region to PCR.

9. The method of claim 1, where the fragment producing amplification reaction comprises subjecting the selected region to RT-PCR.

10. The method of claim 1, where the selected region of the template strands is between about 100 and 1000 nucleotides per strand.

11. The method of claim 1, where the at least four types of non-sequence-terminating nucleotides comprise dATP, dCTP, dGTP and dTTP.

12. The method of claim 1, where the at least four types of non-sequence-terminating nucleotides comprise dATP, dCTP, dGTP and dUTP.

13. The method of claim 1, where one or more than one of the at least four types of the non-sequence-terminating nucleotides comprise an alpha thio dNTP analog.

14. The method of claim 1, where two of the at least four types of the non-sequence-terminating nucleotides comprise an alpha thio dNTP analog, two of the at least four types of the non-sequence-terminating nucleotides comprise dNTPs, and the two sequence-terminating non-Watson-Crick-pairing nucleotides comprise ddNTPs corresponding to the two alpha thio phosphate dNTPs.

15. The method of claim 14, where the two alpha thio phosphate non-sequence-terminating nucleotides are present in an initial concentration of between about 10% and 50% of that of the initial concentration of the two dNTP non-sequence-terminating nucleotides.

16. The method of claim 1, where one or more than one of the two types of the sequence-terminating non-Watson-Crick-pairing nucleotides comprises an alpha thio dNTP analog.

17. The method of claim 1, where one or more than one of the two types of the sequence-terminating non-Watson-Crick-pairing nucleotides is a 2′

deoxnucleotide triphosphates analog having an extension blocking moiety at the 3′

position.

18. The method of claim 17, where the extension blocking moiety is selected from the group consisting of an azide moiety, an amino moiety, a deoxy moiety, a fluoro moiety and a methoxy moiety.

19. The method of claim 1, where one or more than one of the sequence-terminating non-Watson-Crick-pairing nucleotides has an acyclo analog of a nucleotide sugar moiety.

20. The method of claim 1, where the first terminator comprises a pyrimidine nucleotide and where the second terminator comprises a purine nucleotide.

21. The method of claim 1, where the first terminator and the second terminator are selected from the group consisting of ddATP:

ddCTP, ddATP;

ddGTP, ddCTP;

ddTTP, ddGTP;

ddTTP, ddCTP;

ddUTP and ddGTP;

ddUTP and one of the foregoing pairs where one or both members of the pair is a nucleotide analog.

22. The method of claim 1, where one or more than one of the first primer label, the second primer label, the first nucleotide label, the first terminator label and the second terminator label are selected from the group consisting of fluorescent labels, fluorescent energy transfer labels, luminescent labels, chemiluminescent labels, phosphorescent labels and photoluminescent labels.

23. The method of claim 1, where the portion of one of the types of non-sequence-terminating nucleotides that is labeled with a first nucleotide label comprises between about 1% and about 10% of the total concentration of unlabeled nucleotide triphosphates.

24. The method of claim 1, further comprising purifying the labeled reaction products from the fragment producing reaction before determining the location and identity of the bases in the selected region of the first polynucleotide.

25. The method of claim 2, where the sequence of the corresponding region of the second polynucleotide is determined by:

a) providing a sample of the second polynucleotide;

b) selecting a region of the second polynucleotide which corresponds to the region of the first polynucleotide potentially containing the variation;

c) subjecting the corresponding region of the second polynucleotide to a template producing amplification reaction to produce a second plurality of double stranded polynucleotide templates which includes the corresponding region;

d) producing a second family of labeled, linear polynucleotide fragments from both strands of the template simultaneously by a fragment producing reaction including,

i) a set of at least two primers comprising a third primer and a fourth primer,

ii) at least four types of non-sequence-terminating nucleotides, comprising at least two different sets of two Watson-Crick-pairing nucleotides or nucleotide analogs, and

iii) two types of sequence-terminating non-Watson-Crick-pairing nucleotides or nucleotide analogs, comprising a third terminator and a fourth terminator;

where the third primer and the fourth primer flank the selected region of the template strands;

where each of the second family of fragments are terminated by either the third terminator or the fourth terminator at the 3′

end of the fragment; and

where the second family of fragments includes one or more than one fragment terminating at each possible base, represented by the either the third terminator or the fourth terminator, of that portion of the selected region of both template strands flanked by a primer;

e) determining the location and identity of at least some of the bases in the corresponding region of the second polynucleotide.

26. The method of claim 25, where the location and identity of the bases of the corresponding region of the second polynucleotide is determined simultaneously with determining the location and identity of the bases in the selected region of the first polynucleotide.

