Поиск по международным и национальным патентным фондам
Некоторое содержание этого приложения в настоящий момент недоступно.
Если эта ситуация сохраняется, свяжитесь с нами по адресуОтзывы и контакты
1. (WO2000050642) SEQUENCING BY INCORPORATION
Примечание: Текст, основанный на автоматизированных процессах оптического распознавания знаков. Для юридических целей просьба использовать вариант в формате PDF

WHAT IS CLAIMED IS:

1. A method of sequencing a nucleic acid in a microfluidic device, the method comprising:
(i) flowing a nucleic acid template and a primer through a microscale
channel;
(ii) flowing a polymerase and one or more nucleotides or nucleotide
analogs through the microscale channel, thereby adding at least one of the one or more nucleotides or nucleotide analogs to the primer,
resulting in an extended primer;
(iii) detecting the at least one of the one or more nucleotides or nucleotide analogs; and,
(iv) repeating steps (ii) through (iv), thereby sequencing the nucleic acid.

2. The method of claim 1, wherein the one or more nucleotides or nucleotide analogs each comprise a 3 '-blocking group, wherein the extended primer comprises a non-extendable primer, and wherein the method further comprises removing the 3 '-blocking group after step (ii) and prior to step (iv), thereby resulting in an extendable primer.

3. The method of claim 2, wherein the 3 '-blocking group comprises a phosphate group or a carbamate group.

4. The method of claim 2, wherein the one or more nucleotide analogs comprise a compound having formula (I) or (II),



wherein R4 comprises one or more of a linker moiety and a detectable label and B comprises one or more of a nitrogenous base and the detectable label.

5. The method of claim 1, wherein the one or more nucleotides or nucleotide analogs each comprise a fluorescent label,

6. The method of claim 5, wherein step (iii) further comprises photobleaching the fluorescent label after detecting the at least one of the one or more nucleotides or nucleotide analogs.

7. The method of claim 1, wherein step (iii) comprises detecting an intercalating dye, which intercalating dye intercalates into the extended primer.

8. The method of claim 1, wherein the one or more nucleotides or nucleotide analogs comprise chain-terminating labeled nucleotides or nucleotide analogs and non-terminating, non-labeled nucleotides or nucleotide analogs.

9. The method of claim 8, comprising providing a low concentration of chain-terminating labeled nucleotides or nucleotide analogs in comparison to non-terminating, non-labeled nucleotides or nucleotide analogs.

10. The method of claim 8, comprising providing about a 1/1000 ratio of chain-terminating labeled nucleotides or nucleotide analogs to non-terminating, non-labeled nucleotides or nucleotide analogs.

11. A method for sequencing a nucleic acid, the method comprising: (i) providing a nucleic acid template and a primer;

(ii) incubating the nucleic acid template and the primer with a polymerase and one or more nucleotide analogs having formula (III),
< πi )



thereby adding at least one of the one or more nucleotide analogs to the
primer, resulting in an extended primer, wherein R1 comprises a
nucleoside, a nucleotide, a nucleotide analog, or a nucleoside analog, R comprises a blocking group, which blocking group comprises a
detectable label, and R3 comprises a hydrogen or a negative charge; (iii) detecting the detectable label, thereby detecting the at least one of the one or more nucleotide analogs and removing the blocking group from the extended primer; or, removing the blocking group from the
extended primer and detecting the detectable label, thereby detecting the at least one of the one or more nucleotide analogs;
(iv) removing a 3 '-phosphate from the extended primer, thereby producing an extendable primer; and,
(v) repeating steps (ii) through (v), thereby sequencing the nucleic acid
template.

12. The method of claim 11, wherein the nucleic acid template is DNA or RNA.

13. The method of claim 11, wherein the detectable label is a fluorescent moiety or a chemiluminescent moiety.

14. The method of claim 11, R1 comprising a nucleoside 5'-triphosphate.

15. The method of claim 14, wherein the nucleoside 5 '-triphosphate comprises deoxyadenosine 5 '-triphosphate, deoxyguanosine 5'-triphosphate, deoxycytidine 5 '-triphosphate, deoxythymidine 5 '-triphosphate, deoxyuridine 5'-triphosphate, adenosine 5 '-triphosphate, guanosine 5 '-triphosphate, cytidine 5'-triphosphate, uridine 5 '-triphosphate, or an analog thereof.

