METHOD FOR QUANTIFYING NUMBER OF MOLECULES OF TARGET NUCLEIC ACID CONTAINED IN A SAMPLE

• US 20080171324A1
• Filed: 08/13/2007
• Published: 07/17/2008
• Est. Priority Date: 04/17/1997
• Status: Abandoned Application

• Current Assignee: United States Department Of Health And Human Services (Government of the United States of America), Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Cytonix, The United States of America As Represented By The Secretary of Agriculture
• Inventors: BROWN, James F., SILVER, Jonathan E.
• Application Number:
  US11/837,569
• Publication Number:
  US 20080171324A1
• 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 comprises loading one or more sample portions into respective sample chambers, subjecting each of the sample portions to an amplification step, for each sample portion, determining whether the sample portion contains at least one molecule of a target nucleic acid; and then quantifying a number of the sample portions which contain at least one molecule of the target nucleic acid, and/or quantifying a number of peaks indicative of the detectable concentration, and/or detecting an intensity of at least one peak indicative of a concentration of the target nucleic acid. Any of the sample portions which contains a single molecule of the target nucleic acid would attain a detectable concentration of the target nucleic acid after a single round of amplification.

Claims

1. A method for quantifying a number of molecules of at least a first target nucleic acid contained in a first sample, said method comprising:

loading a plurality of first sample portions into respective sample chambers, each of said first sample portions comprising part of a first sample, whereby any of said first sample portions which contains at least a single molecule of said first target nucleic acid would attain a detectable concentration of said first target nucleic acid after a single round of amplification;

subjecting each said first sample portion in said respective sample chambers to at least a first amplification step;

for each said first sample portion, determining whether said first sample portion contains at least one molecule of said first target nucleic acid; and

then

quantifying a number of said first sample portions which contain at least one molecule of said first target nucleic acid.

2. A method as recited in claim 1, wherein said determining whether said first sample portion contains at least one molecule of said first target nucleic acid is performed by carrying out a procedure which generates signals having magnitude which is higher where said detectable concentration is present than where said detectable concentration is not present.

3. A method as recited in claim 2, wherein said procedure comprises detecting fluorescence from fluor-labeled materials.

4. A method as recited in claim 2, wherein said procedure comprises detecting at least one chemical property which changes upon hybridization.

5. A method as recited in claim 2, wherein said procedure comprises evaluating at least one property selected from among the group consisting of agglutination, turbidity, phosphorescence, light scattering, light absorbance, fluorescence energy transfer, fluorescence quenching, fluorescence dequenching, time-delayed fluorescence, chemiluminescence and calorimetric evaluation.

6. A method as recited in claim 1, wherein said first sample chamber comprises at least a portion of an inside of a first microcapillary device, and wherein said peaks are generated by detecting regions within said first microcapillary device in which said detectable concentration is present.

7. A method as recited in claim 1, wherein each of said sample chambers has a volume of about 1 picoliter or less.

8. A method as recited in claim 1, wherein each of said sample chambers has a volume in the range of from about 1 picoliter to about 1 microliter.

9. A method as recited in claim 1, wherein each of said sample chambers has a volume of about 10 picoliters or less.

10. A method as recited in claim 1, wherein each of said sample chambers has a volume of about 100 picoliters.

11. A method as recited in claim 1, wherein each of said sample chambers has a volume of about 1 nanoliter.

12. A method as recited in claim 1, wherein each of said sample chambers has a volume of 10 nanoliters or less.

13. A method as recited in claim 1, wherein each of said sample chambers has a volume of about 10 nanoliters.

14. A method as recited in claim 1, wherein each of said first sample chambers has at least one dimension of 2 mm or less.

15. A method as recited in claim 1, wherein each of said first sample chambers has at least one dimension of 1 mm or less.

16. A method as recited in claim 1, wherein each of said first sample chambers has at least one dimension of 100 microns or less.

17. A method as recited in claim 1, wherein each of said first sample chambers has at least one dimension of 20 microns or less.