27. The method of claim 25, where producing the first family of labeled, linear polynucleotide fragments and producing the second family of labeled, linear polynucleotide fragments is performed in one reaction.

28. The method of claim 25, where the third primer has a third primer label and the fourth primer has a fourth primer label, and where the third primer label and the fourth primer label are distinguishable from each other.

29. The method of claim 25, where at least a portion of one of the types of non-sequence-terminating nucleotides in the production of the second family of labeled, linear polynucleotide fragments is labeled with a second nucleotide label.

30. The method of claim 25, where the third terminator is labeled with a third terminator label and the fourth terminator is labeled with a fourth terminator label, and where the third terminator label and the fourth terminator label are distinguishable from each other.

31. The method of claim 25, where the third primer has a third primer label, the fourth primer has a fourth primer label and at least a portion of one of the types of non-sequence-terminating nucleotides in the production of the second family of labeled, linear polynucleotide fragments is labeled with a second nucleotide label, and where the third primer label, the fourth primer label and the second nucleotide label are all distinguishable from each other.

32. The method of claim 25, where the third primer has a third primer label, the fourth primer has a fourth primer label, the third terminator is labeled with a third terminator label and the fourth terminator is labeled with a fourth terminator label, and where the third primer label, the fourth primer label, the third terminator label and the fourth terminator label are all distinguishable from each other.

33. The method of claim 25, where at least a portion of one of the types of non-sequence-terminating nucleotides in the production of the second family of labeled, linear polynucleotide fragments is labeled with a second nucleotide label, where the third terminator is labeled with a third terminator label and the fourth terminator is labeled with a fourth terminator label, and where the second nucleotide label, the third terminator label and the fourth terminator label are all distinguishable from each other.

34. The method of claim 25, where the third primer has a third primer label, the fourth primer has a fourth primer label, at least a portion of one of the types of non-sequence-terminating nucleotides in the production of the second family of labeled, linear polynucleotide fragments is labeled with a second nucleotide label, the third terminator is labeled with a third terminator label and the fourth terminator is labeled with a fourth terminator label, and where the third primer label, the fourth primer label, the second nucleotide label, the third terminator label and the fourth terminator label are all distinguishable from each other.

35. A method for determining the presence, location or identity, or a combination of these, of the nucleotides in a first polynucleotide, or for determining the presence, location or identity, or a combination of these, of one or more than one nucleotide difference between a first polynucleotides and a second polynucleotide, comprising:

a) providing a sample of the first polynucleotide;

b) selecting a region of the first polynucleotide potentially containing the variation;

c) subjecting the selected region to a template producing amplification reaction to produce a first plurality of double stranded polynucleotide templates which includes the selected region;

d) selecting a region of the templates potentially containing the variation;

e) producing a first family of labeled, linear polynucleotide fragments from both strands of the templates simultaneously by a fragment producing reaction including,

i) a set of at least two primers comprising a first primer and a second primer,

ii) at least four types of non-sequence-terminating nucleotides, comprising at least two different sets of two Watson-Crick-pairing nucleotides or nucleotide analogs, and

iii) two types of sequence-terminating non-Watson-Crick-pairing nucleotides or nucleotide analogs, comprising a first terminator and a second terminator;

where one or more than one of the non-sequence-terminating nucleotides or the sequence-terminating non-Watson-Crick-pairing nucleotides is a nucleotide analog;

where the first primer and the second primer flank the selected region of the template strands;

where each of the first family of fragments are terminated by either the first terminator or the second terminator at the 3′

end of the fragment; and

where the first family of fragments includes one or more than one fragment terminating at each possible base, represented by the either the first terminator or the second terminator, of that portion of the selected region of both template strands flanked by a primer; and

f) determining the location and identity of the bases in the selected region.

36. The method of claim 35, additionally comprising comparing the location and identity of the bases determined with the location and identity of bases from a second polynucleotide, thereby identifying the presence and identity of a variation in a nucleotide sequence between the selected region of the first polynucleotide and a corresponding region of the second polynucleotide, after determining the location and identity of the bases in the selected region of the first polynucleotide.

37. The method of claim 35, where the first primer has a first primer label and the second primer has a second primer label, and where the first primer label and the second primer label are distinguishable from each other.

38. The method of claim 35, where at least a portion of one of the types of non-sequence-terminating nucleotides is labeled with a first nucleotide label.

39. The method of claim 35, where the first terminator is labeled with a first terminator label and the second terminator is labeled with a second terminator label, and where the first terminator label and the second terminator label are distinguishable from each other.