16. The method of claim 11, R1 comprising a compound having formula (IV)



wherein B comprises a nitrogenous base.

17. The method of claim 16, wherein the nitrogenous base comprises adenine, guanine, thymine, cytosine, or uracil.

18. The method of claim 11, R comprising a chain terminating blocking group.

19. The method of claim 18, wherein the chain-terminating blocking group comprises a reversible chain-terminating blocking group.

20. The method of claim 18, R2 comprising a compound having formula (V) or formula (VI)
( V )

^^^SR4
( VI )



wherein R4 comprises one or more of: a linker moiety and the detectable label.

21. The method of claim 20, comprising providing the linker moiety to comprise an acyl, an S-acyl, an alkyl, an aromatic, or an acetyl group.

22. The method of claim 20, wherein removing the blocking group from the extended primer comprises contacting the extended primer with a reducing agent.

23. The method of claim 22, wherein removing the blocking group from the extended primer comprises contacting the extended primer with one or more of: diborane, dithiothreitol, glutathione, TCEP, and disulfide reductase.

24. The method of claim 11, wherein removing the blocking group from the extended primer produces a compound having formula (VII):
(vπ )


25. The method of claim 24, wherein the compound having formula (VII) self-cleaves to produce a compound having formula (VIII):
( VIE )

o
O=P-Ό
O

26. The method of claim 11, comprising detecting the detectable label after removing the blocking group from the extended primer.

27. The method of claim 11, comprising detecting the detectable label prior to removing the blocking group from the extended primer.

28. The method of claim 11, comprising removing the blocking group from the extended primer concurrently with removing the 3 '-phosphate from the extended primer.

29. The method of claim 11, wherein step (iv) comprises
enzymatically or chemically removing the 3'-phosphate from the extended primer.

30. The method of claim 11, wherein step (iv) comprises incubating the extended primer with a phosphatase.

31. The method of claim 30, wherein the phosphatase is an alkaline phosphatase.

32. A method for sequencing a nucleic acid, the method comprising:

(i) providing a nucleic acid template and a primer;

(ii) incubating the nucleic acid template and the primer with a polymerase and one or more nucleotide analogs having formula (IX),
( IX )
R1 rl

o
thereby adding at least one of the one or more nucleotide analogs to the
primer, resulting in an extended primer; wherein R1 comprises a
nucleoside, a nucleotide, a nucleoside analog, or a nucleotide analog, R2 comprises a linker moiety, and either R1 or R2 further comprises a detectable label;
(iii) detecting the detectable label, thereby detecting the at least one of the one or more nucleotide analogs and removing R2 and a carbamate
group from the extended primer; or, removing R and the carbamate group from the extended primer and detecting the detectable label,
thereby detecting the at least one of the one or more nucleotide
analogs; and,
(iv) repeating steps (ii) and (iv), thereby sequencing the nucleic acid
template.

33. The method of claim 32, wherein the nucleic acid template is DNA or RNA.

34. The method of claim 32, R1 comprising a nucleoside 5'-triphosphate.

35. The method of claim 34, wherein the nucleoside 5 '-triphosphate comprises deoxyadenosine 5 '-triphosphate, deoxyguanosine 5 '-triphosphate, deoxycytidine 5'-triphosphate, deoxythymidine 5 '-triphosphate, deoxyuridine 5'-triphosphate, adenosine 5'-triphosphate, guanosine 5 '-triphosphate, cytidine 5'-triphosphate, uridine 5'-triphosphate, or an analog thereof.

36. The method of claim 32, R2 comprising an alkyl, an acetyl, an acyl, an aromatic, or a heterocyclic aromatic group.

37. The method of claim 32, R2 comprising a compound having formula (X):
( X )


38. The method of claim 32, wherein removing R2 and the carbamate group from the extended primer comprises incubating the extended primer with a reducing agent.

