Apparatus and method for sequencing a nucleic acid

• US 20030068629A1
• Filed: 03/21/2002
• Published: 04/10/2003
• Est. Priority Date: 03/21/2001
• Status: Abandoned Application

• Current Assignee: 454 CORPORATION
• Original Assignee: 454 CORPORATION
• Inventors: Weiner, Michael P., Rothberg, Jonathan M., Berka, Jan, McDade, Keith, Simpson, John W., Colangelo, Christopher M., Dewell, Scott B., Bader, Joel S.
• Application Number:
  US10/104,280
• Publication Number:
  US 20030068629A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0822 Slides
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/0893 having a very large number of wells, microfabricated wells
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 9/527 for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• 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 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/101 : Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2565/107 : Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2565/301 : Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/519 : characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: G01N 21/0303 : Optical path conditioning in cuvettes, e.g. windows; adapted optical elements or systems; path modifying or adjustment G01N21/031 - G01N21/15 take precedence
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/7703 : using reagent-clad optical fibres or optical waveguides using measurement of total internal reflection or attenuated total reflection G01N21/552; optical fibres or waveguides per se G02B

Abstract

Disclosed herein are methods and apparatuses for sequencing a nucleic acid. These methods permit a very large number of independent sequencing reactions to be arrayed in parallel, permitting simultaneous sequencing of a very large number (>10,000) of different oligonucleotides.

Claims

1. An array comprising a planar surface with a plurality of reaction chambers disposed thereon, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m and each chamber has a width in at least one dimension of between 0.3 μ

m and 100 μ

m.

115. The method of claim 111 additionally comprising repeating steps (a) and (b) sequentially.

164. The method of claim 159, wherein either or both of the sulfurylase and luciferase are immobilized on one or more mobile solid supports disposed at each reaction site.

2. An array comprising a planar surface with a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m and each cavity has a width in at least one dimension of between 0.3 μ

m and 100 μ

m.

3. An array comprising a planar surface with a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m and each reaction chamber has no more than one single stranded circular nucleic acid disposed therein.

4. The array of claim 3 wherein each single stranded circular nucleic acid contains at least 100 copies of a nucleic acid sequence, each copy covalently linked end to end.

5. The array of claim 3 wherein the single stranded circular nucleic acid is immobilized in the reaction chamber.

6. The array of claim 5 wherein the single stranded circular nucleic acid is immobilized on a mobile solid support disposed in the reaction chamber.

7. The array of claim 3, wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 100 μ

m.

8. The array of claim 3, wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 20 μ

m.

9. The array of claim 3. wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 10 μ

m.

10. The array of claim 3, wherein each reaction chamber has a width in at least one dimension of between 20 μ

m and 70 μ

m.

11. The array of claim 3, wherein the cavities number greater than 400,000.

12. The array of claim 3, wherein the cavities number between 400,000 and 20,000,000.

13. The array of claim 3, wherein the cavities number between 1,000,000 and 16,000,000.

14. The array of claim 3, wherein the shape of each cavity is substantially hexagonal.

15. The array of claim 3 wherein the center to center spacing is between 10 to 150 μ

m.

16. The array of claim 3 wherein the center to center spacing is between 50 to 100 μ

m.

17. The array of claim 3, wherein each cavity has a depth of between 10 μ

m and 100 μ

m.

18. The array of claim 3, wherein each cavity has a depth that is between 0.25 and 5 times the size of the width of the cavity.

19. The array of claim 3, wherein each cavity has a depth that is between 0.3 and 1 times the size of the width of the cavity.

20. An array comprising a planar top surface and a planar bottom surface wherein the planar top surface has at least 1,000 cavities thereon, each cavity forming an analyte reaction chamber, the planar bottom surface is optically conductive such that optical signals from the reaction chambers can be detected through the bottom planar surface, wherein the distance between the top surface and the bottom surface is no greater than 10 cm, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m and each chamber having a width in at least one dimension of between 0.3 μ

m and 100 μ

m.

21. The array of claim 20 wherein the center to center spacing is between 10 to 150 μ

m.

22. The array of claim 20 wherein the center to center spacing is between 50 to 100 μ

m.