40. The method of claim 35, where the first primer has a first primer label, the second primer has a second primer label and at least a portion of one of the types of non-sequence-terminating nucleotides is labeled with a first nucleotide label, and where the first primer label, the second primer label and the first nucleotide label are all distinguishable from each other.

41. The method of claim 35, where the first primer has a first primer label, the second primer has a second primer label, the first terminator is labeled with a first terminator label and the second terminator is labeled with a second terminator label, and where the first primer label, the second primer label, the first terminator label and the second terminator label are all distinguishable from each other.

42. The method of claim 35, where at least a portion of one of the types of non-sequence-terminating nucleotides is labeled with a first nucleotide label, where the first terminator is labeled with a first terminator label and the second terminator is labeled with a second terminator label, and where the first nucleotide label, the first terminator label and the second terminator label are all distinguishable from each other.

43. The method of claim 35, where the first primer has a first primer label, the second primer has a second primer label, at least a portion of one of the types of non-sequence-terminating nucleotides is labeled with a first nucleotide label, the first terminator is labeled with a first terminator label and the second terminator is labeled with a second terminator label, and where the first primer label, the second primer label, the first nucleotide label, the first terminator label and the second terminator label are all distinguishable from each other.

44. The method of claim 35, where the selected region of the first polynucleotide comprises a plurality of discontinuous sequences on the first polynucleotide.

45. The method of claim 35, where the template producing amplification reaction comprises subjecting the selected region to PCR.

46. The method of claim 35, where the template producing amplification reaction comprises subjecting the selected region to RT-PCR.

47. The method of claim 35, where the first plurality of double stranded polynucleotide templates comprises double stranded nucleic acid strands of between about 50 and 50,000 nucleotides per strand.

48. The method of claim 35, further comprising purifying the temples to remove other amplification reaction components after subjecting the selected region to a template producing amplification reaction.

49. The method of claim 35, where the fragment producing amplification reaction comprises subjecting the selected region to PCR.

50. The method of claim 35, where the fragment producing amplification reaction comprises subjecting the selected region to RT-PCR.

51. The method of claim 35, where the selected region of the template strands is between about 100 and 1000 nucleotides per strand.

52. The method of claim 35 where the at least four types of non-sequence-terminating nucleotides comprise dATP, dCTP, dGTP and dTTP.

53. The method of claim 35 where the at least four types of non-sequence-terminating nucleotides comprise dATP, dCTP, dGTP and dUTP.

54. The method of claim 35 where one or more than one of the at least four types of the non-sequence-terminating nucleotides comprise an alpha thio dNTP analog.

55. The method of claim 35, where two of the at least four types of the non-sequence-terminating nucleotides comprise an alpha thio dNTP analog, two of the at least four types of the non-sequence-terminating nucleotides comprise dNTPs, and the two sequence-terminating non-Watson-Crick-pairing nucleotides comprise ddNTPs corresponding to the two alpha thio phosphate dNTPs.

56. The method of claim 55, where the two alpha thio phosphate non-sequence-terminating nucleotides are present in an initial concentration of between about 10% and 50% of that of the initial concentration of the two dNTP non-sequence-terminating nucleotides.

57. The method of claim 35 where one or more than one of the two types of the sequence-terminating non-Watson-Crick-pairing nucleotides comprises an alpha thio dNTP analog.

58. The method of claim 35 where one or more than one of the two types of the sequence-terminating non-Watson-Crick-pairing nucleotides is a 2′

deoxnucleotide triphosphates analog having an extension blocking moiety at the 3′

position.

59. The method of claim 58, where the extension blocking moiety is selected from the group consisting of an azide moiety, an amino moiety, a deoxy moiety, a fluoro moiety and a methoxy moiety.

60. The method of claim 35 where one or more than one of the sequence-terminating non-Watson-Crick-pairing nucleotides has an acyclo analog of a nucleotide sugar moiety.

61. The method of claim 35 where the first terminator comprises a pyrimidine nucleotide and where the second terminator comprises a purine nucleotide.

62. The method of claim 35 where the first terminator and the second terminator are selected from the group consisting of ddATP:

ddCTP, ddATP;

ddGTP, ddCTP;

ddTTP, ddGTP;

ddTTP, ddCTP;

ddUTP, ddGTP;

ddUTP and one of the foregoing pairs where one or both members of the pair is a nucleotide analog.

63. The method of claim 35, where one or more than one of the first primer label, the second primer label, the first nucleotide label, the first terminator label and the second terminator label are selected from the group consisting of fluorescent labels, fluorescent energy transfer labels, luminescent labels, chemiluminescent labels, phosphorescent labels and photoluminescent labels.

