Methods and products for analyzing polymers

• US 6,355,420 B1
• Filed: 08/13/1998
• Issued: 03/12/2002
• Est. Priority Date: 02/12/1997
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: U.S. Genomics, Inc (Toxic Reports LLC)
• Inventors: Chan, Eugene Y.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Taylor, Janell E.
• Application Number:
  US09/134,411
• Time in Patent Office: 1,307 Days
• Field of Search: 435/6, 435/94, 435/149, 435/287.1, 435/287.2, 435/970, 435/973, 250/340, 250/341
• US Class Current: 435/6.12
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• 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: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 2021/0346 Capillary cells; Microcells
• CPC Class Codes: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2021/6482 Sample cells, cuvettes
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte

Abstract

Methods and products for analyzing polymers are provided. The methods include methods for determining various other structural properties of the polymers.

Claims

1. A method for analyzing a polymer of linked units comprising

moving a plurality of individual units of a polymer of linked units through a channel and exposing the plurality of individual units to an agent selected from the group consisting of electromagnetic radiation, a quenching source and a fluorescence excitation source as the units move past the agent, individual units interacting with the agent to produce a detectable signal within the channel or at the edge of the channel, and

detecting sequentially the signals resulting from said interaction to analyze the polymer.

2. The method of claim 1, wherein the signal is electromagnetic radiation.

3. The method of claim 2, wherein the agent is electromagnetic radiation.

4. The method of claim 3, wherein a portion of the plurality of individual units of the polymer is labeled with a fluorophore.

5. The method of claim 3, wherein the plurality of individual units of the polymer are sequentially exposed to electromagnetic radiation by bringing the plurality of individual units in proximity to a light emissive compound and exposing the light emissive compound to electromagnetic radiation, and wherein the plurality of individual units of the polymer detectably affect emission of electromagnetic radiation from the light emissive compound.

6. The method of claim 3 wherein the plurality of individual units of the polymer are sequentially exposed to electromagnetic radiation, and wherein the electromagnetic radiation detectably affects emission of electromagnetic radiation from the plurality of individual units of the polymer to produce the detectable signal.

7. The method of claim 5, wherein the individual units detectably affecting emission of electromagnetic radiation from the light emissive compound are labeled with a fluorophore.

8. The method of claim 5, wherein light emissive compound is attached to a solid material.

9. The method of claim 3 wherein the plurality of individual units of the polymer are sequentially exposed to the agent by moving the polymer through a nanochannel in a wall material and exposing the plurality of individual units of the polymer to the agent at an interaction station at the nanochannel.

10. The method of claim 9, wherein the polymer is moved through a nanochannel in a wall material that has a light-emissive compound embedded in the wall material, adjacent the nanochannel, whereby the plurality of individual units interact with the light emissive compound as the polymer moves through the nanochannel.

11. The method of claim 9, wherein said wall material comprises a plurality of nanochannels, an interaction station at the nanochannel, and further comprising moving a plurality of polymers through said nanochannel, only one polymer per nanochannel at any given time, and detecting simultaneously the signals resulting from the interaction of individual units of the polymers and the agent at said interaction station.

12. The method of claim 1 further comprising storing in a database said signals which are detected.

13. The method of claim 2 further comprising comparing the signals detected to a pattern of signals from another polymer to determine the relatedness of the two polymers.

14. The method of claim 2 further comprising comparing the detected signals to a known pattern of signals characteristic of a known polymer to determine the relatedness of the polymer being analyzed to the known polymer.

15. The method of claim 2, wherein the plurality of individual units are two units, a first unit at a first end of the polymer and a second unit at an opposite second end of the polymer.

16. The method of claim 15 further comprising measuring the length of time elapsed between detection of a first signal from the first unit and a second signal from the second unit.

17. The method of claim 2 further comprising detecting the time elapsed between the sequential detection of the signals.

18. The method of claim 2, wherein a first of said individual units interacts with the agent to produce a first detectable signal and a second of said individual units interacts with the agent to produce a second detectable signal different from the first detectable signal.