23. The array of claim 20, wherein the cavities number greater than 400,000.

24. The array of claim 20, wherein the cavities number between 400,000 and 20,000,000.

25. The array of claim 20, wherein the cavities number between 1,000,000 and 16,000,000.

26. The array of claim 20, wherein the shape of each cavity is substantially hexagonal.

27. The array of claim 20, wherein each cavity has a width in at least one dimension of between 0.3 μ

m and 10 μ

m.

28. The array of claim 20, wherein each cavity has a width in at least one dimension of between 0.3 μ

m and 20 μ

m.

29. The array of claim 20, wherein each cavity has a width in at least one dimension of between 20 μ

m and 70 μ

m.

30. The array of claim 20, wherein each cavity has a depth of between 10 μ

m and 100 μ

m.

31. The array of claim 20, wherein each cavity has a depth that is between 0.25 and 5 times the size of the width of the cavity.

32. The array of claim 20, wherein each cavity has a depth that is between 0.3 and 1 times the size of the width of the cavity.

33. The array of claim 20, wherein each cavity has at least one irregular wall surface.

34. The array of claim 20, wherein each cavity has a smooth wall surface.

35. The array of claim 20, wherein the cavities are formed from a fiber optic bundle.

36. The array of claim 35, wherein the cavities are formed by etching one end of the fiber optic bundle.

37. The array of claim 20, wherein each cavity contains reagents for analyzing a nucleic acid or protein.

38. The array of claim 20 further comprising a second surface spaced apart from the planar array and in opposing contact therewith such that a flow chamber is formed over the array.

39. An array means for carrying out separate parallel common reactions in an aqueous environment, wherein the array means comprises a substrate comprising at least 1,000 discrete reaction chambers containing a starting material that is capable of reacting with a reagent, each of the reaction chambers being dimensioned such that when one or more fluids containing at least one reagent is delivered into each reaction chamber, the diffusion time for the reagent to diffuse out of the well exceeds the time required for the starting material to react with the reagent to form a product.

40. The array of claim 39 wherein the reaction chambers are formed by generating a plurality of cavities on the substrate.

41. The array of claim 39 wherein the reaction chambers are formed by generating discrete patches on a planar surface, said patches having a different surface chemistry than the surrounding planar surface.

42. The array of claims 39 or 41, wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 100 μ

m.

43. The array of claims 39 or 41, wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 20 μ

m.

44. The array of claims 39 or 41, wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 10 μ

m.

45. The array of claims 39 or 41, wherein each reaction chamber has a width in at least one dimension of between 20 μ

m and 70 μ

m.

46. The array of claims 39 or 41 wherein the center to center spacing is between 5 to 200 μ

m.

47. The array of claims 39 or 41 wherein the center to center spacing is between 10 to 150 μ

m.

48. The array of claims 39 or 41 wherein the center to center spacing is between 50 to 100 μ

m.

49. The array of claims 39 or 41, wherein the cavities number greater than 400,000.

50. The array of claims 39 or 41, wherein the cavities number between 400,000 and 20,000,000.

51. The array of claims 39 or 41, wherein the cavities number between 1,000,000 and 16,000,000.

52. The array of claims 39 or 41, wherein the shape of each cavity is substantially hexagonal.

53. The array of claims 39 or 41, wherein each cavity has a depth of between 10 μ

m and 100 μ

m.

54. The array of claims 39 or 41, wherein each cavity has a depth that is between 0.25 and 5 times the size of the width of the cavity.

55. The array of claims 39 or 41, wherein each cavity has a depth that is between 0.3 and 1 times the size of the width of the cavity.

56. The array of claims 39 or 41, wherein each cavity has at least one irregular wall surface.

57. The array of claims 39 or 41, wherein each cavity has a smooth wall surface.

58. The array of claims 39 or 41, wherein the cavities are formed from a fiber optic bundle.

59. The array of claim 58, wherein the cavities are formed by etching one end of the fiber optic bundle.

60. The array of claims 39 or 41, wherein each cavity contains reagents for analyzing a nucleic acid or protein.

61. The array of claims 39 or 41 further comprising a population of mobile solid supports disposed in the reaction chambers, each mobile solid support having one or more bioactive agents attached thereto.