39. The method of claim 32, wherein the detectable label comprises a fluorescent or chemiluminescent moiety.

40. The method of claim 32, comprising detecting the detectable label prior to removing R and the carbamate group from the extended primer.

41. The method of claim 32, comprising detecting the detectable label after removing R2 and the carbamate group from the extended primer.

42. The method of claim 11 or 32, comprising performing the method in a microfluidic device.

43. A method of sequencing a nucleic acid, the method comprising:

(i) providing a nucleic acid template and a primer;
(ii) incubating the nucleic acid template and the primer with a polymerase and one or more fluorescently labeled nucleotides, thereby adding at
least one of the one or more fluorescently labeled nucleotides to the
primer, resulting in an extended primer;
(iii) photobleaching the at least one of the one or more fluorescently
labeled nucleotides; and,
(iv) repeating steps (ii) and (iii) thereby sequencing the nucleic acid.

44. The method of claim 43, wherein the nucleic acid template comprises DNA or RNA.

45. The method of claim 43, wherein the one or more fluorescently labeled nucleotides comprise one or more of: deoxyadenosine 5 '-triphosphate, deoxyguanosine 5 '-triphosphate, deoxycytidine 5'-triphosphate, deoxythymidine 5'-triphosphate, deoxyuridine 5 '-triphosphate, adenosine 5 '-triphosphate, guanosine 5'-triphosphate, cytidine 5'-triphosphate, uridine 5 '-triphosphate, and an analog thereof.

46. The method of claim 43, wherein the one or more fluorescently labeled nucleotides comprise a series of four detectably different nucleotides, which four different nucleotides are selected from: deoxyadenosine 5'-triphosphate, deoxyguanosine 5 '-triphosphate, deoxycytidine 5 '-triphosphate, deoxythymidine 5'-triphosphate, deoxyuridine 5 '-triphosphate, adenosine 5 '-triphosphate, guanosine 5'-triphosphate, cytidine 5 '-triphosphate, uridine 5'-triphosphate, and an analog thereof.

47. The method of claim 43, wherein incubating the nucleic acid template and the primer with the polymerase and the one or more fluorescently labeled nucleotides further comprises removing unincorporated fluorescently labeled nucleotides prior to photobleaching.

48. The method of claim 43, wherein step (ii) comprises incubating the nucleic acid template and the primer with a first nucleotide and wherein step (iv) comprises repeating steps (ii) and (iii) at least a first time for at least a second nucleotide, a third nucleotide, and a fourth nucleotide.

49. The method of claim 48, wherein the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide each comprise a different nucleotide.

50. The method of claim 49, wherein the different nucleotide is selected from: deoxyadenosine 5 '-triphosphate, deoxyguanosine 5 '-triphosphate, deoxycytidine 5'-triphosphate, deoxythymidine 5'-triphosphate, deoxyuridine 5'-triphosphate, adenosine 5'-triphosphate, guanosine 5 '-triphosphate, cytidine 5'-triphosphate, uridine 5 '-triphosphate, and an analog thereof.

51. The method of claim 43, wherein step (iii) comprises applying a light pulse, which light pulse destroys or reduces to an acceptable level the fluorescence of the at least one of the one or more fluorescently labeled nucleotides.

52. The method of claim 51, comprising applying the light pulse for about 20 seconds or less, about 10 seconds or less, about 2 seconds or less, about 1 second or less, or about 0.1 second or less.

53. The method of claim 51, wherein the light pulse has a wavelength equal to a wavelength of light absorbed by the at least one of the one or more fluorescently labeled nucleotides.

54. The method of claim 43, wherein the photobleaching step reduces the fluorescence of the extended primer to a background level.

55. The method of claim 43, wherein the photobleaching step reduces the fluorescence of the at least one of the one or more fluorescently labeled nucleotides to a background level.

56. The method of claim 43, wherein step (iii) comprises
photobleaching the at least one or more fluorescently labeled nucleotides for about 20 seconds or less, about 10 seconds or less, about 2 seconds or less, about 1 second or less, or about 0.1 second or less.