18. A method as recited in claim 1, wherein each of said first sample chambers has at least one dimension of a few microns or less.

19. A method as recited in claim 1, wherein each of said first sample portions has a volume of about 1 picoliter or less.

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

21. A method as recited in claim 1, wherein each of said first sample portions has a volume of about 10 picoliters or less.

22. A method as recited in claim 1, wherein each of said first sample portions has a volume which is nanoliter-sized.

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

24. A method as recited in claim 1, wherein each of said first sample portions has a volume of about 10 nanoliters or less.

25. A method as recited in claim 1, wherein each of said first sample portions has a volume of about 100 nanoliters or less.

26. A method as recited in claim 1, wherein each of said first sample portions has a volume of about 1 microliter or less.

27. A method as recited in claim 1, wherein each of said first sample portions is confined in at least one dimension by opposing barriers separated by about 2 mm or less.

28. A method as recited in claim 1, wherein each of said first sample portions is confined in at least one dimension by opposing barriers separated by about 1 mm or less.

29. A method as recited in claim 1, wherein each of said first sample portions is confined in at least one dimension by opposing barriers separated by about 500 microns or less.

30. A method as recited in claim 1, wherein each of said first sample portions is confined in at least one dimension by opposing barriers separated by about 100 microns or less.

31. A method as recited in claim 1, wherein each of said first sample portions is confined in at least one dimension by opposing barriers separated by about 20 microns or less.

32. A method as recited in claim 1, wherein each of said first sample portions is confined in at least one dimension by opposing barriers separated by a few microns or less.

33. A method as recited in claim 1, wherein each of said sample chambers contains at least one amplification targeting reagent.

34. A method as recited in claim 1, wherein at least one of said first sample chambers further comprises constituents for enabling amplification of said first target nucleic acid.

35. A method as recited in claim 1, wherein at least one of said sample portions further comprises constituents for enabling amplification of said first target nucleic acid.

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

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

38. A method as recited in claim 1, wherein any of said first sample portions which contains at least a single molecule of said first target nucleic acid would attain a detectable concentration of said first target nucleic acid after a single amplification step.

39. A method as recited in claim 1 wherein said sample chambers are positioned in a device which comprises from about 10,000 to over 100,000 sample chambers.

40. A method as recited in claim 1, wherein said first target nucleic acid comprises at least one nucleic acid sequence of at least one pathogenic organism.

41. A method as recited in claim 1, wherein said first sample comprises at least one forensic sample.

42. A method as recited in claim 1, wherein said first target nucleic acid is indicative of the likelihood or presence of at least one genetic disorder.

43. A method as recited in claim 1, wherein said method for detecting whether at least one molecule of said first target nucleic acid is present in said first sample is part of a procedure selected from among the group consisting of analyzing mutations in activated oncogenes, molecular cloning, analyzing DNA, and detecting at least one sequence difference.

44. A method as recited in claim 43, wherein said procedure comprises at least one task selected from among the group consisting of generating specific sequences of DNA for cloning or use as probes, detecting segments of DNA for genetic mapping, detecting expressed sequences by amplification of particular segments of cDNA, analyzing expressed sequences by amplification of particular segments of cDNA, generating libraries of cDNA from small amounts of mRNA, generating large amounts of DNA for sequencing, analyzing mutations, and chromosome crawling.

45. A method as recited in claim 43, wherein said sequence difference is selected from among insertions, deletions and changes.

46. A method as recited in claim 1, wherein said method for detecting whether at least one molecule of said first target nucleic acid is present in said first sample is part of a procedure selected from among the group consisting of fertility procedures, immunology procedures, cytology procedures, gas analysis procedures and pharmaceutical screening procedures.

47. A method for quantifying a number of molecules of at least a first target nucleic acid contained in a first sample, said method comprising:

loading at least a first sample portion into a first sample chamber, said first sample portion comprising at least a part of a first sample, whereby if said first sample portion contains at least a single molecule of a first target nucleic acid, said first sample portion would attain a detectable concentration of said first target nucleic acid within a portion of said first sample portion after a single round of amplification;

subjecting said first sample portion to at least a first amplification step;

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

then

quantifying a number of peaks indicative of said detectable concentration.