19. The method of claim 1, wherein the polymer is a nucleic acid.

20. The method of claim 1, wherein only a portion of the units of the polymer are labeled.

21. The method of claim 1, wherein only a portion of the units of the polymer are labeled and wherein the labeling is random.

22. The methods of claim 1, wherein at least two units of the polymer are labeled differently so as to produce two different detectable signals.

23. The method of claim 1, wherein the plurality of individual units of the polymer are exposed to at least two agents, wherein the interaction between the units of the polymer and the at least two agents produces at least two signals.

24. The method of claim 23, wherein the at least two agents are positioned in distinct regions of a channel through which the polymer passes.

25. The method of claim 23, wherein the at least two signals are different signals.

26. The method of claim 23, wherein the at least two signals are the same signals.

27. The method of claim 9, wherein the nanochannel is fixed in the wall.

28. The method of claim 1, wherein the unit is exposed to the agent at a station and wherein the station is a non-ligand material.

81. The method of claim 1, wherein the polymer is double stranded DNA.

82. The method of claim 1, wherein the polymer is labeled with a unit-specific marker.

29. A method for analyzing a polymer of linked units comprising:

moving a plurality of individual units of a polymer of linked units through a channel and past a station; and

,

detecting sequentially signals arising from a detectable physical change in the polymer or the station as individual units pass the station within the channel or at the edge of the channel to analyze the polymer.

30. The method of claim 29, wherein the station is an interaction station and wherein individual units are exposed at the interaction station to an agent that interacts with the individual unit to produce a detectable electromagnetic radiation signal characteristic of said interaction.

31. The method of claim 29, wherein the station is a signal generation station and the characteristic signal produced is a polymer dependent impulse.

120. The method of claim 29, wherein the polymer is labeled with a unit specific marker.

123. The apparatus of claim 29, wherein the polymer is labeled with a unit specific marker.

32. A method for analyzing a polymer of linked units, comprising:

exposing a plurality of individual units of a polymer to a station to produce a non-ion conductance signal resulting from the exposure of the units of the polymer to the station, and wherein the station is attached to a wall material having a surface defining a channel, and wherein the polymer is not fixed to the surface.

84. The method of claim 32, wherein the wall material is a solid wall material.

85. The method of claim 32, wherein the station is an interaction station.

86. The method of claim 32, wherein only a portion of the individual units of the polymer are labeled.

87. The method of claim 86, wherein the units are labeled with a unit-specific marker.

121. The method of claim 32, wherein the polymer is labeled with a unit specific marker.

33. An apparatus for analyzing a polymer of linked units by detecting a signal comprising

an interaction station positioned within a channel constructed and arranged to receive a polymer of linked units sequentially passing through said station, selected units of said polymer being labeled with a light sensitive label;

a source of electromagnetic radiation constructed and arranged to irradiate, at said interaction station, said labeled units;

a sensor constructed and arranged to detect sequentially electromagnetic radiation providing characteristic signals resulting from interaction with said label or said unit; and

a processor constructed and arranged to analyze said polymer based on said sequentially detect radiation.

34. The apparatus of claim 33 wherein said interaction station includes an optical waveguide.

35. The apparatus of claim 33 wherein said channel is a nanochannel and wherein the nanochannel is between 1 and 500 Angstroms.

36. The apparatus of claim 33 wherein said interaction station includes electrodes establishing electric field for passing said units of said polymer.

37. The apparatus of claim 33 further includes a memory for collecting and storing signals from said sensor corresponding to said characteristic signals.

38. The apparatus of claim 37 wherein said processor is further arranged to access records stored in said memory for a selected one of the plurality of the polymers according to a unique identifier associated with the selected polymer.

39. The apparatus of claim 33 further includes a memory for storing, for each of the plurality of the polymers and in a manner accessible using a unique identifier for the polymer, records including information indicative of said sequentially detected radiation;

and said processor being arranged to access the records stored in the memory for a selected one of the plurality of the polymers according to a unique identifier associated with the selected polymer.