57. The method of claim 43, the method further comprising detecting the at least one of the one or more fluorescently labeled nucleotides prior to or concuoent with photobleaching the at least one of the one or more fluorescently labeled nucleotides.

58. The method of claim 43, the method further comprising detecting the at least one of the one or more fluorescently labeled nucleotides by the photobleaching the at least one of the one or more fluorescently labeled nucleotides.

59. The method of claim 43, wherein the nucleic acid template comprises at least about 500 or more, about 1000 or more, about 2000 or more nucleotides, or about 10,000 or more nucleotides.

60. The method of claim 43, comprising sequencing a nucleic acid template with at least about 80%, at least about 90%, or at least about 95% accuracy, which nucleic acid template comprises at least about 500 or more, about 1000 or more, about 2000 or more, or about 10,000 or more nucleotides.

61. The method of claim 43, comprising performing steps (i) through (iv) in a microscale channel.

62. The method of claim 43, the method further comprising washing the microscale channel after step (ii), thereby removing or rendering unincorporable any unincorporated nucleotides from the microscale channel.

63. A method of sequencing a nucleic acid, the method comprising: (i) hybridizing a nucleic acid template and a primer, producing a hybridized nucleic acid comprising a double-stranded region;
(ii) incubating the hybridized nucleic acid with a polymerase and a series of nucleotides, thereby adding at least one nucleotide to the primer, resulting in an extended double-stranded region, wherein the incubating is performed in the presence of an intercalating dye, which intercalating dye intercalates into the extended double stranded region;
(iii) detecting the intercalating dye, thereby detecting the addition of the at least one nucleotide to the primer; and,
(iv) repeating steps (ii) through (iv) thereby sequencing the nucleic acid.

64. The method of claim 63, wherein the nucleic acid is DNA or

RNA.

65. The method of claim 63, wherein the series of nucleotides comprises one or more of: deoxyadenosine 5'-triphosphate, deoxyguanosine 5'-triphosphate, deoxycytidine 5 '-triphosphate, deoxythymidine 5 '-triphosphate, deoxyuridine 5 '-triphosphate, adenosine 5'-triphosphate, guanosine 5'-triphosphate, cytidine 5'-triphosphate, uridine 5 '-triphosphate, and an analog thereof.

66. The method of claim 63, wherein the intercalating dye comprises ethidium, ethidium bromide, an acridine dye, an intercalating nucleic acid stain, a cyanine dye, proflavin, propidium iodide, acriflavin, proflavin, actinomycin, anthracyclines, or nogalamycin.

67. The method of claim 63, wherein the intercalating dye intercalates into the double stranded region and into the extended double-stranded region.

68. The method of claim 63, wherein the detecting step comprises detecting a signal difference between the double stranded region and the extended double stranded region.

69. The method of claim 63, wherein the detecting step further comprises photobleaching the intercalating dye after detecting the intercalating dye or approximately concuoent with detecting the intercalating dye.

70. The method of claim 63, comprising performing the method in a microscale channel.

71. The method of claim 70, wherein the incubating step comprises:
(a) incubating the nucleic acid double stranded region with a first
nucleotide;
(b) washing unincorporated nucleotides from the microscale channel and detecting the intercalating dye;
(c) repeating steps (a) and (b) for a second nucleotide, a third
nucleotide, and a fourth nucleotide.

72. The method of claim 71, wherein the first nucleotide, the second, nucleotide, the third nucleotide and the fourth nucleotide each comprise a different nucleotide.

73. The method of claim 72, wherein the different nucleotide is selected from: deoxyadenosine 5 '-triphosphate, deoxyguanosine 5 '-triphosphate, deoxycytidine 5 '-triphosphate, deoxythymidine 5'-triphosphate, deoxyuridine 5'-triphosphate, adenosine 5'-triphosphate, guanosine 5'-triphosphate, cytidine 5'-triphosphate, uridine 5'-triphosphate, and an analog thereof.

74. The method of claim 11, claim 32, claim 43, or claim 63, comprising providing a set of particles, which set of particles comprises one or more of: the nucleic acid template and the primer.