48. A method as recited in claim 47, wherein said peaks are produced by carrying out a procedure which generates signals having magnitude which is higher where said detectable concentration is present than where said detectable concentration is not present.

49. A method as recited in claim 48, wherein said procedure comprises detecting fluorescence from fluor-labeled materials.

50. A method as recited in claim 48, wherein said procedure comprises detecting at least one chemical property which changes upon hybridization.

51. A method as recited in claim 48, wherein said procedure comprises evaluating at least one property selected from among the group consisting of agglutination, turbidity, phosphorescence, light scattering, light absorbance, fluorescence energy transfer, fluorescence quenching, fluorescence dequenching, time-delayed fluorescence, chemiluminescence and calorimetric evaluation.

52. A method as recited in claim 47, wherein said first sample chamber comprises at least a portion of an inside of a first microcapillary device, and wherein said peaks are generated by detecting regions within said first microcapillary device in which said detectable concentration is present.

53. A method as recited in claim 47, wherein said first sample chamber has a volume of about 1 picoliter or less.

54. A method as recited in claim 47, wherein said first sample chamber has a volume in the range of from about 1 picoliter to about 1 microliter.

55. A method as recited in claim 47, wherein said first sample chamber has a volume of about 10 picoliters or less.

56. A method as recited in claim 47, wherein said first sample chamber has a volume of about 100 picoliters.

57. A method as recited in claim 47, wherein said first sample chamber has a volume of about 1 nanoliter.

58. A method as recited in claim 47, wherein said first sample chamber has a volume of 10 nanoliters or less.

59. A method as recited in claim 47, wherein said first sample chamber has a volume of about 10 nanoliters.

60. A method as recited in claim 47, wherein said first sample chamber has at least one dimension of 2 mm or less.

61. A method as recited in claim 47, wherein said first sample chamber has at least one dimension of 1 mm or less.

62. A method as recited in claim 47, wherein said first sample chamber has at least one dimension of 100 microns or less.

63. A method as recited in claim 47, wherein said first sample chamber has at least one dimension of 20 microns or less.

64. A method as recited in claim 47, wherein said first sample chamber has at least one dimension of a few microns or less.

65. A method as recited in claim 47, wherein said first sample portion has a volume of about 1 picoliter or less.

66. A method as recited in claim 47, wherein said first sample portion has a volume in the range of from about 1 picoliter to about 1 microliter.

67. A method as recited in claim 47, wherein said first sample portion has a volume of about 10 picoliters or less.

68. A method as recited in claim 47, wherein said first sample portion has a volume which is nanoliter-sized.

69. A method as recited in claim 47, wherein said first sample portion has a volume of about 1 nanoliter or less.

70. A method as recited in claim 47, wherein said first sample portion has a volume of about 10 nanoliters or less.

71. A method as recited in claim 47, wherein said first sample portion has a volume of about 100 nanoliters or less.

72. A method as recited in claim 47, wherein said first sample portion has a volume of about 1 microliter or less.

73. A method as recited in claim 47, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 2 mm or less.

74. A method as recited in claim 47, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 1 mm or less.

75. A method as recited in claim 47, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 500 microns or less.

76. A method as recited in claim 47, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 100 microns or less.

77. A method as recited in claim 47, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 20 microns or less.

78. A method as recited in claim 47, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by a few microns or less.

79. A method as recited in claim 47, wherein said first sample chamber contains at least one amplification targeting reagent.

80. A method as recited in claim 47, wherein said first sample chamber further comprises constituents for enabling amplification of said first target nucleic acid.

81. A method as recited in claim 47, wherein said first sample portion further comprises constituents for enabling amplification of said first target nucleic acid.