62. The array of claim 40, wherein the cavities are formed in the substrate via etching, molding or micromaching.

63. The array of claim 40, wherein the substrate is a fiber optic bundle.

64. The array of claim 40, wherein the cavities are cylindrical.

65. The array of claim 40, wherein the bottom of each of the cavities is planar.

66. The array of claim 40, wherein the bottom of each of the cavities is concave.

67. An array comprising a planar surface with a plurality of reaction chambers disposed thereon, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m and each chamber has a width in at least one dimension of between 0.3 μ

m and 100 μ

m wherein at least one reaction chamber has a mobile solid support having at least one reagent immobilized thereon, wherein the reagent is suitable for use in a nucleic acid sequencing reaction.

68. The array of claim 67, wherein the diameter of each mobile solid support is between 0.01 to 0.1 times the width of each cavity.

69. The array of claim 67, wherein at least 5% to 20% of the reaction chambers have a mobile solid support having at least one reagent immobilized thereon.

70. The array of claim 67, wherein at least 20% to 60% of the reaction chambers have a mobile solid support having at least one reagent immobilized thereon.

71. The array of claim 67, wherein at least 50% to 100% of the reaction chambers have a mobile solid support having at least one reagent immobilized thereon.

72. The array of claim 67, wherein the mobile solid support has at least one reagent immobilized thereon, wherein the reagent is a polypeptide with sulfurylase activity.

73. The array of claim 67, wherein the mobile solid support has at least one reagent immobilized thereon, wherein the reagent is a polypeptide with luciferase activity.

74. The array of claim 67, wherein the mobile solid support has at least one reagent immobilized thereon, wherein the reagent is a chimeric polypeptide having both sulfurylase and luciferase activity.

75. The array of claim 67, wherein the mobile solid support has at least a first reagent and a second reagent immobilized thereon, wherein the first reagent is a polypeptide with sulfurylase activity, and the second reagent is a polypeptide with luciferase activity.

76. The array of claim 67 wherein the center to center spacing is between 10 to 150 μ

m.

77. The array of claim 67 wherein the center to center spacing is between 50 to 100 μ

m.

78. The array of claim 67, wherein the cavities number greater than 400,000.

79. The array of claim 67, wherein the cavities number between 400,000 and 20,000,000.

80. The array of claim 67, wherein the cavities number between 1,000,000 and 16,000,000.

81. The array of claim 67, wherein the shape of each cavity is substantially hexagonal.

82. The array of claim 67, wherein each cavity has a width in at least one dimension of between 0.3 μ

m and 10 μ

m.

83. The array of claim 67, wherein each cavity has a width in at least one dimension of between 0.3 μ

m and 20 μ

m.

84. The array of claim 67, wherein each cavity has a width in at least one dimension of between 20 μ

m and 70 μ

m.

85. The array of claim 67, wherein each cavity has a depth of between 10 μ

m and 100 μ

m.

86. The array of claim 67, wherein each cavity has a depth that is between 0.25 and 5 times the size of the width of the cavity.

87. The array of claim 67, wherein each cavity has a depth that is between 0.3 and 1 times the size of the width of the cavity.

90. The method of using the mobile solid support of claim 67 to detect ATP activity.

91. The method of claim 90, wherein the ATP is detected as part of a nucleic acid sequencing reaction.

88. An array comprising a planar surface with a plurality of reaction chambers disposed thereon, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m and each chamber has a width in at least one dimension of between 0.3 μ

m and 100 μ

m wherein at least one reaction chamber has a mobile solid support having at least one reagent immobilized thereon, wherein the reagent(s) is a nucleic acid, said nucleic acid comprising a single stranded concatamer.

89. The array of claim 88, wherein the nucleic acid is suitable for use in a pyrosequencing reaction.

92. The array of claim 88, wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 20 μ

m.

93. The array of claim 88, wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 10 μ

m.

94. The array of claim 88, wherein each reaction chamber has a width in at least one dimension of between 20 μ

m and 70 μ

m.

95. The array of claim 88, wherein the cavities number greater than 400,000.

96. The array of claim 88, wherein the cavities number between 400,000 and 20,000,000.

97. The array of claim 88, wherein the cavities number between 1,000,000 and 16,000,000.

98. The array of claim 88 wherein the center to center spacing is between 10 to 150 μ

m.

99. The array of claim 88 wherein the center to center spacing is between 50 to 100 μ

m.

