COMPOSITIONS AND KITS FOR MOLECULAR COUNTING

• US 20150133319A1
• Filed: 02/27/2013
• Published: 05/14/2015
• Est. Priority Date: 02/27/2012
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Fu, Glenn K., Fodor, Stephen P.A., Wilhelmy, Julie
• Granted Patent: US 10,941,396 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2320/10 in screening processes
• 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 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

Methods, kits and systems are disclosed for analyzing one or more molecules in a sample. Analyzing the one or more molecules may comprise quantitation of the one or more molecules. Individual molecules may quantitated by PCR, arrays, beads, emulsions, droplets, or sequencing. Quantitation of individual molecules may further comprise stochastic labeling of the one or more molecules with a plurality of oligonucleotide tags to produce one or more stochastically labeled molecules. The methods may further comprise amplifying, sequencing, detecting, and/or quantifying the stochastically labeled molecules. The molecules may be DNA, RNA and/or proteins.

Claims

1. A digital reverse transcription method comprising:

a. contacting a sample comprising a plurality of RNA molecules with a plurality of oligonucleotide tags to produce a labeled-RNA molecule, wherein;

i. the plurality of RNA molecules comprise at least 2 mRNA molecules of different sequences;

ii. the plurality of oligonucleotide tags comprises at least 2 oligonucleotide tags of different sequences; and

iii. the plurality of oligonucleotide tags comprises an oligodT sequence;

b. conducting a first strand synthesis reaction by contacting the labeled-RNA molecules with a reverse transcriptase enzyme to produce a labeled-cDNA molecule; and

c. detecting the labeled-cDNA molecule by hybridizing the labeled-cDNA molecule to a solid support.

2. The method of claim 1, wherein the oligonucleotide tag further comprises a unique identifier region.

3. The method of claim 2, wherein the unique identifier region is at least one nucleotide in length.

4. The method of claim 1, wherein the oligonucleotide tag further comprises a universal primer binding site.

5. The method of claim 1, wherein the oligonucleotide tag is at least one nucleotide in length.

6. The method of claim 1, wherein the solid support is an array.

7. The method of claim 1, wherein the solid support is an addressable array.

8. The method of claim 1, wherein the solid support is an Affymetrix 3K tag array, Arrayjet non-contact printed array, or Applied Microarrays Inc (AMI) array.

9. The method of claim 1, wherein the solid support is a bead.

10. The method of claim 1, further comprising conducting a polymerase chain reaction on the labeled-cDNA molecule of step (b) to produce a double-stranded labeled-cDNA molecule.

11. The method of claim 10, wherein conducting the polymerase chain reaction comprises annealing a first target specific primer to the labeled-cDNA molecule.

12. The method of claim 10, wherein conducting the polymerase chain reaction further comprises annealing a universal primer to the universal primer binding site of the oligonucleotide tag.

13. The method of claim 10, wherein the polymerase chain reaction comprises absolute PCR, HD-PCR, Next Gen PCR, digital RTA, or any combination thereof.

14. The method of claim 10, further comprising conducting a nested PCR reaction on the double-stranded labeled-cDNA molecule.

15. The method of claim 14, wherein conducting the nested PCR reaction comprises denaturing the double-stranded labeled cDNA molecule to produce a denatured single-stranded labeled-cDNA molecule.

16. The method of claim 14, wherein conducting the nested PCR reaction further comprises annealing a second target specific primer to the denatured single-stranded labeled-cDNA molecule.

17. The method of claim 14, wherein conducting the nested PCR reaction further comprises annealing a universal primer to the universal primer binding site of the oligonucleotide tag.

18. The method of claim 1, further comprising conducting a sequencing reaction to determine the sequence of at least a portion of the oligonucleotide tag, at least a portion of the labeled-cDNA molecule, a complement thereof, a reverse complement thereof, or any combination thereof.

19. The method of claim 1, wherein detecting the labeled-cDNA molecules comprises an array detector, fluorescent reader, non-fluorescent detector, CR reader, or scanner.

20. The method of claim 19, wherein the array detector is a flatbed scanner.

21. The method of claim 19, wherein the fluorescent reader is a Sensovation, AG fluorescent reader.

22. The method of claim 1, wherein detecting the labeled-cDNA molecules comprises detecting the labeled-cDNA molecules hybridized to the solid support.

23. The method of claim 1, further comprising conducting a hybridization chain reaction.

24. The method of claim 1, wherein the oligonucleotide tag comprises one or more secondary structures.

25. The method of claim 24, wherein the one or more secondary structures is a hairpin.

26. The method of claim 1, wherein the oligonucleotide tag is linear.

127. The method of any of claims 1 and 75-76, wherein labeled-molecules are produced by stochastic labeling.

27. A kit comprising:

a. a plurality of oligonucleotide tags, wherein the oligonucleotide tag of the plurality of oligonucleotide tags comprises;

i. a target specific region; and

ii. a unique identifier region;

b. an enzyme.

