METHOD AND DEVICE FOR DETECTING THE PRESENCE OF A SINGLE TARGET NUCLEIC ACID IN A SAMPLE

• US 20080171327A1
• Filed: 08/13/2007
• Published: 07/17/2008
• Est. Priority Date: 04/17/1997
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated), United States Department Of Health And Human Services
• Original Assignee: Cytonix, The United States of America As Represented By The Secretary of Agriculture
• Inventors: Silver, Jonathan E., Brown, James F.
• Granted Patent: US 8,278,071 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/06 Fluid handling related problems
• CPC Class Codes: B01L 2200/0642 Filling fluids into wells by specific techniques
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• 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 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: B01L 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 3/5088 confining liquids at a location by surface tension, e.g. virtual wells on plates, wires B01L3/50857 takes precedence
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 : Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: Y10S 436/805 : Optical property
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A method comprising subjecting one or more sample portion(s) to a single amplification step, thereby amplifying a single molecule in the sample portion to a detectable level, and, in some embodiments, then determining whether the sample portion contains at least one molecule of the target nucleic acid. In some embodiments, the sample portion is in a porous sample structure, or in a sample chamber which comprises means for minimizing diffusion of the sample portion, or in a sample chamber which is inside a microcapillary device, or in a sample retaining means.

Claims

1. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

subjecting said first sample portion to a single amplification step, thereby amplifying a single molecule in said first sample portion to a detectable level; and

then determining whether said first sample portion contains at least one molecule of said target nucleic acid.

2. A method as recited in claim 1, wherein said single amplification step is a thermocycle step.

3. A method as recited in claim 1, wherein said single amplification step is a homogeneous amplification step.

4. A method as recited in claim 1, wherein said method comprises subjecting said first sample portion to a single round of amplification.

5. A method as recited in claim 1, wherein said first sample portion is confined in a first volume sufficiently small to produce a detectable product when subjected to said single amplification step when said at least one molecule of a target nucleic acid is present.

6. A method as recited in claim 5, wherein said first volume is located in at least one flow-through channel positioned in a microfludic device.

7. A method as recited in claim 5, wherein said first volume is located in a microfludic device.

8. A method as recited in claim 5, wherein said first sample portion is confined in said first volume and said first volume is in contact at least in part with a fluid in which said first volume is immiscible.

9. A method as recited in claim 8, wherein said first volume is located in said fluid.

10. A method as recited in claim 8, wherein said fluid comprises mineral oil.

11. A method as recited in claim 8, wherein said fluid comprises a fluorinated oil.

12. A method as recited in claim 8, wherein said fluid comprises a curable liquid.

13. A method as recited in claim 8, wherein said fluid is a gas.

14. A method as recited in claim 5, wherein said first volume is about 1 microliter or less.

15. A method as recited in claim 5, wherein said first volume is about 100 nanoliter or less.

16. A method as recited in claim 5, wherein said first volume is about 10 nanoliter or less.

17. A method as recited in claim 5, wherein said first volume is about 2 nanoliter or less

18. A method as recited in claim 5, wherein said first volume is from about 1 picoliter to about 1 microliter.

19. A method as recited in claim 5, wherein said first sample portion is confined at least in part by opposing barriers separated by 500 microns or less.

20. A method as recited in claim 19, wherein at least one of said opposing barriers is a liquid with which said first volume is immiscible.

21. A method as recited in claim 19, wherein at least one of said opposing barriers is a solid.

22. A method as recited in claim 19, wherein at least one of said opposing barriers is a gas.

23. A method as recited in claim 1, wherein said at least one molecule of a target nucleic acid and the amplification product from said single amplification step are restrained in a first region sufficiently small for said amplification product to be detected.

24. A method as recited in claim 23, wherein said at least one molecule of a target nucleic acid and the amplification product from said single amplification step are restrained in said first region and said first region is positioned in at least one flow-through channel positioned in a microfludic device.

25. A method as recited in claim 23, wherein said first region is positioned on a surface, and said at least one molecule of a target nucleic acid and said amplification product from said single amplification step are restrained by an affinity to at least one prepositioned chemical substance on said first region of said surface.

26. A method as recited in claim 25, wherein said prepositioned chemical substance comprises nucleic acids.

27. A method as recited in claim 25, wherein said prepositioned chemical substance comprises a set of at least one compound selected from among the group consisting of sets of primers and sets of probes.

28. A method as recited in claim 25, wherein said prepositioned chemical substance is one of a plurality of different prepositioned chemical substances.

29. A method as recited in claim 25, wherein said surface is contained in a device in which at least about 10,000 prepositioned chemical substances are included.

30. A method as recited in claim 25, wherein said prepositioned chemical substance has a specific affinity for said at least one molecule of a target nucleic acid and said amplification product from said single amplification step

31. A method as recited in claim 25, wherein said first region has at least one dimension of 2 mm or less.

32. A method as recited in claim 25, wherein said first region has at least one dimension of 1 mm or less.

33. A method as recited in claim 25, wherein said first region has at least one dimension of 100 microns or less.

34. A method as recited in claim 25, wherein said first region has at least one dimension of 20 microns or less.

35. A method as recited in claim 25, wherein said first region has at least one dimension of a few microns or less.

36. A method as recited in claim 23, wherein said first region is located in a first sample chamber.

37. A method as recited in claim 36, wherein said first sample chamber has opposing barriers separated by 500 microns or less.

38. A method as recited in claim 36, wherein said first sample chamber has a volume of about 1 picoliter or less.

39. A method as recited in claim 36, wherein said first sample chamber has a volume in the range of from about 1 picoliter to about 1 microliter.