100. The array of claim 88, wherein each cavity has a depth of between 10 μ

m and 100 μ

m.

101. The array of claim 88, wherein each cavity has a depth that is between 0.25 and 5 times the size of the width of the cavity.

102. The array of claim 88, wherein each cavity has a depth that is between 0.3 and 1 times the size of the width of the cavity.

103. A method for delivering a bioactive agent to an array, comprising dispersing over the array a plurality of mobile solid supports, each mobile solid support having at least one reagent immobilized thereon, wherein the reagent is suitable for use in a nucleic acid sequencing reaction, where the array comprises a planar surface with a plurality of reaction chambers disposed thereon, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m and each chamber has a width in at least one dimension of between 0.3 μ

m and 100 μ

m.

104. An apparatus for simultaneously monitoring an array of reaction chambers for light indicating that a reaction is taking place at a particular site, the apparatus comprising:

(a) an array of reaction chambers formed from a planar substrate comprising a plurality of cavitated surfaces, each cavitated surface forming a reaction chamber adapted to contain analytes, and wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m, the array comprising more than 400,000 discrete reaction chambers;

(b) an optically sensitive device arranged so that in use the light from a particular reaction chamber will impinge upon a particular predetermined region of said optically sensitive device;

(c) means for determining the light level impinging upon each of said predetermined regions and

(d) means to record the variation of said light level with time for each of said reaction chamber.

105. An analytic sensor, comprising:

(a) an array formed from a first bundle of optical fibers with a plurality of cavitated surfaces at one end thereof, each cavitated surface forming a reaction chamber adapted to contain analytes, and wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m, the array comprising more than 400,000 discrete reaction chambers;

(b) an enzymatic or fluorescent means for generating light in the reaction chambers;

(c) light detection means comprising a light capture means and a second fiber optic bundle for transmitting light to the light detecting means, the second fiber optic bundle being in optical contact with the array, such that light generated in an individual reaction chamber is captured by a separate fiber or groups of separate fibers of the second fiber optic bundle for transmission to the light capture means.

106. The sensor of claim 105, wherein said sensor is suitable for use in a biochemical assay.

107. The sensor of claim 105, wherein said sensor is suitable for use in a cell-based assay.

108. The sensor of claim 105, wherein the light capture means is a CCD camera.

109. The sensor of claim 105, wherein the reaction chambers contain one or more mobile solid supports with a bioactive agent immobilized thereon.

110. A composition comprising a plurality of optical fibers in an fused optical fiber array with a plurality of cavitated surfaces at one end thereof, each cavitated surface forming a reaction chamber adapted to contain analytes, and wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m, the array comprising more than 400,000 discrete reaction chambers, each reaction chamber containing one or more mobile solid supports, wherein the diameter of the mobile solid supports is between 0.01 to 0.1 times the width of each reaction chamber.

111. A method for carrying out separate parallel common reactions in an aqueous environment, comprising:

(a) delivering a fluid containing at least one reagent to an array, wherein the array comprises a substrate comprising at least 400,000 discrete reaction chambers containing a starting material that is capable of reacting with the reagent, each of the reaction chambers being dimensioned such that when the fluid is delivered into each reaction chamber, the diffusion time for the reagent to diffuse out of the well exceeds the time required for the starting material to react with the reagent to form a product; and

(b) washing the fluid from the array in the time period (i) after the starting material has reacted with the reagent to form a product in each reaction chamber but (ii) before the reagent delivered to any one reaction chamber has diffused out of that reaction chamber into any other reaction chamber.

112. The method of claim 111 wherein the product formed in any one reaction chamber is independent of the product formed in any other reaction chamber, but is generated using one or more common reagents.

113. The method of claim 111 wherein the starting material is a nucleic acid sequence and at least one reagent in the fluid is a nucleotide or nucleotide analog.

114. The method of claim 113 wherein the fluid additionally comprises a polymerase capable of reacting the nucleic acid sequence and the nucleotide or nucleotide analog.

116. A method for delivering nucleic acid sequencing enzymes to an array, said array having a planar surface with a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m;

the method comprising dispersing over the array a plurality of mobile solid supports having one or more nucleic acid sequencing enzymes immobilized thereon, such that at least one mobile solid support is contained within each reaction chamber.