75. The method of claim 74, wherein the set of particles comprises an ordered aoay.

76. The method of claim 74, wherein the set of particles comprises about 1 or more particles, about 10 or more particles, about 100 or more particles, about 1000 or more particles, or about 10,000 or more particles.

77. The method of claim 74, wherein the set of particles comprises a set of beads, which beads are selected from: polymer beads, silica beads, ceramic beads, clay beads, glass beads, magnetic beads, metallic beads, paramagnetic beads, inorganic beads, and organic beads; and wherein the beads have a shape, which shape is selected from one or more of: spherical, helical, cylindrical, spheroid, ioegular, rod shaped, cone shaped, cubic, and polyhedral.

78. The method of claim 74, comprising positioning the set of particles within a microscale channel.

79. The method of claim 74, wherein the incubating step comprises flowing the polymerase across the set of particles or flowing the set of particles through the polymerase.

80. A method of sequencing a nucleic acid, comprising:
(i) providing a first set of particles comprising at least one set of nucleic acid templates in a first microfluidic channel;
(ii) flowing a train of reagents comprising a plurality of sequencing reagents across the first set of particles, or flowing the first set of particles through the reagent train, thereby sequentially contacting the at least one set of nucleic acid templates with the plurality of sequencing reagents;
(iii) detecting a signal resulting from exposure of the first set of particles to the reagent train, thereby providing a portion of sequence of the nucleic acid template; and,
repeating steps (ii) and (iii), thereby sequencing the nucleic acid.

81. The method of claim 80, wherein the nucleic acid templates comprise DNA or RNA.

82. The method of claim 80, wherein the train of reagents comprises one or more of: a template, a primer, a polymerase, a sufurylase, an apyrase, an inorganic phosphate, ATP, a thermostable polymerase, luciferin, luciferase, an endonuclease, an exonuclease, Mg++, a molecular crowding agent, a buffer, a dNTP, a salt, a phosphatase, a reducing agent, a modified dNTP, a nucleotide, a nucleotide analog, a nucleotide analog comprising a 3 '-blocking group, a nucleotide analog comprising a 3 '-phosphate group, a nucleotide analog comprising a 3 '-carbamate group, a chain-terminating nucleotide analog, a reversible chain terminating nucleotide analog, a fluorescently labeled nucleotide, and an intercalating dye.

83. The method of claim 80, wherein the plurality of sequencing reagents comprises at least one of: a compound having formula (I), and a compound having formula (II):



wherein R comprises one or more of a linker moiety and a detectable label and B comprises one or more of a nitrogenous base and the detectable label.

84. The method of claim 83, wherein the linker moiety comprises an acyl, an S-acyl, an alkyl, an aromatic, or an heteroaromatic group.

85. The method of claim 83, wherein the nitrogenous base comprises adenine, guanine, thymine, cytosine, or uracil.

86. The method of claim 80, wherein step (ii) comprises:
(a) flowing at least one set of nucleic acid primers across the set of
particles or flowing the set of particles through at least one set of
nucleic acid primers, thereby contacting the nucleic acid templates
with the nucleic acid primers and forming a double-stranded region;
and,
(b) flowing a polymerase and one or more nucleotides or nucleotide
analogs across the set of particles or flowing the set of particles
through the polymerase and the one or more nucleotides or nucleotide analogs, thereby adding at least one of the one or more nucleotides or nucleotide analogs to at least one of the nucleic acid primers, resulting in an extended double-stranded region.

87. The method of claim 86, wherein the one or more nucleotide analogs comprise chain-terminating nucleotide analogs.

88. The method of claim 86, wherein the chain-terminating nucleotide analogs comprise reversible chain-terminating nucleotide analogs.

89. The method of claim 86, wherein the one or more nucleotide analogs comprise a 3 '-blocking group.

90. The method of claim 89, wherein the 3'-blocking group comprises a carbamate group or a phosphate group.

91. The method of claim 89, the method further comprising removing the 3'-blocking group from the extended double-stranded region, resulting in an extendable double-stranded region.