64. The method of claim 35, where the portion of one of the types of non-sequence-terminating nucleotides that is labeled with a first nucleotide label comprises between about 1% and about 10% of the total concentration of unlabeled non-sequence-terminating nucleotides.

65. The method of claim 35, further comprising purifying the labeled reaction products from the fragment producing reaction before determining the location and identity of the bases in the selected region of the first polynucleotide.

66. The method of claim 35, where one or more of the first primer, the second primer, a portion of one of the types of non-sequence-terminating nucleotides, the first terminator and the second terminator is labeled, and where determining the location and identity of the bases in the selected region of the first polynucleotide is accomplished by detecting the label or labels.

67. The method of claim 36, where the sequence of the corresponding region of the second polynucleotide is determined by:

a) providing a sample of the second polynucleotide;

b) selecting a region of the second polynucleotide which corresponds to the region of the first polynucleotide potentially containing the variation;

c) subjecting the corresponding region of the second polynucleotide to a template producing amplification reaction to produce a second plurality of double stranded polynucleotide templates which includes the corresponding region;

d) producing a second family of labeled, linear polynucleotide fragments from both strands of the template simultaneously by a fragment producing reaction including,

i) a set of at least two primers comprising a third primer and a fourth primer,

ii) at least four types of non-sequence-terminating nucleotides, comprising at least two different sets of two Watson-Crick-pairing nucleotides or nucleotide analogs, and

iii) two types of sequence-terminating non-Watson-Crick-pairing nucleotides or nucleotide analogs, comprising a third terminator and a fourth terminator;

where the third primer and the fourth primer flank the selected region of the template strands;

where each of the second family of fragments are terminated by either the third terminator or the fourth terminator at the 3′

end of the fragment; and

where the second family of fragments includes one or more than one fragment terminating at each possible base, represented by the either the third terminator or the fourth terminator, of that portion of the selected region of both template strands flanked by a primer;

e) determining the location and identity of at least some of the bases in the corresponding region of the second polynucleotide.

68. The method of claim 67, where the location and identity of the bases of the corresponding region of the second polynucleotide is determined simultaneously with determining the location and identity of the bases in the selected region of the first polynucleotide.

69. The method of claim 67, where producing the first family of labeled, linear polynucleotide fragments and producing the second family of labeled, linear polynucleotide fragments is performed in one reaction.

70. The method of claim 67, where the third primer has a third primer label and the fourth primer has a fourth primer label, and where the third primer label and the fourth primer label are distinguishable from each other.

71. The method of claim 67, where at least a portion of one of the types of non-sequence-terminating nucleotides in the production of the second family of labeled, linear polynucleotide fragments is labeled with a second nucleotide label.

72. The method of claim 67, where the third terminator is labeled with a third terminator label and the fourth terminator is labeled with a fourth terminator label, and where the third terminator label and the fourth terminator label are distinguishable from each other.

73. The method of claim 67, where the third primer has a third primer label, the fourth primer has a fourth primer label and at least a portion of one of the types of non-sequence-terminating nucleotides in the production of the second family of labeled, linear polynucleotide fragments is labeled with a second nucleotide label, and where the third primer label, the fourth primer label and the second nucleotide label are all distinguishable from each other.

74. The method of claim 67, where the third primer has a third primer label, the fourth primer has a fourth primer label, the third terminator is labeled with a third terminator label and the fourth terminator is labeled with a fourth terminator label, and where the third primer label, the fourth primer label, the third terminator label and the fourth terminator label are all distinguishable from each other.

75. The method of claim 67, where at least a portion of one of the types of non-sequence-terminating nucleotides in the production of the second family of labeled, linear polynucleotide fragments is labeled with a second nucleotide label, where the third terminator is labeled with a third terminator label and the fourth terminator is labeled with a fourth terminator label, and where the second nucleotide label, the third terminator label and the fourth terminator label are all distinguishable from each other.

76. The method of claim 67, where the first primer has a first primer label, the second primer has a second primer label, the third primer has a third primer label, the second primer has a second primer label, at least a portion of one of the types of non-sequence-terminating nucleotides in the production of the first family of labeled, linear polynucleotide fragments is labeled with a first nucleotide label, at least a portion of one of the types of non-sequence-terminating nucleotides in the production of the second family of labeled, linear polynucleotide fragments is labeled with a second nucleotide label, the first terminator is labeled with a first terminator label, the second terminator is labeled with a second terminator label, the third terminator is labeled with a third terminator label and the fourth terminator is labeled with a fourth terminator label, and where the first primer label, the second primer label, the third primer label, the fourth primer label, the first nucleotide label, the second nucleotide label, the first terminator label, the second terminator label, the third terminator label and the fourth terminator label are all distinguishable from each other.