117. The method of claim 116 wherein one of the nucleic acid sequencing enzymes is a polypeptide with sulfurylase activity.

118. The method of claim 116 wherein one of the nucleic acid sequencing enzymes is a polypeptide with luciferase activity.

119. The method of claim 116 wherein one of the nucleic acid sequencing enzymes is a polypeptide with both sulfurylase and luciferase activity.

120. The method of claim 116 wherein the plurality of mobile solid supports include mobile solid supports having either a polypeptide with sulfurylase activity immobilized thereon or a polypeptide with luciferase activity immobilized thereon, or both a polypeptide with sulfurylase activity immobilized thereon and a polypeptide with luciferase activity immobilized thereon.

127. The method of any one of claims 116-126 wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 100 μ

m.

128. The method of any one of claims 116-126 wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 20 μ

m.

129. The method of any one of claims 116-126 wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 10 μ

m.

130. The method of any one of claims 116-126 wherein each reaction chamber has a width in at least one dimension of between 20 μ

m and 70 μ

m.

131. The method of any one of claims 116-126 wherein the cavities number greater than 400,000.

132. The method of any one of claims 116-126 wherein the cavities number between 400,000 and 20,000,000.

133. The method of any one of claims 116-126 wherein the cavities number between 1,000,000 and 16,000,000.

134. The method of any one of claims 116-126 wherein the center to center spacing is between 10 to 150 μ

m.

135. The method of any one of claims 116-126 wherein the center to center spacing is between 50 to 100 μ

m.

136. The method of any one of claims 116-126, wherein each cavity has a depth of between 10 μ

m and 100 μ

m.

137. The method of any one of claims 116-126 wherein each cavity has a depth that is between 0.25 and 5 times the size of the width of the cavity.

138. The method of any one of claims 116-126 wherein each cavity has a depth that is between 0.3 and 1 times the size of the width of the cavity.

121. A method for delivering a nucleic acid sequence to an array, said array having a planar surface with a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m;

the method comprising dispersing over the array a plurality of mobile solid supports having one or more nucleic sequences immobilized thereon.

122. The method of claim 121 wherein the nucleic acid sequence is a single stranded circular nucleic acid.

123. The method of claim 122 wherein each single stranded circular nucleic acid contains at least 100 copies of a nucleic acid sequence, each copy covalently linked end to end.

139. The method of claims 121 or 124 wherein the nucleic acid sequence is further amplified to produce multiple copies of said nucleic acid sequence after being disposed in the reaction chamber.

140. The method of claim 139 wherein the nucleic acid sequence is amplified using an amplification technology selected from the group consisting of polymerase chain reaction, ligase chain reaction and isothermal DNA amplification.

124. A method for delivering a nucleic acid sequence to an array, said array having a planar surface with a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m;

the method comprising immobilizing within each cavity no more than one nucleic acid sequence.

125. The method of claim 124 wherein the nucleic acid sequence is a single stranded circular nucleic acid.

126. The method of claim 124 wherein each single stranded circular nucleic acid contains at least 100 copies of a nucleic acid sequence, each copy covalently linked end to end.

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

(a) providing one or more nucleic acid anchor primers;

(b) providing a plurality of single-stranded circular nucleic acid templates disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m;

(c) annealing an effective amount of the nucleic acid anchor primer to at least one of the single-stranded circular templates to yield a primed anchor primer-circular template complex;

(d) combining the primed anchor primer-circular template complex with a polymerase to form an extended anchor primer covalently linked to multiple copies of a nucleic acid complementary to the circular nucleic acid template;

(e) annealing an effective amount of a sequencing primer to one or more copies of said covalently linked complementary nucleic acid;

(f) extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto the 3′

end of said sequencing primer, a sequencing reaction byproduct; and

(g) identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid.

142. The method of claim 141 wherein each single stranded circular nucleic acid contains at least 100 copies of a nucleic acid sequence, each copy covalently linked end to end.

143. The method of claim 141 wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 100 μ

m.

144. The method of claim 141 wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 20 μ

m.

145. The method of claim 141 wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 10 μ

m.

146. The method of claim 141 wherein each reaction chamber has a width in at least one dimension of between 20 μ

m and 70 μ

m.