82. A method as recited in claim 47, wherein said first amplification step is a homogeneous amplification step.

83. A method as recited in claim 47, wherein said first amplification step is a thermocycle step

84. A method as recited in claim 47, wherein if said first sample portion contains at least a single molecule of said first target nucleic acid, said first sample portion would attain a detectable concentration of said first target nucleic acid after a single amplification step.

85. A method as recited in claim 47 wherein said sample chambers are positioned in a device which comprises from about 10,000 to over 100,000 sample chambers.

86. A method as recited in claim 47, wherein said first target nucleic acid comprises at least one nucleic acid sequence of at least one pathogenic organism.

87. A method as recited in claim 47, wherein said first sample comprises at least one forensic sample.

88. A method as recited in claim 47, wherein said first target nucleic acid is indicative of the likelihood or presence of at least one genetic disorder.

89. A method as recited in claim 47, wherein said method for detecting whether at least one molecule of said first target nucleic acid is present in said first sample is part of a procedure selected from among the group consisting of analyzing mutations in activated oncogenes, molecular cloning, analyzing DNA, and detecting at least one sequence difference.

90. A method as recited in claim 89, wherein said procedure comprises at least one task selected from among the group consisting of generating specific sequences of DNA for cloning or use as probes, detecting segments of DNA for genetic mapping, detecting expressed sequences by amplification of particular segments of cDNA, analyzing expressed sequences by amplification of particular segments of cDNA, generating libraries of cDNA from small amounts of mRNA, generating large amounts of DNA for sequencing, analyzing mutations, and chromosome crawling.

91. A method as recited in claim 89, wherein said sequence difference is selected from among insertions, deletions and changes.

92. A method as recited in claim 47, wherein said method for detecting whether at least one molecule of said first target nucleic acid is present in said first sample is part of a procedure selected from among the group consisting of fertility procedures, immunology procedures, cytology procedures, gas analysis procedures and pharmaceutical screening procedures.

93. A method for quantifying a number of molecules of at least a first target nucleic acid contained in a first sample, said method comprising:

loading at least a first sample portion into a first sample chamber, said first sample portion comprising at least a part of said first sample, whereby if said first sample portion contains at least a single molecule of said first target nucleic acid, said first sample portion would attain a detectable concentration of said first target nucleic acid within a portion of said first sample portion after a single round of amplification;

subjecting said first sample portion in said first sample chamber to at least a first amplification step; and

then detecting an intensity of at least one peak indicative of said detectable concentration.

94. A method as recited in claim 93, wherein said peak is produced by carrying out a procedure which generates signals having magnitude which is higher where said detectable concentration is present than where said detectable concentration is not present.

95. A method as recited in claim 94, wherein said procedure comprises detecting fluorescence from fluor-labeled materials.

96. A method as recited in claim 94, wherein said procedure comprises detecting at least one chemical property which changes upon hybridization.

97. A method as recited in claim 94, wherein said procedure comprises evaluating at least one property selected from among the group consisting of agglutination, turbidity, phosphorescence, light scattering, light absorbance, fluorescence energy transfer, fluorescence quenching, fluorescence dequenching, time-delayed fluorescence, chemiluminescence and calorimetric evaluation.

98. A method as recited in claim 93, wherein said first sample chamber comprises at least a portion of an inside of a microcapillary device, and wherein said peaks are generated by detecting regions within said microcapillary device in which said detectable concentration is present.

99. A method as recited in claim 93, wherein said first sample chamber has a volume of about 1 picoliter or less.

100. A method as recited in claim 93, wherein said first sample chamber has a volume in the range of from about 1 picoliter to about 1 microliter.

101. A method as recited in claim 93, wherein said first sample chamber has a volume of about 10 picoliters or less.

102. A method as recited in claim 93, wherein said first sample chamber has a volume of about 100 picoliters.

103. A method as recited in claim 93, wherein said first sample chamber has a volume of about 1 nanoliter.

104. A method as recited in claim 93, wherein said first sample chamber has a volume of 10 nanoliters or less.

105. A method as recited in claim 93, wherein said first sample chamber has a volume of about 10 nanoliters.

106. A method as recited in claim 93, wherein said first sample chamber has at least one dimension of 2 mm or less.

107. A method as recited in claim 93, wherein said first sample chamber has at least one dimension of 1 mm or less.

108. A method as recited in claim 93, wherein said first sample chamber has at least one dimension of 100 microns or less.