40. A method as recited in claim 36, wherein said first sample chamber has a volume of about 10 picoliters or less.

41. A method as recited in claim 36, wherein said first sample chamber has a volume of about 100 picoliters or less.

42. A method as recited in claim 36, wherein said first sample chamber has a volume of about 1 nanoliter or less.

43. A method as recited in claim 36, wherein said first sample chamber has a volume of 10 nanoliters or less.

44. A method as recited in claim 36, wherein said first sample chamber has a volume of about 10 nanoliters.

45. A method as recited in claim 36 wherein said first sample chamber is positioned in a device which comprises from about 10,000 to over 100,000 sample chambers.

46. A method as recited in claim 23, wherein said first sample portion has at least one dimension of 2 millimeters or less.

47. A method as recited in claim 23, wherein said first sample portion has at least one dimension of 1 millimeter or less.

48. A method as recited in claim 23, wherein said first sample portion has at least one dimension of 500 microns or less.

49. A method as recited in claim 23, wherein said first sample portion has at least one dimension of 100 microns or less.

50. A method as recited in claim 23, wherein said first sample portion has at least one dimension of 20 microns or less.

51. A method as recited in claim 23, wherein said first sample portion has at least one dimension of a few microns or less.

52. A method as recited in claim 23, wherein said first sample portion has a volume of about 1 picoliter or less.

53. A method as recited in claim 23, wherein said first sample portion has a volume in the range of from about 1 picoliter to about 1 microliter.

54. A method as recited in claim 23, wherein said first sample portion has a volume of about 10 picoliters or less.

55. A method as recited in claim 23, wherein said first sample portion has a volume which is nanoliter-sized.

56. A method as recited in claim 23, wherein said first sample portion has a volume of about 1 nanoliter or less.

57. A method as recited in claim 23, wherein said first sample portion has a volume of about 10 nanoliters or less.

58. A method as recited in claim 23, wherein said first sample portion has a volume of about 100 nanoliters or less.

59. A method as recited in claim 23, wherein said first sample portion has a volume of about 1 microliter or less.

60. A method as recited in claim 1, wherein:

said first sample portion is located in a first sample chamber and said first sample chamber is positioned in a microfludic device which comprises at least one flow-through channel which communicates with said first sample chamber, and

said method further comprises supplying at least one displacing fluid to said flow-through

channel after loading a portion of said first sample into said flow-through channel, whereby said displacing fluid displaces said first sample from said flow-through channel.

61. A method as recited in claim 1, wherein:

said first sample portion is located in a first sample chamber and said first sample chamber is positioned in a microfludic device which comprises at least one flow-through channel which communicates with said first sample chamber, and

said method further comprises supplying at least one sealing fluid to said flow-through channel, whereby said sealing fluid seals said first sample portion within said first sample chamber.

62. A method as recited in claim 61, wherein said method further comprises curing said sealing fluid after said supplying said sealing fluid to said flow-through channel.

63. A method as recited in claim 61, wherein said sealing fluid comprises at least one adhesive.

64. A method as recited in claim 61, wherein said sealing fluid is immiscible with said first sample portion.

65. A method as recited in claim 64, wherein said sealing fluid displaces a second portion of said sample from said flow-through channel.

66. A method as recited in claim 64, wherein said sealing fluid comprises mineral oil.

67. A method as recited in claim 64, wherein said sealing fluid comprises a fluorinated oil.

68. A method as recited in claim 61, wherein said sealing fluid displaces part of said first sample portion from said flow-through channel as said sealing fluid flows through said flow-through channel.

69. A method for detecting a single molecule of a target nucleic acid in a first sample portion, said method comprising:

amplifying said single molecule to a detectable level in a single amplification step.

70. A method as recited in claim 69, wherein said method further comprises detecting the presence of said target nucleic acid in said first sample portion.

71. A method as recited in claim 69, wherein said single amplification step is a thermocycle step.

72. A method as recited in claim 69, wherein said single amplification step is a homogeneous amplification step.

73. A method as recited in claim 69, wherein said method comprises subjecting said first sample portion to a single round of amplification.

74. A method as recited in claim 69, wherein said first sample portion is confined in a first volume sufficiently small to produce a detectable product when subjected to said single amplification step when said single molecule of a target nucleic acid is present.

75. A method as recited in claim 74, wherein said first volume is located in at least one flow-through channel positioned in a microfludic device.

76. A method as recited in claim 74, wherein said first volume is located in a microfludic device.

77. A method as recited in claim 74, wherein said first sample portion is confined in said first volume and said first volume is in contact at least in part with a fluid in which said first volume is immiscible.

78. A method as recited in claim 77, wherein said first volume is located in said fluid.

79. A method as recited in claim 77, wherein said fluid comprises mineral oil.

80. A method as recited in claim 77, wherein said fluid comprises a fluorinated oil.

81. A method as recited in claim 77, wherein said fluid comprises a curable liquid.

82. A method as recited in claim 77, wherein said fluid is a gas.

83. A method as recited in claim 74, wherein said first volume is about 1 microliter or less.

84. A method as recited in claim 74, wherein said first volume is about 100 nanoliter or less.

85. A method as recited in claim 74, wherein said first volume is about 10 nanoliter or less.

86. A method as recited in claim 74, wherein said first volume is about 2 nanoliter or less

87. A method as recited in claim 74, wherein said first volume is from about 1 picoliter to about 1 microliter.

88. A method as recited in claim 74, wherein said first sample portion is confined at least in part by opposing barriers separated by 500 microns or less.

89. A method as recited in claim 88, wherein at least one of said opposing barriers is a liquid with which said first volume is immiscible.

90. A method as recited in claim 88, wherein at least one of said opposing barriers is a solid.

91. A method as recited in claim 88, wherein at least one of said opposing barriers is a gas.

92. A method as recited in claim 69, wherein said single molecule of a target nucleic acid and the amplification product from said single amplification step are restrained in a first region sufficiently small for said amplification product to be detected.