28. The kit of claim 27, wherein the enzyme is a reverse transcriptase enzyme.

29. The kit of claim 27, wherein the enzyme is a ligase.

30. The kit of claim 27, wherein the enzyme is a polymerase.

31. The kit of claim 27, wherein the enzyme is an RNase.

32. The kit of claim 27, wherein the enzyme is a DNase.

33. The kit of claim 27, wherein the enzyme is an endonuclease.

34. The kit of claim 27, wherein the oligonucleotide tag is at least 25 nucleotides in length.

35. The kit of claim 27, wherein the unique identifier region is at least 10 nucleotides in length.

36. The kit of claim 27, wherein the target specific region is at least 10 nucleotides in length.

37. The kit of claim 27, wherein the target specific region comprises an oligodT sequence.

38. The kit of claim 27, wherein the oligonucleotide tag further comprises a universal primer binding site.

39. The kit of claim 27, further comprising a support.

40. The kit of claim 39, wherein the support is a semi-solid support.

41. The kit of claim 39, wherein the support is a solid support.

42. The kit of claim 41, wherein the solid support is an array.

43. The kit of claim 39, wherein the support is an addressable array.

44. The kit of claim 39, wherein the support is an Affymetrix 3K tag array, Arrayjet non-contact printed array, or Applied Microarrays Inc (AMI) array.

45. The kit of claim 39, the support is a bead.

46. The kit of claim 27, further comprising a primer.

47. The kit of claim 46, wherein the primer is a universal primer.

48. The kit of claim 46, wherein the primer binds to the oligonucleotide tag.

49. The kit of claim 46, wherein the primer binds to the universal primer binding site of the oligonucleotide tag.

50. The kit of claim 27, further comprising a control oligo.

51. The kit of claim 50, wherein the control oligo comprises at least 15 nucleotides.

52. The kit of claim 50, wherein the control oligo is a bright hybridization control oligo.

53. The kit of claim 50, wherein the control oligo is a spike-in template control.

54. The kit of claim 27, wherein the oligonucleotide tag further comprises a label.

55. The kit of claim 46, wherein the primer further comprises a label.

56. The kit of claim 50, wherein the control oligo further comprises a label.

57. The kit of any of claims 54-56, wherein the label is a dye label.

58. The kit of any of claims 54-56, wherein the label is a Cy3 dye.

59. The kit of any of claims 54-56, wherein the label is a Tye563 dye.

60. The kit of claim 27, further comprising a buffer.

61. The kit of claim 27, further comprising a carrier.

62. The kit of claim 27, further comprising a detergent.

63. The kit of claim 27, further comprising a thermal cycler.

64. The kit of claim 27, further comprising a sequencer.

65. The kit of claim 27, further comprising a hybridization chamber.

66. The kit of claim 27, further comprising a detector.

67. The kit of claim 27, further comprising an array detector, fluorescent reader, non-fluorescent detector, CR reader, or scanner.

68. The kit of claim 67, wherein the fluorescent reader is a Sensovation or AG fluorescent reader.

69. The kit of claim 67, wherein the scanner is a flatbed scanner.

70. The kit of claim 27, further comprising a computer.

71. The kit of claim 70, wherein the computer comprises a memory device.

72. The kit of claim 71, wherein the memory device is capable of storing data.

73. The kit of claim 27, further comprising a software program.

74. The kit of claim 27, further comprising a computer-readable program.

75. A cell analysis method comprising:

a. contacting a sample comprising a plurality of molecules with a plurality of oligonucleotide tags to produce a labeled-molecule, wherein;

i. the plurality of molecules comprise at least 2 molecules of different sequences;

ii. the plurality of oligonucleotide tags comprises at least 2 oligonucleotide tags of different sequences; and

iii. the sample is from at least one cell; and

b. detecting the labeled-molecule by hybridizing the labeled-molecule to a solid support.

77. The method of any of claims 75-76, wherein the oligonucleotide tag further comprises a unique identifier region.

78. The method of claim 77, wherein the unique identifier region is at least 10 nucleotides in length.

79. The method of claim 77, wherein the unique identifier region is cannot hybridize to the molecule.

80. The method of any of claims 75-76, wherein the oligonucleotide tag further comprises a universal primer binding site.