92. The method of claim 91, wherein the removing the 3 '-blocking group comprises flowing a reducing agent and a phosphatase across the set of particles or flowing the set of particles through the reducing agent and the
phosphatase.

93. The method of claim 92, wherein the reducing agent is selected from diborane, dithiothreitol, glutathione, TCEP, and disulfide reductase.

94. The method of claim 86, wherein the one or more nucleotides comprise a series of nucleoside 5'-triphosphates and the method further comprises flowing an intercalating dye across the set of particles or flowing the set of particles through an intercalating dye.

95. The method of claim 94, wherein detecting the signal comprises detecting the intercalating dye.

96. The method of claim 94, the method further comprising photobleaching the intercalating dye after detecting the intercalating dye or concuoent with detecting the intercalating dye.

97. The method of claim 80, wherein step (iii) comprises
photobleaching the at least one of the one or more nucleotides or nucleotide analogs.

98. The method of claim 80, wherein one or more of: the first set of particles and the reagent train, is flowed through the microscale channel by one or more of: pressure, centripetal force, centrifugal force, a moving magnetic field, and an electrokinetic force.

99. The method of claim 80, wherein the first set of particles comprises an ordered aoay.

100. The method of claim 80, wherein the first set of particles comprises about 1 or more particles, about 10 or more particles, about 100 or more particles, about 1000 or more particles, or about 10,000 or more particles.

101. The method of claim 80, wherein the first set of particles comprises a set of beads, which beads are selected from: polymer beads, silica beads, ceramic beads, clay beads, glass beads, magnetic beads, metallic beads, paramagnetic beads, inorganic beads, and organic beads; and wherein the beads have a shape, which shape is selected from one or more of: spherical, helical, cylindrical, spheroid, ioegular, rod-shaped, cone-shaped, cubic, and polyhedral.

102. The method of claim 80, wherein the train of reagents comprises reagents for sequencing the nucleic acid templates by pyrosequencing, by
incorporation, by synthesis, by photobleaching, or by intercalation.

103. The method of claim 80, further comprising reiteratively repeating steps (ii) and (iii) until at least one reagent in the reagent train becomes depleted, the method further comprising flowing a second train of reagents comprising a plurality of sequencing reagents across the first set of particles, or flowing the first set of particles through the second reagent train.

104. The method of claim 80, wherein the first set of particles is present in an array of particle sets, the method further comprising flowing the train of reagents across the array of particle sets, or flowing the array of particle sets through the reagent train.

105. A microfluidic device for sequencing a nucleic acid, the device comprising:
(i) a body structure having a microscale cavity disposed therein;
(ii) a set of particles disposed within the microscale cavity, the set of
particles comprising at least one set of nucleic acid templates and at
least one set of nucleic acid primers; and
(iii) one or more nucleotides or nucleotide analogs disposed within the
microscale cavity.

106. The microfluidic device of claim 105, wherein the set of particles comprises an ordered array.

107. The microfluidic device of claim 105, wherein the set of particles comprises about 1 or more particles, about 10 or more particles, about 100 or more particles, about 1000 or more particles, or about 10,000 or more particles.

108. The microfluidic device of claim 105, wherein the set of particles comprises a set of beads, which beads are selected from: polymer beads, silica beads, ceramic beads, clay beads, glass beads, magnetic beads, metallic beads, paramagnetic beads, inorganic beads, and organic beads; and wherein the beads have a shape, which shape is selected from one or more of: spherical, helical, cylindrical, spheroid, ioegular, rod shaped, cone shaped, cubic, and polyhedral.

109. The microfluidic device of claim 105, wherein the one or more nucleotides analogs comprise one or more of: a compound having formula (I), and a compound having formula (II):




wherein R comprises a linker moiety and a detectable label and B comprises a detectable nitrogenous base or a nitrogenous base.

110. The microfluidic device of claim 105, wherein the one or more nucleotides or nucleotide analogs comprise fluorescently labeled nucleotides or nucleotide analogs.

111. The microfluidic device of claim 105, the device further comprising an intercalating dye.