93. A method as recited in claim 92, wherein said single molecule of a target nucleic acid and the amplification product from said single amplification step are restrained in said first region and said first region is positioned in at least one flow-through channel positioned in a microfludic device.

94. A method as recited in claim 92, wherein said first region is positioned on a surface, and said single molecule of a target nucleic acid and said amplification product from said single amplification step are restrained by an affinity to at least one prepositioned chemical substance on said first region of said surface.

95. A method as recited in claim 94, wherein said prepositioned chemical substance comprises nucleic acids.

96. A method as recited in claim 94, wherein said prepositioned chemical substance comprises a set of at least one compound selected from among the group consisting of sets of primers and sets of probes.

97. A method as recited in claim 94, wherein said prepositioned chemical substance is one of a plurality of different prepositioned chemical substances.

98. A method as recited in claim 94, wherein said surface is contained in a device in which at least about 10,000 prepositioned chemical substances are included.

99. A method as recited in claim 94, wherein said prepositioned chemical substance has a specific affinity for said single molecule of a target nucleic acid and said amplification product from said single amplification step

100. A method as recited in claim 94, wherein said first region has at least one dimension of 2 mm or less.

101. A method as recited in claim 94, wherein said first region has at least one dimension of 1 mm or less.

102. A method as recited in claim 94, wherein said first region has at least one dimension of 100 microns or less.

103. A method as recited in claim 94, wherein said first region has at least one dimension of 20 microns or less.

104. A method as recited in claim 94, wherein said first region has at least one dimension of a few microns or less.

105. A method as recited in claim 92, wherein said first region is located in a first sample chamber.

106. A method as recited in claim 105, wherein said first sample chamber has opposing barriers separated by 500 microns or less.

107. A method as recited in claim 105, wherein said first sample chamber has a volume of about 1 picoliter or less.

108. A method as recited in claim 105, wherein said first sample chamber has a volume in the range of from about 1 picoliter to about 1 microliter.

109. A method as recited in claim 105, wherein said first sample chamber has a volume of about 10 picoliters or less.

110. A method as recited in claim 105, wherein said first sample chamber has a volume of about 100 picoliters or less.

111. A method as recited in claim 105, wherein said first sample chamber has a volume of about 1 nanoliter or less.

112. A method as recited in claim 105, wherein said first sample chamber has a volume of 10 nanoliters or less.

113. A method as recited in claim 105, wherein said first sample chamber has a volume of about 10 nanoliters.

114. A method as recited in claim 105 wherein said first sample chamber is positioned in a device which comprises from about 10,000 to over 100,000 sample chambers.

115. A method as recited in claim 92, wherein said first sample portion has at least one dimension of 2 millimeters or less.

116. A method as recited in claim 92, wherein said first sample portion has at least one dimension of 1 millimeter or less.

117. A method as recited in claim 92, wherein said first sample portion has at least one dimension of 500 microns or less.

118. A method as recited in claim 92, wherein said first sample portion has at least one dimension of 100 microns or less.

119. A method as recited in claim 92, wherein said first sample portion has at least one dimension of 20 microns or less.

120. A method as recited in claim 92, wherein said first sample portion has at least one dimension of a few microns or less.

121. A method as recited in claim 92, wherein said first sample portion has a volume of about 1 picoliter or less.

122. A method as recited in claim 92, wherein said first sample portion has a volume in the range of from about 1 picoliter to about 1 microliter.

123. A method as recited in claim 92, wherein said first sample portion has a volume of about 10 picoliters or less.

124. A method as recited in claim 92, wherein said first sample portion has a volume which is nanoliter-sized.

125. A method as recited in claim 92, wherein said first sample portion has a volume of about 1 nanoliter or less.

126. A method as recited in claim 92, wherein said first sample portion has a volume of about 10 nanoliters or less.

127. A method as recited in claim 92, wherein said first sample portion has a volume of about 100 nanoliters or less.

128. A method as recited in claim 92, wherein said first sample portion has a volume of about 1 microliter or less.

129. A method as recited in claim 69, wherein:

said first sample portion is located in a first sample chamber and said first sample chamber is positioned in a microfludic device which comprises at least one flow-through channel which communicates with said first sample chamber, and

said method further comprises supplying at least one displacing fluid to said flow-through

channel after loading a portion of said first sample into said flow-through channel, whereby said displacing fluid displaces said first sample from said flow-through channel.

130. A method as recited in claim 69, wherein:

said first sample portion is located in a first sample chamber and said first sample chamber is positioned in a microfludic device which comprises at least one flow-through channel which communicates with said first sample chamber, and

said method further comprises supplying at least one sealing fluid to said flow-through channel, whereby said sealing fluid seals said first sample portion within said first sample chamber.

131. A method as recited in claim 130, wherein said method further comprises curing said sealing fluid after said supplying said sealing fluid to said flow-through channel.

132. A method as recited in claim 130, wherein said sealing fluid comprises at least one adhesive.

133. A method as recited in claim 130, wherein said sealing fluid is immiscible with said first sample portion.

134. A method as recited in claim 133, wherein said sealing fluid displaces a second portion of said sample from said flow-through channel.

135. A method as recited in claim 133, wherein said sealing fluid comprises mineral oil.

136. A method as recited in claim 133, wherein said sealing fluid comprises a fluorinated oil.

137. A method as recited in claim 130, wherein said sealing fluid displaces part of said first sample portion from said flow-through channel as said sealing fluid flows through said flow-through channel.

138. A method comprising:

amplifying at least one target nucleic acid molecule to a detectable level in a single amplification step.

139. A method as recited in claim 138, wherein said method further comprises detecting the presence of said target nucleic acid in a first sample portion.