40. The apparatus of claim 33 wherein said processor is further arranged to compare said sequentially detected signals of said analyzed polymer to a known pattern of signals characteristic of a known polymer to determine relatedness of said analyzed polymer to said known polymer.

41. The apparatus of claim 33 wherein said light sensitive label include fluorophore, wherein said source is constructed to emit an excitation wavelength of said fluorophores and said sensor is arranged to sequentially detect fluorescent radiation emitted from said fluorophore.

42. The apparatus of claim 41 wherein said interaction station includes an optical waveguide constructed to deliver said excitation wavelength to a nanochannel constructed and arranged to pass said polymer.

43. The apparatus of claim 42 wherein said nanochannel is between 1 and 500 Angstroms.

44. The apparatus of claim 41 wherein said processor compares said sequentially detected signals of said analyzed polymer to a known pattern of signals characteristic of a known polymer to determine relatedness of said analyzed polymer to said known polymer.

45. The apparatus of claim 33 wherein said label includes at least three distinct fluorophores, wherein said source is constructed to emit excitation wavelengths of said fluorophores and said sensor is constructed to detect sequentially fluorescent radiation emitted from said fluorophores.

46. The apparatus of claim 33 wherein the plurality of individual units of said polymer are labeled by at least two labels, wherein the interaction between the units of the polymer and said at least two labels produces at least two said characteristic signals.

47. The apparatus of claim 46 wherein said processor determines the distance between two unit specific labels of said polymer, the identity of each unit specific label being indicative of the identity of at least one unit of said polymer and determines the distance between said characteristic signals as an indication of the distance between the two unit specific labels.

48. The apparatus of claim 33 wherein said processor sequences said polymer by obtaining polymer dependent signals from a plurality of overlapping polymers, at least a portion of each of the polymers having a sequence of linked units identical to the other of the polymers, and comparing said polymer dependent signals to obtain a sequence of linked units which is identical in the plurality of polymers.

80. The apparatus of claim 33 wherein said optical waveguide is used to irradiate said units passing through said nanochannel.

119. The apparatus of claim 80, wherein the channel is a microchannel.

49. A method for identifying an individual unit of a polymer comprising

transiently exposing the individual unit of the polymer to an agent selected from the group consisting of electromagnetic radiation, a quenching source and a fluorescence excitation source, the identity of the individual unit being unknown, to generate an interaction with a detectable electromagnetic radiation signal characteristic of said individual unit,

detecting said electromagnetic radiation signal, and

distinguishing said signal from signals generated from adjacent signal generating units of the polymer as an indication of the identity of the individual unit.

88. The method of claim 49, wherein the agent is one or more fluorophores and the individual unit is transiently exposed by positioning the individual unit within energy transfer proximity of the agent, and wherein said signal is detected by detecting fluorescence energy transfer between the agent and the individual unit.

89. The method of claim 88, wherein the agent is at least three fluorophores.

90. The method of claims 49, wherein the individual unit of the polymer is exposed to the agent by positioning the individual unit at an interaction station comprising a nanochannel in a wall material.

91. The method of claim 90, wherein the wall material comprises two layers, one of the layers being conductive and the other being nonconductive and wherein the nanochannel traverses both layers.

117. The method of claim 49, wherein the channel is a microchannel.

50. A method for determining the order of two individual units of a polymer of linked units comprising:

involving the polymer of linked units linearly with respect to a station,

exposing one of the individual units to the station to produce a signal arising from a detectable physical change in the unit or the station,

exposing the other of the individual units to the station to produce a second detectable signal arising from a detectable physical change in the unit or the station, different from the first signal, wherein at least one of the two individual units is an internal unit and wherein at least one of the two individual units has an extrinsic label; and

determining the order of the signals as an indication of the order of the two individual units.