147. The method of claim 141 wherein the cavities number greater than 400,000.

148. The method of claim 141 wherein the cavities number between 400,000 and 20,000,000.

149. The method of claim 141 wherein the cavities number between 1,000,000 and 16,000,000.

150. The method of claim 141 wherein the center to center spacing is between 10 to 150 μ

m.

151. The method of claim 141 wherein the center to center spacing is between 50 to 100 μ

m.

152. The method of claim 141, wherein each cavity has a depth of between 10 μ

m and 100 μ

m.

153. The method of claim 141 wherein each cavity has a depth that is between 0.25 and 5 times the size of the width of the cavity.

154. The method of claim 141 wherein each cavity has a depth that is between 0.3 and 1 times the size of the width of the cavity.

155. The method of claim 141 wherein the nucleic acid sequence is further amplified to produce multiple copies of said nucleic acid sequence after being disposed in the reaction chamber.

156. The method of claim 155 wherein the nucleic acid sequence is amplified using an amplification technology selected from the group consisting of polymerase chain reaction, ligase chain reaction and isothermal DNA amplification.

157. The method of claim 141 wherein the single stranded circular nucleic acid is immobilized in the reaction chamber.

158. The method of claim 141 wherein the single stranded circular nucleic acid is immobilized on one or more mobile solid supports disposed in the reaction chamber.

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

(a) providing at least one nucleic acid anchor primer;

(b) providing a plurality of single-stranded circular nucleic acid templates in an array having at least 400,000 discrete reaction sites;

(c) annealing a first amount of the nucleic acid anchor primer to at least one of the single-stranded circular templates to yield a primed anchor primer-circular template complex;

(d) combining the primed anchor primer-circular template complex with a polymerase to form an extended anchor primer covalently linked to multiple copies of a nucleic acid complementary to the circular nucleic acid template;

(e) annealing a second amount of a sequencing primer to one or more copies of the covalently linked complementary nucleic acid;

(f) extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, when the predetermined nucleotide triphosphate is incorporated onto the 3′

end of the sequencing primer, to yield a sequencing reaction byproduct; and

(g) identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid at each reaction site that contains a nucleic acid template.

160. The method of claim 159, wherein the anchor primer is linked to a particle.

161. The method of claim 159, wherein the anchor primer is linked to the particle prior to formation of the extended anchor primer.

162. The method of claim 159, wherein the anchor primer is linked to the particle after formation of the extended anchor primer.

163. The method of claim 159, wherein the sequencing reaction byproduct is PPi and a coupled sulfurylase/luciferase reaction is used to generate light for detection.

165. A method of determining the base sequence of a plurality of nucleotides on an array, the method comprising:

(a) providing a plurality of sample DNAs, each disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m,

(b) adding an activated nucleotide 5′

-triphosphate precursor of one known nitrogenous base to a reaction mixture in each reaction chamber, each reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer strand at least one nucleotide residue shorter than the templates to form at least one unpaired nucleotide residue in each template at the 3′

-end of the primer strand, under reaction conditions which allow incorporation of the activated nucleoside 5′

-triphosphate precursor onto the 3′

-end of the primer strands, provided the nitrogenous base of the activated nucleoside 5′

-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the templates;

(c) detecting whether or not the nucleoside 5′

-triphosphate precursor was incorporated into the primer strands in which incorporation of the nucleoside 5′

-triphosphate precursor indicates that the unpaired nucleotide residue of the template has a nitrogenous base composition that is complementary to that of the incorporated nucleoside 5′

-triphosphate precursor; and

(d) sequentially repeating steps (b) and (c), wherein each sequential repetition adds and, detects the incorporation of one type of activated nucleoside 5′

-triphosphate precursor of known nitrogenous base composition; and

(e) determining the base sequence of the unpaired nucleotide residues of the template in each reaction chamber from the sequence of incorporation of said nucleoside precursors.