140. A method as recited in claim 138, wherein said single amplification step is a thermocycle step.

141. A method as recited in claim 138, wherein said single amplification step is a homogeneous amplification step.

142. A method as recited in claim 138, wherein said target nucleic acid molecule is contained in a first sample portion and said method comprises subjecting a first sample portion to a single round of amplification.

143. A method as recited in claim 138, wherein said target nucleic acid molecule is contained in a first sample portion and said first sample portion is confined in a first volume sufficiently small to produce a detectable product when subjected to said single amplification step when said at least one molecule of a target nucleic acid is present.

144. A method as recited in claim 143, wherein said first volume is located in at least one flow-through channel positioned in a microfludic device.

145. A method as recited in claim 143, wherein said first volume is located in a microfludic device.

146. A method as recited in claim 143, wherein said first sample portion is confined in said first volume and said first volume is in contact at least in part with a fluid in which said first volume is immiscible.

147. A method as recited in claim 146, wherein said first volume is located in said fluid.

148. A method as recited in claim 146, wherein said fluid comprises mineral oil.

149. A method as recited in claim 146, wherein said fluid comprises a fluorinated oil.

150. A method as recited in claim 146, wherein said fluid comprises a curable liquid.

151. A method as recited in claim 146, wherein said fluid is a gas.

152. A method as recited in claim 143, wherein said first volume is about 1 microliter or less.

153. A method as recited in claim 143, wherein said first volume is about 100 nanoliter or less.

154. A method as recited in claim 143, wherein said first volume is about 10 nanoliter or less.

155. A method as recited in claim 143, wherein said first volume is about 2 nanoliter or less

156. A method as recited in claim 143, wherein said first volume is from about 1 picoliter to about 1 microliter.

157. A method as recited in claim 143, wherein said first sample portion is confined at least in part by opposing barriers separated by 500 microns or less.

158. A method as recited in claim 157, wherein at least one of said opposing barriers is a liquid with which said first volume is immiscible.

159. A method as recited in claim 157, wherein at least one of said opposing barriers is a solid.

160. A method as recited in claim 157, wherein at least one of said opposing barriers is a gas.

161. A method as recited in claim 138, wherein said at least one molecule of a target nucleic acid and a product of said amplification step are restrained in a first region sufficiently small for said amplification product to be detected.

162. A method as recited in claim 161, wherein said at least one molecule of a target nucleic acid and the amplification product from said single amplification step are restrained in said first region and said first region is positioned in at least one flow-through channel positioned in a microfludic device.

163. A method as recited in claim 161, wherein said first region is positioned on a surface, and said at least one molecule of a target nucleic acid and said amplification product from said single amplification step are restrained by an affinity to at least one prepositioned chemical substance on said first region of said surface.

164. A method as recited in claim 163, wherein said prepositioned chemical substance comprises nucleic acids.

165. A method as recited in claim 163, wherein said prepositioned chemical substance comprises a set of at least one compound selected from among the group consisting of sets of primers and sets of probes.

166. A method as recited in claim 163, wherein said prepositioned chemical substance is one of a plurality of different prepositioned chemical substances.

167. A method as recited in claim 163, wherein said surface is contained in a device in which at least about 10,000 prepositioned chemical substances are included.

168. A method as recited in claim 163, wherein said prepositioned chemical substance has a specific affinity for said at least one molecule of a target nucleic acid and said amplification product from said single amplification step

169. A method as recited in claim 163, wherein said first region has at least one dimension of 2 mm or less.

170. A method as recited in claim 163, wherein said first region has at least one dimension of 1 mm or less.

171. A method as recited in claim 163, wherein said first region has at least one dimension of 100 microns or less.

172. A method as recited in claim 163, wherein said first region has at least one dimension of 20 microns or less.

173. A method as recited in claim 163, wherein said first region has at least one dimension of a few microns or less.

174. A method as recited in claim 161, wherein said first region is located in a first sample chamber.

175. A method as recited in claim 174, wherein said first sample chamber has opposing barriers separated by 500 microns or less.

176. A method as recited in claim 174, wherein said first sample chamber has a volume of about 1 picoliter or less.

177. A method as recited in claim 174, wherein said first sample chamber has a volume in the range of from about 1 picoliter to about 1 microliter.

178. A method as recited in claim 174, wherein said first sample chamber has a volume of about 10 picoliters or less.

179. A method as recited in claim 174, wherein said first sample chamber has a volume of about 100 picoliters or less.

180. A method as recited in claim 174, wherein said first sample chamber has a volume of about 1 nanoliter or less.

181. A method as recited in claim 174, wherein said first sample chamber has a volume of 10 nanoliters or less.

182. A method as recited in claim 174, wherein said first sample chamber has a volume of about 10 nanoliters.

183. A method as recited in claim 174 wherein said first sample chamber is positioned in a device which comprises from about 10,000 to over 100,000 sample chambers.

184. A method as recited in claim 161, wherein said target nucleic acid molecule is contained in a first sample portion.

185. A method as recited in claim 184, wherein said first sample portion has at least one dimension of 2 millimeters or less.

186. A method as recited in claim 184, wherein said first sample portion has at least one dimension of 1 millimeter or less.

187. A method as recited in claim 184, wherein said first sample portion has at least one dimension of 500 microns or less.

188. A method as recited in claim 184, wherein said first sample portion has at least one dimension of 100 microns or less.

189. A method as recited in claim 184, wherein said first sample portion has at least one dimension of 20 microns or less.

190. A method as recited in claim 184, wherein said first sample portion has at least one dimension of a few microns or less.

191. A method as recited in claim 184, wherein said first sample portion has a volume of about 1 picoliter or less.

192. A method as recited in claim 184, wherein said first sample portion has a volume in the range of from about 1 picoliter to about 1 microliter.