81. The method of any of claims 75-76, wherein the oligonucleotide tag is at least 20 nucleotides in length.

82. The method of any of claims 75-76, wherein the oligonucleotide tag further comprises a target specific region.

83. The method of claim 82, wherein the target specific region comprises an oligodT sequence.

84. The method of claim 82, wherein the target specific region is at least 10 nucleotides in length.

85. The method of any of claims 75-76, wherein the solid support is an array.

86. The method of any of claims 75-76, wherein the solid support is an addressable array.

87. The method of any of claims 75-76, wherein the solid support is an Affymetrix 3K tag array, Arrayjet non-contact printed array, or Applied Microarrays Inc (AMI) array.

88. The method of any of claims 75-76, wherein the solid support is a bead.

89. The method of any of claims 75-76, further comprising conducting a first strand synthesis reaction to produce a labeled-cDNA molecule.

90. The method of claim 75, further comprising conducting a polymerase chain reaction on the labeled-molecule or any product thereof to produce a double-stranded labeled-molecule.

92. The method of any of claims 90-91, wherein conducting the polymerase chain reaction comprises annealing a first target specific primer to the labeled-molecule or any product thereof.

93. The method of any of claims 90-91, wherein conducting the polymerase chain reaction further comprises annealing a universal primer to the universal primer binding site of the oligonucleotide tag.

94. The method of any of claims 90-91, wherein the polymerase chain reaction comprises absolute PCR, HD-PCR, Next Gen PCR, digital RTA, or any combination thereof.

95. The method of any of claims 75-76, further comprising conducting a nested PCR reaction on the double-stranded labeled-cDNA molecule.

96. The method of claim 95, wherein conducting the nested PCR reaction comprises denaturing the labeled-molecule or any product thereof to produce a denatured single-stranded labeled-molecule or any product thereof.

97. The method of any of claims 95-96, wherein conducting the nested PCR reaction further comprises annealing a second target specific primer to the denatured single-stranded labeled-molecule or any product thereof.

98. The method of any of claims 95-96, wherein conducting the nested PCR reaction further comprises annealing a universal primer to the universal primer binding site of the oligonucleotide tag.

99. The method of any of claims 75-76, further comprising conducting a sequencing reaction to determine the sequence of at least a portion of the oligonucleotide tag, at least a portion of the labeled-molecule, a product thereof, a complement thereof, a reverse complement thereof, or any combination thereof.

100. The method of any of claims 75-76, wherein detecting the labeled-molecules or any products thereof comprises an array detector, fluorescent reader, non-fluorescent detector, CR reader, or scanner.

101. The method of claim 100, wherein the fluorescent reader is a Sensovation, or a AG fluorescent reader.

102. The method of claim 100, wherein the scanner is a flatbed scanner.

103. The method of any of claims 75-76, wherein detecting the labeled-molecules or any products thereof comprises detecting the labeled-molecules hybridized to the solid support.

104. The method of any of claims 75-76, wherein the molecule is a nucleic acid molecule.

105. The method of claim 104, wherein the nucleic acid molecule is a DNA molecule.

106. The method of claim 104, wherein the nucleic acid molecule is an RNA molecule.

107. The method of any of claims 75-76, wherein the molecule is a peptide.

108. The method of claim 107, wherein the peptide is a polypeptide.

110. The method of claim 75, wherein the sample is from a single cell.

111. The method of claim 75, wherein the sample is from less than about 100 cells.

112. The method of claim 75, wherein the sample is from less than about 50 cells.

113. The method of claim 75, wherein the sample is from less than about 20 cells.

114. The method of claim 75, wherein the sample is from less than about 10 cells.

115. The method of claim 75, wherein the sample is from less than about 5 cells.

116. The method of claim 75, wherein the cell is a mammalian cell.

117. The method of claim 75, wherein the cell is a human cell.

118. The method of claim 75, wherein the cell is from a subject suffering from a disease or condition.

119. The method of claim 118, wherein the disease or condition is cancer.

120. The method of claim 118, wherein the disease or condition is a pathogenic infection.

121. The method of claim 118, wherein the disease or condition is a genetic disorder.

76. A clonal amplification method comprising:

a. stochastically labeling a plurality of molecules with a plurality of oligonucleotide tags to produce a labeled-molecule, wherein;

i. the plurality of molecules comprise at least 2 molecules of different sequences; and

ii. the plurality of oligonucleotide tags comprises at least 2 oligonucleotide tags of different sequences;

b. amplifying the labeled-molecules to produce a labeled-amplicon; and

c. detecting the labeled-amplicon.