109. A method as recited in claim 93, wherein said first sample chamber has at least one dimension of 20 microns or less.

110. A method as recited in claim 93, wherein said first sample chamber has at least one dimension of a few microns or less.

111. A method as recited in claim 93, wherein said first sample portion has a volume of about 1 picoliter or less.

112. A method as recited in claim 93, wherein said first sample portion has a volume in the range of from about 1 picoliter to about 1 microliter.

113. A method as recited in claim 93, wherein said first sample portion has a volume of about 10 picoliters or less.

114. A method as recited in claim 93, wherein said first sample portion has a volume which is nanoliter-sized.

115. A method as recited in claim 93, wherein said first sample portion has a volume of about 1 nanoliter or less.

116. A method as recited in claim 93, wherein said first sample portion has a volume of about 10 nanoliters or less.

117. A method as recited in claim 93, wherein said first sample portion has a volume of about 100 nanoliters or less.

118. A method as recited in claim 93, wherein said first sample portion has a volume of about 1 microliter or less.

119. A method as recited in claim 93, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 2 mm or less.

120. A method as recited in claim 93, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 1 mm or less.

121. A method as recited in claim 93, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 500 microns or less.

122. A method as recited in claim 93, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 100 microns or less.

123. A method as recited in claim 93, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by about 20 microns or less.

124. A method as recited in claim 93, wherein said first sample portion is confined in at least one dimension by opposing barriers separated by a few microns or less.

125. A method as recited in claim 93, wherein said first sample chamber contains at least one amplification targeting reagent.

126. A method as recited in claim 93, wherein said first sample chamber further comprises constituents for enabling amplification of said first target nucleic acid.

127. A method as recited in claim 93, wherein said first sample portion further comprises constituents for enabling amplification of said first target nucleic acid.

128. A method as recited in claim 93, wherein said first amplification step is a homogeneous amplification step.

129. A method as recited in claim 93, wherein said first amplification step is a thermocycle step

130. A method as recited in claim 93, wherein if said first sample portion contains at least a single molecule of said first target nucleic acid, said first sample portion would attain a detectable concentration of said first target nucleic acid after a single amplification step.

131. A method as recited in claim 93 wherein said sample chambers are positioned in a device which comprises from about 10,000 to over 100,000 sample chambers.

132. A method as recited in claim 93, wherein said first target nucleic acid comprises at least one nucleic acid sequence of at least one pathogenic organism.

133. A method as recited in claim 93, wherein said first sample comprises at least one forensic sample.

134. A method as recited in claim 93, wherein said first target nucleic acid is indicative of the likelihood or presence of at least one genetic disorder.

135. A method as recited in claim 93, wherein said method for detecting whether at least one molecule of said first target nucleic acid is present in said first sample is part of a procedure selected from among the group consisting of analyzing mutations in activated oncogenes, molecular cloning, analyzing DNA, and detecting at least one sequence difference.

136. A method as recited in claim 135, wherein said procedure comprises at least one task selected from among the group consisting of generating specific sequences of DNA for cloning or use as probes, detecting segments of DNA for genetic mapping, detecting expressed sequences by amplification of particular segments of cDNA, analyzing expressed sequences by amplification of particular segments of cDNA, generating libraries of cDNA from small amounts of mRNA, generating large amounts of DNA for sequencing, analyzing mutations, and chromosome crawling.

137. A method as recited in claim 135, wherein said sequence difference is selected from among insertions, deletions and changes.

138. A method as recited in claim 93, wherein said method for detecting whether at least one molecule of said first target nucleic acid is present in said first sample is part of a procedure selected from among the group consisting of fertility procedures, immunology procedures, cytology procedures, gas analysis procedures and pharmaceutical screening procedures.