193. A method as recited in claim 184, wherein said first sample portion has a volume of about 10 picoliters or less.

194. A method as recited in claim 184, wherein said first sample portion has a volume which is nanoliter-sized.

195. A method as recited in claim 184, wherein said first sample portion has a volume of about 1 nanoliter or less.

196. A method as recited in claim 184, wherein said first sample portion has a volume of about 10 nanoliters or less.

197. A method as recited in claim 184, wherein said first sample portion has a volume of about 100 nanoliters or less.

198. A method as recited in claim 184, wherein said first sample portion has a volume of about 1 microliter or less.

199. A method as recited in claim 138, wherein:

said target nucleic acid molecule is contained in a first sample portion which is located in a first sample chamber,

said first sample chamber is positioned in a microfludic device which comprises at least one flow-through channel which communicates with said first sample chamber, and

said method further comprises supplying at least one displacing fluid to said flow-through channel after loading a portion of said first sample into said flow-through channel, whereby said displacing fluid displaces said first sample from said flow-through channel.

200. A method as recited in claim 138, wherein:

said target nucleic acid molecule is contained in a first sample portion which is located in a first sample chamber,

said first sample portion is located in a first sample chamber and said first sample chamber is positioned in a microfludic device which comprises at least one flow-through channel which communicates with said first sample chamber, and

said method further comprises supplying at least one sealing fluid to said flow-through channel, whereby said sealing fluid seals said first sample portion within said first sample chamber.

201. A method as recited in claim 200, wherein said method further comprises curing said sealing fluid after said supplying said sealing fluid to said flow-through channel.

202. A method as recited in claim 200, wherein said sealing fluid comprises at least one adhesive.

203. A method as recited in claim 200, wherein said sealing fluid is immiscible with said first sample portion.

204. A method as recited in claim 203, wherein said sealing fluid displaces a second portion of said sample from said flow-through channel.

205. A method as recited in claim 203, wherein said sealing fluid comprises mineral oil.

206. A method as recited in claim 203, wherein said sealing fluid comprises a fluorinated oil.

207. A method as recited in claim 200, wherein said sealing fluid displaces part of said first sample portion from said flow-through channel as said sealing fluid flows through said flow-through channel.

208. A method comprising:

subjecting a first sample portion in a first porous sample structure to a single amplification step, thereby amplifying a single molecule in said first sample portion to a detectable level.

209. A method as recited in claim 208, wherein said method further comprises determining whether said first sample portion contains at least one molecule of a target nucleic acid.

210. A method comprising:

subjecting a first sample portion in a sample chamber to a single amplification step, thereby amplifying a single molecule in said first sample portion to a detectable level, said sample chamber comprising means for minimizing diffusion of said first sample portion.

211. A method as recited in claim 210, wherein said method further comprises determining whether said first sample portion contains at least one molecule of a target nucleic acid.

212. A method comprising:

subjecting each of a plurality of sample portions to single amplification step, thereby amplifying a single molecule in each of said sample portions to a detectable level.

213. A method as recited in claim 212, wherein said method further comprises, for each of said plurality of said sample portions, determining whether said sample portion contains at least one molecule of a target nucleic acid.

214. A method comprising:

subjecting a first portion of a sample to a single amplification step, thereby amplifying a single molecule in said first portion to a detectable level; and

subjecting a second portion of said sample to a single amplification step, thereby amplifying a single molecule in said second portion to a detectable level.

215. A method as recited in claim 214, wherein said method further comprises determining whether said first portion contains at least one molecule of a first target nucleic acid and determining whether said second portion contains at least one molecule of a second target nucleic acid.

216. A method comprising:

subjecting a portion of a first sample to a single amplification step, thereby amplifying a single molecule in said portion of said first sample to a detectable level; and

subjecting a portion of a second sample to a single amplification step, thereby amplifying a single molecule in said portion of said second sample to a detectable level.

217. A method as recited in claim 216, wherein said method further comprises determining whether said portion of said first sample contains at least one molecule of a first target nucleic acid and determining whether said portion of said second sample contains at least one molecule of a second target nucleic acid.

218. A method comprising:

subjecting a first sample portion in a first sample chamber to a single amplification step, thereby amplifying a single molecule in said first sample portion to a detectable level, said first sample chamber comprising at least a portion of an inside of a first microcapillary device.

219. A method as recited in claim 218, wherein said method further comprises determining whether said first sample portion contains at least one molecule of a target nucleic acid.

220. A method comprising:

subjecting a first sample portion in a first sample chamber to a single amplification step, thereby amplifying a single molecule in said first sample portion to a detectable level, said first sample chamber comprising at least a portion of an inside of a first microcapillary device; and

subjecting a second sample portion in a second sample chamber to a single amplification step, thereby amplifying a single molecule in said second sample portion to a detectable level, said second sample chamber comprising at least a portion of said inside of said first microcapillary device.

221. A method as recited in claim 220, wherein said method further comprises determining whether said first sample portion contains at least one molecule of a target nucleic acid, and determining whether said second sample portion contains at least one molecule of said target nucleic acid.

222. A method comprising:

subjecting a first sample portion in a sample retaining means to a single amplification step, thereby amplifying a single molecule in said first sample portion to a detectable level, said sample retaining means being positioned within a first microcapillary device.

223. A method as recited in claim 222, wherein said method further comprises determining whether said first sample portion contains at least one molecule of a target nucleic acid.

224. A method comprising:

subjecting a first sample portion in a sample retaining means to a single amplification step, thereby amplifying a single molecule in said first sample portion to a detectable level.

225. A method as recited in claim 224, wherein said method further comprises determining whether said first sample portion contains at least one molecule of a target nucleic acid.