91. The method of claim 76, wherein amplifying the labeled-molecule comprises conducting a polymerase chain reaction.

109. The method of claim 76, wherein the plurality of molecules is from a cell.

122. The method of any of claims 109-110, wherein the cell is from a healthy subject.

123. The method of any of claims 109-110, wherein the cell is a diseased cell.

124. The method of claim 123, wherein the diseased cell is a cancerous cell.

125. The method of any of claims 109-110, wherein the cell is a healthy cell.

126. The method of claim 125, wherein the cell is not a diseased or infected cell.

128. A stochastic label-based hybridization chain reaction method, comprising stochastically labeling one or more nucleic acid molecules with a plurality of hairpin oligonucleotide tags, wherein:

(a) the hairpin oligonucleotide tag comprises an overhang; and

(b) the one or more nucleic acid molecules act as initiators for a hybridization chain reaction.

129. The method of claim 128, wherein at least a portion of the hairpin oligonucleotide tag hybridizes to at least a portion of the one or more nucleic acid molecules.

130. The method of claim 128, wherein the hairpin oligonucleotide tag comprises an oligodT sequence.

131. The method of claim 128, wherein the one or more nucleic acid molecules comprise one or more adapters.

132. The method of claim 131, wherein at least a portion of the hairpin oligonucleotide tag hybridizes to at least a portion of the one or more adapters.

133. The method of claim 128, wherein at least one hairpin oligonucleotide tag of the plurality of hairpin oligonucleotide tags comprises one or more labels.

134. The method of claim 128, wherein at least one hairpin oligonucleotide tag of the plurality of hairpin oligonucleotide tags comprises two or more labels.

135. The method of claim 128, wherein each hairpin oligonucleotide tag of the plurality of hairpin oligonucleotide tags comprises one or more labels.

136. The method of claim 128, wherein each hairpin oligonucleotide tag of the plurality of hairpin oligonucleotide tags comprises two or more labels.

137. The method of claim 128, wherein the hairpin oligonucleotide tag does not comprise a label.

138. The method of claim 128, wherein the plurality of hairpin oligonucleotide tags comprises one or more hairpin oligonucleotide tags with a 5′

overhang, hairpin oligonucleotide tags with a 3′

overhang, or a combination thereof.

139. The method of claim 128, wherein the stem portion of the hairpin oligonucleotide tag is one or more nucleotides in length.

140. The method of claim 128, wherein the stem portion of the hairpin oligonucleotide tag is two or more nucleotides in length.

141. The method of claim 128, wherein the stem portion of the hairpin oligonucleotide tag is three or more nucleotides in length.

142. The method of claim 128, wherein the stem portion of the hairpin oligonucleotide tag is four or more nucleotides in length.

143. The method of claim 128, wherein the stem portion of the hairpin oligonucleotide tag is five or more nucleotides in length.

144. The method of claim 128, wherein the stem portion of the hairpin oligonucleotide tag is six or more nucleotides in length.

145. The method of claim 128, wherein the stem portion of the hairpin oligonucleotide tag is seven or more nucleotides in length.

146. The method of claim 128, wherein the stem portion of the hairpin oligonucleotide tag is eight or more nucleotides in length.

147. The method of claim 128, wherein the stem portion of the hairpin oligonucleotide tag is nine or more nucleotides in length.

148. The method of claim 128, wherein the stem portion of the hairpin oligonucleotide tag is ten or more nucleotides in length.

149. The method of claim 128, wherein the loop portion of the hairpin oligonucleotide tag is one or more nucleotides in length.

150. The method of claim 128, wherein the loop portion of the hairpin oligonucleotide tag is two or more nucleotides in length.

151. The method of claim 128, wherein the loop portion of the hairpin oligonucleotide tag is three or more nucleotides in length.

152. The method of claim 128, wherein the loop portion of the hairpin oligonucleotide tag is four or more nucleotides in length.

153. The method of claim 128, wherein the loop portion of the hairpin oligonucleotide tag is five or more nucleotides in length.

154. The method of claim 128, wherein the loop portion of the hairpin oligonucleotide tag is six or more nucleotides in length.

155. The method of claim 128, wherein the loop portion of the hairpin oligonucleotide tag is seven or more nucleotides in length.

156. The method of claim 128, wherein the loop portion of the hairpin oligonucleotide tag is eight or more nucleotides in length.

157. The method of claim 128, wherein the loop portion of the hairpin oligonucleotide tag is nine or more nucleotides in length.

158. The method of claim 128, wherein the loop portion of the hairpin oligonucleotide tag is ten or more nucleotides in length.

159. The method of claim 128, wherein the hairpin oligonucleotide tag comprises a unique identifier region.

160. The method of claim 159, wherein the unique identifier region is in the loop portion of the hairpin oligonucleotide tag.

161. The method of claim 159, wherein the unique identifier region is in the stem portion of the hairpin oligonucleotide tag.

162. The method of claim 159, wherein the unique identifier region is in the overhang portion of the hairpin oligonucleotide tag.

163. The method of claim 133, wherein the label comprises a unique identifier region.