166. A method for determining the nucleic acid sequence in a template nucleic acid polymer, comprising:

(a) introducing a plurality of template nucleic acid polymers into a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m, each reaction chamber having a polymerization environment in which the nucleic acid polymer will act as a template polymer for the synthesis of a complementary nucleic acid polymer when nucleotides are added;

(b) successively providing to the polymerization environment a series of feedstocks, each feedstock comprising a nucleotide selected from among the nucleotides from which the complementary nucleic acid polymer will be formed, such that if the nucleotide in the feedstock is complementary to the next nucleotide in the template polymer to be sequenced said nucleotide will be incorporated into the complementary polymer and inorganic pyrophosphate will be released;

(c) detecting the formation of inorganic pyrophosphate to determine the identify of each nucleotide in the complementary polymer and thus the sequence of the template polymer.

167. A method of identifying the base in a target position in a DNA sequence of sample DNA, wherein:

(a) sample DNA is disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m, said DNA being rendered single stranded either before or after being disposed in the reaction chambers,

(b) an extension primer is provided which hybridizes to said immobilized single-stranded DNA at a position immediately adjacent to said target position;

(c) said immobilized single-stranded DNA is subjected to a polymerase reaction in the presence of a predetermined nucleotide triphosphate, wherein if the predetermined nucleotide triphosphate is incorporated onto the 3′

end of said sequencing primer then a sequencing reaction byproduct is formed; and

(d) identifying the sequencing reaction byproduct, thereby determining the nucleotide complementary to the base at said target position.

168. A method of identifying a base at a target position in a sample DNA sequence comprising:

(a) providing sample DNA disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m, said DNA being rendered single stranded either before or after being disposed in the reaction chambers

(b) providing an extension primer which hybridizes to the sample DNA immediately adjacent to the target position

(c) subjecting the sample DNA sequence and the extension primer to a polymerase reaction in the presence of a nucleotide triphosphate whereby the nucleotide triphosphate will only become incorporated and release pyrophosphate (PPi) if it is complementary to the base in the target position, said nucleotide triphosphate being added either to separate aliquots of sample-primer mixture or successively to the same sample-primer mixture p1 (d) detecting the release of PPi to indicate which nucleotide is incorporated.

169. A method of identifying a base at a target position in a single-stranded sample DNA sequence, the method comprising:

(a) providing an extension primer which hybridizes to sample DNA immediately adjacent to the target position, said sample DNA disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m, said DNA being rendered single stranded either before or after being disposed in the reaction chambers;

(b) subjecting the sample DNA and extension primer to a polymerase reaction in the presence of a predetermined deoxynucleotide or dideoxynucleotide whereby the deoxynucleotide or dideoxynucleotide will only become incorporated and release pyrophosphate (PPi) if it is complementary to the base in the target position, said predetermined deoxynucleotides or dideoxynucleotides being added either to separate aliquots of sample-primer mixture or successively to the same sample-primer mixture,

(c) detecting any release of PPi enzymatically to indicate which deoxynucleotide or dideoxynucleotide is incorporated;

characterized in that, the PPi-detection enzyme(s) are included in the polymerase reaction step and in that in place of deoxy- or dideoxy adenosine triphosphate (ATP) a dATP or ddATP analogue is used which is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for a said PPi-detection enzyme.

170. An apparatus for analyzing a nucleic acid sequence, the apparatus comprising:

(a) a reagent delivery cuvette, wherein the cuvette includes an array comprising a planar surface with a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m, wherein the reagent delivery cuvette contains reagents for use in a sequencing reaction;

(b) a reagent delivery means in communication with the reagent delivery cuvette;

(c) an imaging system in communication with the reagent delivery chamber; and

(d) a data collection system in communication with the imaging system.

171. An apparatus for determining the base sequence of a plurality of nucleotides on an array, the apparatus comprising:

(a) a reagent cuvette containing a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m;

(b) reagent delivery means for adding an activated nucleotide 5′

-triphosphate precursor of one known nitrogenous base to a reaction mixture in each reaction chamber, each reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer strand at least one nucleotide residue shorter than the templates to form at least one unpaired nucleotide residue in each template at the 3′

-end of the primer strand, under reaction conditions which allow incorporation of the activated nucleoside 5′

-triphosphate precursor onto the 3′

-end of the primer strands, provided the nitrogenous base of the activated nucleoside 5′

-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the templates;

(c) detection means for detecting whether or not the nucleoside 5′

-triphosphate precursor was incorporated into the primer strands in which incorporation of the nucleoside 5′

-triphosphate precursor indicates that the unpaired nucleotide residue of the template has a nitrogenous base composition that is complementary to that of the incorporated nucleoside 5′

-triphosphate precursor; and

(d) means for sequentially repeating steps (b) and (c), wherein each sequential repetition adds and, detects the incorporation of one type of activated nucleoside 5′

-triphosphate precursor of known nitrogenous base composition; and

(e) data processing means for determining the base sequence of the unpaired nucleotide residues of the template in each reaction chamber from the sequence of incorporation of said nucleoside precursors.