226. A method as recited in claim 224, wherein said method further comprises forcing a curable fluid across an exposed surface of said sample retaining means, thereby displacing any excess sample from said exposed surface without displacing said first sample portion from said sample retaining means.

227. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

isolating said first sample portion in a region having at least one dimension of from about 2 mm to a few microns or less,

subjecting said first sample portion to at least a first depolymerization step which would depolymerize a molecule of said target nucleic acid if a molecule of said target nucleic acid is present in said first sample portion, and then

subjecting said first sample portion to at least a first polymerization step which would form a detectable polymerization product if said first sample portion contained at least one molecule of said target nucleic acid.

252. A method as recited in any one of claims 227-251, wherein said first sample portion contains at least one molecule of said target nucleic acid.

253. A method as recited in any one of claims 227-239, wherein said region comprises at least one region selected from among the group consisting of an inside of a first microcapillary device, a sample chamber and a porous retaining means.

255. A method as recited in any one of claims 227-251, wherein said first sample portion comprises a set of at least one compound selected from among the group consisting of sets of primers and sets of probes.

256. A method as recited in any one of claims 227-239, wherein said region comprises a set of at least one compound selected from among the group consisting of sets of primers and sets of probes.

228. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

isolating said first sample portion in a region having at least one dimension of from about 2 mm to a few microns or less,

subjecting said first sample portion to at least a first dehybridization step which would dehybridize a molecule of said target nucleic acid if a molecule of said target nucleic acid is present in said first sample portion, and then

subjecting said first sample portion to at least a first polymerization step which would form a detectable polymerization product if said first sample portion contained at least one molecule of said target nucleic acid.

229. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

isolating said first sample portion in a region having at least one dimension of from about 2 mm to a few microns or less,

subjecting said first sample portion to at least a first polymerization step which would form a detectable polymerization product if said first sample portion contained at least one molecule of said target nucleic acid.

230. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

loading said first sample portion into a device, and locating said target nucleic acid in a region having at least one dimension of from about 2 mm to a few microns or less,

subjecting said first sample portion to at least a first dehybridization step which would dehybridize a molecule of said target nucleic acid if a molecule of said target nucleic acid is present in said first sample portion, and then

subjecting said first sample portion to at least a first polymerization step which would form a detectable polymerization product if said first sample portion contained at least one molecule of said target nucleic acid.

231. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

isolating said first sample portion in a region having at least one dimension of from about 2 mm to a few microns or less,

subjecting said first sample portion to at least a first dehybridization step which would dehybridize a molecule of said target nucleic acid if a molecule of said target nucleic acid is present in said first sample portion, and then

subjecting said first sample portion to at least a first hybridization step which would form a detectable hybridization product if said first sample portion contained at least one molecule of said target nucleic acid.

232. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

isolating said first sample portion in a region having at least one dimension of from about 2 mm to a few microns or less,

subjecting said first sample portion to at least a first hybridization step which would form a detectable hybridization product if said first sample portion contained at least one molecule of said target nucleic acid.

233. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

isolating said first sample portion in a region having at least one dimension of from about 2 mm to a few microns or less,

subjecting said first sample portion to at least a first enzymatic step which would form a detectable product if said first sample portion contained at least one molecule of said target nucleic acid.

234. A method for detecting whether a first target nucleotide is present in a single nucleic acid molecule in a first sample portion, said method comprising:

isolating said first sample portion in a region having at least one dimension of from about 2 mm to a few microns or less,

subjecting said first sample portion to at least a first dehybridization step which would dehybridize said single target nucleic acid molecule, and then

subjecting said first sample portion to at least a first polymerization step which would form a detectable polymerization product if said first sample portion contained said first target nucleotide of said single nucleic acid molecule.

235. A method for detecting whether a first target nucleotide is present in a single nucleic acid molecule in a first sample portion, said method comprising:

isolating said first sample portion in a region having at least one dimension of from about 2 mm to a few microns or less,

subjecting said first sample portion to at least a first enzymatic step which would form a detectable product if said first sample portion contained said first target nucleotide of said single nucleic acid molecule.

236. A method for detecting whether a first target nucleotide is present in a single nucleic acid molecule in a first sample portion, said method comprising:

isolating said first sample portion in a region having at least one dimension of from about 2 mm to a few microns or less,

subjecting said first sample portion to at least a first dehybridization step which would dehybridize said single target nucleic acid molecule, and then

subjecting said first sample portion to at least a first hybridization step with a first probe which would form a detectable hybridization product if said first sample portion contained said first target nucleotide of said nucleic acid.

237. A method for detecting whether a first target nucleotide is present in a single nucleic acid molecule in a first sample portion, said method comprising:

isolating said first sample portion in a region having at least one dimension of from about 2 mm to a few microns or less,

subjecting said first sample portion to at least a first hybridization step with a first probe which would form a detectable hybridization product if said first sample portion contained said first target nucleotide of said single nucleic acid.

238. A method for detecting whether a first target nucleotide is present in a single nucleic acid molecule in a first sample portion, said method comprising:

isolating said first sample portion in a region having at least one dimension of from about 2 mm to a few microns or less,

subjecting said first sample portion to at least a first polymerization step with a first probe which would form a detectable hybridization product if said first sample portion contained said first target nucleotide of said single nucleic acid.

239. A method for detecting whether a first target nucleotide is present in a single nucleic acid molecule in a first sample portion, said method comprising:

isolating said first sample portion in a region having at least one dimension of from about 2 mm to a few microns or less,

subjecting said first sample portion to at least a first enzymatic step with a first probe which would form a detectable hybridization product if said first sample portion contained said first target nucleotide of said single nucleic acid.

240. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

isolating said first sample portion in a volume of from about one microliter to a picoliter or less,

subjecting said first sample portion to at least a first depolymerization step which would depolymerize a molecule of said target nucleic acid if a molecule of said target nucleic acid is present in said first sample portion, and then

subjecting said first sample portion to at least a first polymerization step which would form a detectable polymerization product if said first sample portion contained at least one molecule of said target nucleic acid.

254. A method as recited in any one of claims 240-251, wherein said volume comprises at least one region selected from among the group consisting of an inside of a first microcapillary device, a sample chamber and a porous retaining means.

257. A method as recited in any one of claims 240-251, wherein said volume comprises a set of at least one compound selected from among the group consisting of sets of primers and sets of probes.

241. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

isolating said first sample portion in a volume of from about one microliter to a picoliter or less,

subjecting said first sample portion to at least a first dehybridization step which would dehybridize a molecule of said target nucleic acid if a molecule of said target nucleic acid is present in said first sample portion, and then

subjecting said first sample portion to at least a first polymerization step which would form a detectable polymerization product if said first sample portion contained at least one molecule of said target nucleic acid.

242. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

isolating said first sample portion in a volume of from about one microliter to a picoliter or less,

subjecting said first sample portion to at least a first polymerization step which would form a detectable polymerization product if said first sample portion contained at least one molecule of said target nucleic acid.

243. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

isolating said first sample portion in a volume of from about one microliter to a picoliter or less,

subjecting said first sample portion to at least a first dehybridization step which would dehybridize a molecule of said target nucleic acid if a molecule of said target nucleic acid is present in said first sample portion, and then

subjecting said first sample portion to at least a first hybridization step which would form a detectable hybridization product if said first sample portion contained at least one molecule of said target nucleic acid.

244. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

isolating said first sample portion in a volume of from about one microliter to a picoliter or less,

subjecting said first sample portion to at least a first hybridization step which would form a detectable hybridization product if said first sample portion contained at least one molecule of said target nucleic acid.

245. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample portion, said method comprising:

isolating said first sample portion in a volume of from about one microliter to a picoliter or less,

subjecting said first sample portion to at least a first enzymatic step which would form a detectable product if said first sample portion contained at least one molecule of said target nucleic acid.

246. A method for detecting whether a first target nucleotide is present in a single nucleic acid molecule in a first sample portion, said method comprising:

isolating said first sample portion in a volume of from about one microliter to a picoliter or less,

subjecting said first sample portion to at least a first dehybridization step which would dehybridize said single target nucleic acid molecule, and then

subjecting said first sample portion to at least a first polymerization step which would form a detectable polymerization product if said first sample portion contained said first target nucleotide of said single nucleic acid molecule.

247. A method for detecting whether a first target nucleotide is present in a single nucleic acid molecule in a first sample portion, said method comprising:

isolating said first sample portion in a volume of from about one microliter to a picoliter or less,

subjecting said first sample portion to at least a first enzymatic step which would form a detectable product if said first sample portion contained said first target nucleotide of said single nucleic acid molecule.

248. A method for detecting whether a first target nucleotide is present in a single nucleic acid molecule in a first sample portion, said method comprising:

isolating said first sample portion in a volume of from about one microliter to a picoliter or less,

subjecting said first sample portion to at least a first dehybridization step which would dehybridize said single target nucleic acid molecule, and then

subjecting said first sample portion to at least a first hybridization step with a first probe which would form a detectable hybridization product if said first sample portion contained said first target nucleotide of said nucleic acid.

249. A method for detecting whether a first target nucleotide is present in a single nucleic acid molecule in a first sample portion, said method comprising:

isolating said first sample portion in a volume of from about one microliter to a picoliter or less,

subjecting said first sample portion to at least a first hybridization step with a first probe which would form a detectable hybridization product if said first sample portion contained said first target nucleotide of said single nucleic acid.

250. A method for detecting whether a first target nucleotide is present in a single nucleic acid molecule in a first sample portion, said method comprising:

isolating said first sample portion in a volume of from about one microliter to a picoliter or less,

subjecting said first sample portion to at least a first polymerization step with a first probe which would form a detectable hybridization product if said first sample portion contained said first target nucleotide of said single nucleic acid.

251. A method for detecting whether a first target nucleotide is present in a single nucleic acid molecule in a first sample portion, said method comprising:

isolating said first sample portion in a volume of from about one microliter to a picoliter or less,

subjecting said first sample portion to at least a first enzymatic step with a first probe which would form a detectable hybridization product if said first sample portion contained said first target nucleotide of said single nucleic acid.

258. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample, said method comprising:

isolating a first part of said first sample portion in a second fluid, said first part of said first sample portion having a a volume of from about one microliter to a picoliter or less;

subjecting said first part of said first sample portion to at least one dehybridization step which would dehybridize a molecule of said target nucleic acid if a molecule of said target nucleic acid is present in said first part of said first sample portion,

subjecting said first part of said first sample portion to at least one polymerization step which would form a detectable polymerization product if said first sample portion contained at least one molecule of said target nucleic acid.

259. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample, said method comprising:

isolating a first part of said first sample portion in a second fluid, said first part of said first sample portion having a a volume of from about one microliter to a picoliter or less,

subjecting said first part of said first sample portion to at least one enzymatic step which would form a detectable polymerization product if said first sample portion contained at least one molecule of said target nucleic acid.

260. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample, said method comprising:

isolating a first portion of a first sample in a second fluid, said first portion of said first sample portion having a a volume of from about one microliter to a picoliter or less,

subjecting said first portion of said first sample to at least a first amplification step which would amplify a single target molecule in said first portion of said first sample to a detectable level if a single molecule of said target nucleic acid is present in said first portion of said first sample; and

then

determining whether said first portion of said first sample contains at least one molecule of said target nucleic acid.

261. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample, said method comprising:

isolating a first portion of a first sample in a second fluid, said first portion of said first sample portion having a a volume of from about one microliter to a picoliter or less,

subjecting said first portion of said first sample to at least a first amplification step to amplify at least a single target molecule in said first portion of said first sample to a detectable level; and

then

determining whether said first portion of said first sample contains at least one molecule of said target nucleic acid.

262. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample, said method comprising:

isolating a first portion of a first sample in a second fluid, said first portion of said first sample portion having a a volume of from about one microliter to a picoliter or less,

determining whether said first portion of said first sample contains at least one molecule of at least one target nucleic acid.

263. A method for detecting whether at least one molecule of a target nucleic acid is present in a first sample, said method comprising:

isolating a first portion of a first sample in a sample retaining means, said first portion of said first sample portion having a a volume of from about one microliter to a picoliter or less,

determining whether said first portion of said first sample contains at least one molecule of at least one target nucleic acid.

264. A method as recited in claim 263, wherein said sample retaining means comprises an inside of a first microcapillary device, a sample chamber or a porous retaining means.

274. A method as recited in claim 263, wherein said means for retaining nucleic acid sample comprises an inside of a first microcapillary device, a sample chamber or a porous retaining means.

265. A method comprising:

isolating a first portion of a first sample, said first portion of said first sample portion having a volume of from about one microliter to a picoliter or less,

amplifying at least one molecule of a target nucleic acid to a detectable level in a single amplification step.

266. A method for detecting whether at least one molecule of a target nucleic is present in a first sample portion, said method comprising:

subjecting said first sample portion to a single amplification step, thereby amplifying a single molecule of said target nucleic acid in said first sample portion to a detectable level; and

then

determining whether said first sample portion contains at least one molecule of said target nucleic acid.

267. A composition comprising:

a retaining means having a volume of from about one microliter to a picoliter or less, and

at least one nucleic acid molecule,

at least one polymerase molecule, and

at least one probe contained therein.

268. A composition comprising:

a retaining means having a volume of from about one microliter to a picoliter or less, and

a single nucleic acid molecule,

at least one polymerase molecule, and

at least one probe contained therein.

269. A composition comprising:

a sample retaining means having a region with at least one dimension of from about 2 mm to a few microns or less, and

a single nucleic acid molecule,

at least one polymerase molecule, and

at least one probe molecule contained therein.

270. A composition comprising:

a nucleic acid retaining means having a region with at least one dimension of from about 2 mm to a few microns or less, and

a single nucleic acid molecule,

at least one polymerase molecule, and

at least one probe molecule contained therein.

271. A composition comprising:

a nucleic acid retaining means having a region with at least one dimension of from about 2 mm to a few microns or less, and

a single nucleic acid molecule,

at least one polymerization enzyme molecule, and

at least one probe molecule contained therein.

272. A device comprising:

at least one means for retaining fluid

at least one nucleic acid molecule positioned in said means for retaining fluid;

at least one polymerase molecule positioned in said means for retaining fluid; and

at least one primer molecule positioned in said means for retaining fluid;

said retaining fluid having a volume of from about one microliter to a picoliter or less,

273. A device comprising:

at least one means for retaining a nucleic acid sample

a single nucleic acid molecule positioned in said means for retaining nucleic acid sample;

at least one polymerase molecule positioned in said means for retaining nucleic acid sample; and

at least one primer molecule positioned in said means for retaining nucleic acid sample.

275. A composition comprising:

a single nucleic acid molecule;

at least one polymerase molecule;

at least one primer molecule; and

a retaining fluid,

said nucleic acid molecule, said polymerase molecule and said primer molecule being isolated within said retaining fluid having a volume of from about one microliter to a picoliter or less.

276. A composition comprising:

at least one nucleic acid molecule;

at least one polymerase molecule;

at least one primer molecule; and

a retaining fluid,

said nucleic acid molecule, said polymerase molecule and said primer molecule being isolated within said retaining fluid having a volume of from about one microliter to a picoliter or less.

277. A method comprising:

providing constituents comprising a single nucleic acid molecule, at least one polymerase molecule, and at least one probe molecule;

loading said constituents into a retaining means having a volume of from about one microliter to a picoliter or less, and

carrying out at least one amplification step.

278. A method comprising:

providing constituents comprising a single nucleic acid molecule, at least one polymerase molecule, and at least one probe molecule;

loading said constituents into a retaining means having a volume of from about one microliter to a picoliter or less, and

carrying out at least one polymerization step.

279. A method comprising:

providing constituents comprising a single nucleic acid molecule, at least one polymerase molecule, and at least one probe molecule;

loading said constituents into a retaining means having a volume of from about one microliter to a picoliter or less, and

carrying out at least one hybridization step.

280. A method comprising:

providing constituents comprising a single nucleic acid molecule, at least one polymerase molecule, and at least one probe molecule;

loading said constituents into a retaining means having a volume of from about one microliter to a picoliter or less, and

carrying out at least one polymerase step.

281. A method comprising:

providing constituents comprising a single nucleic acid molecule, at least one polymerase molecule, and at least one probe molecule;

loading said constituents into a retaining means having a volume of from about one microliter to a picoliter or less,

carrying out at least one amplification step, and then

determining the presence of amplified nucleic acid product.

282. A method comprising:

providing constituents comprising a single nucleic acid molecule, at least one polymerase molecule, and at least one probe molecule;

loading said constituents into a retaining means having at least one dimension of from about 2 mm to a few microns or less,

carrying out at least one amplification step, and then

determining the presence of amplified nucleic acid product.

283. A method comprising:

providing constituents comprising a single nucleic acid molecule, at least one polymerase molecule, and at least one probe molecule;

loading said constituents into a retaining means having at least one dimension of from about 2 mm to a few microns or less,

carrying out at least one polymerization step, and then

determining the presence of polymerized nucleic acid product.