172. An apparatus for determining the nucleic acid sequence in a template nucleic acid polymer, comprising:

(a) an array comprising a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m,

(b) nucleic acid delivery means for introducing a template nucleic acid polymers into the reaction chambers;

(c) nucleic acid delivery means to deliver reagents to the reaction chambers to create a polymerization environment in which the nucleic acid polymers will act as a template polymers for the synthesis of complementary nucleic acid polymers when nucleotides are added;

(d) reagent delivery means for successively providing to the polymerization environment a series of feedstocks, each feedstock comprising a nucleotide selected from among the nucleotides from which the complementary nucleic acid polymer will be formed, such that if the nucleotide in the feedstock is complementary to the next nucleotide in the template polymer to be sequenced said nucleotide will be incorporated into the complementary polymer and inorganic pyrophosphate will be released;

(e) detection means for detecting the formation of inorganic pyrophosphate enzymatically; and

(f) data processing means to determine the identity of each nucleotide in the complementary polymers and thus the sequence of the template polymers.

173. An apparatus for processing a plurality of analytes, the apparatus comprising:

(a) a flow chamber having disposed therein a substrate comprising a plurality of cavitated surfaces, each cavitated surface forming a reaction chamber adapted to contain analytes, and wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m;

(b) fluid means for delivering processing reagents from one or more reservoirs to the flow chamber so that the analytes disposed therein are exposed to the reagents; and

(c) detection means for detecting a sequence of optical signals from each of the reaction chambers, each optical signal of the sequence being indicative of an interaction between a processing reagent and the analyte disposed in the reaction chamber, wherein the detection means is in communication with the cavitated surfaces.

174. The apparatus of claim 173, wherein the detection means is a CCD camera.

175. The apparatus of claim 173, wherein the analyte is nucleic acid.

176. The apparatus of claim 173, wherein the analytes are immobilized on one or more mobile solid supports that are disposed in the reaction chamber.

177. The apparatus of claim 173 wherein the processing reagents are immobilized on one or more mobile solid supports.

178. An array comprising a planar surface with a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m, each cavity being formed such that the diffusion coefficient for any aqueous solution leaving the reaction chamber is 1.6667×

10<sup>−

9</sup> m²/s.

179. A method of determining the base sequence of a plurality of nucleotides on an array, the method comprising:

(a) providing a plurality of sample DNAs, each disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m,

(c) detecting the light level emitted from a plurality of reaction sites on respective portions of an optically sensitive device;

(d) converting the light impinging upon each of said portions of said optically sensitive device into an electrical signal which is distinguishable from the signals from all of said other regions;

(e) determining a light intensity for each of said discrete regions from the corresponding electrical signal;

(f) recording the variations of said electrical signals with time.

180. Method for sequencing a nucleic acid, the method comprising:

(a) providing one or more nucleic acid anchor primers;

(b) providing a plurality of single-stranded circular nucleic acid templates disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m;

(c) detecting the light level emitted from a plurality of reaction sites on respective portions of an optically sensitive device;

(d) converting the light impinging upon each of said portions of said optically sensitive device into an electrical signal which is distinguishable from the signals from all of said other regions;

(e) determining a light intensity for each of said discrete regions from the corresponding electrical signal;

(f) recording the variations of said electrical signals with time.

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

(a) providing at least one nucleic acid anchor primer;

(b) providing a plurality of single-stranded circular nucleic acid templates in an array having at least 400,000 discrete reaction sites;

(c) detecting the light level emitted from a plurality of reaction sites on respective portions of an optically sensitive device;

(d) converting the light impinging upon each of said portions of said optically sensitive device into an electrical signal which is distinguishable from the signals from all of said other regions;

(e) determining a light intensity for each of said discrete regions from the corresponding electrical signal;

(f) recording the variations of said electrical signals with time.