92. The method of claim 50, wherein the station is an interaction station and wherein one of the individual units is exposed at the interaction station to an agent that interacts with the one individual unit to produce a first detectable electromagnetic radiation signal, and wherein the other of the individual units is exposed at the interaction station to an agent that interacts with the other of the individual units to produce a second detectable electromagnetic radiation signal.

93. The method of claim 92, wherein the agent is selected from the group consisting of electromagnetic radiation, a quenching source and a fluorescence excitation source.

94. The method of claim 50, wherein the station is a signal generation station and the signal produced is a polymer dependent impulse.

95. The method of claim 50, wherein the polymer is a nucleic acid.

96. The method of claim 50, wherein the individual units of the polymer are labeled with a fluorophore.

97. The method of claims 50, wherein the two individual units are randomly labeled individual units of the polymer.

98. The method of claims 92, wherein the interaction station comprises a nanochannel in a wall material.

118. The method of claim 50, wherein the channel is a microchannel.

51. A method for analyzing a polymer of linked units comprising:

(1) providing a polymer of linked units, labeled with a unit specific marker

(2) detecting sequentially signals from unit specific markers of less than all of the linked units that the unit specific markers are capable of identifying, and

(3) storing a signature of said signals detected to analyze the polymer.

99. The method of claim 51, wherein the signals are detected linearly.

100. The method of claim 51, wherein the signature of signals is at least 10 signals.

101. The method of claim 51, wherein the signature of signals defines the order of unit specific markers.

102. The method of claim 51, wherein the signature of signals defines the distance between unit specific markers.

103. The method of claim 51, wherein the signature of signals defines the number of unit specific markers.

104. The method of claim 51, wherein all of the unit specific markers are detected.

105. The method of claim 51, wherein only a portion of the unit specific markers are detected.

106. The method of claim 51, wherein the polymer is partially and randomly labeled with a unit specific marker.

107. The method of claim 105, wherein all of the units of the polymer are labeled with a unit specific marker.

108. The method of claim 51, wherein the labeled polymer of linked units is exposed to an agent selected from the group consisting of electromagnetic radiation, a quenching source and a fluorescence excitation source and wherein the signals are produced by the interaction between a unit specific marker of the polymer and the agent.

109. The method of claim 51, wherein the labeled polymer of linked units is moved with respect to a station and wherein the signals are generated upon exposure of a unit specific marker of the polymer to the station.

110. The method of claim 51, wherein the method is a method for identifying a unit specific marker of the polymer, the identity of the unit specific marker being indicative of the identity of at least one unit of the polymer, and wherein the unit specific marker is transiently exposed to a station to produce signals characteristic of said unit specific marker, and further comprising the step of distinguishing said signal from signals generated from adjacent signal generating unit specific markers of the polymer as an indication of the identity of the unit specific marker.

111. The method of claim 110, wherein the station is an agent selected from the group consisting of electromagnetic radiation, a quenching source and a fluorescence excitation source and wherein the signals are detectable electromagnetic radiation signals.

112. The method of claim 51, wherein the method is a method for determining the proximity of two unit specific markers of the polymer wherein the proximity of the two unit specific markers is the signature of said signals, the identity of each unit specific marker being indicative of the identity of at least one unit of the polymer, wherein the labeled polymer is moved relative to a station to expose the two unit specific markers to the station to produce a characteristic signal arising from a detectable physical change in the unit specific marker or the station, and further comprising the step of measuring the amount of time elapsed between detecting each characteristic signal, the amount of time elapsed being indicative of the proximity of the two unit specific markers.

113. The method of claim 51, wherein the method is a method for determining the order of two sequence unit markers of the polymer, the identity of each unit specific marker being indicative of the identity of at least one unit of the polymer wherein the order of the two unit specific markers is the signature of said signals, wherein the labeled polymer is moved linearly with respect to a station, to expose one of the unit specific markers to the station to produce a signal which is a signature of signals, and to expose the other of the unit specific markers to the station to produce a second detectable which is a signature of said signals, different from the first signal, and further comprising the step of determining the order of the signals as an indication of the order of the two unit specific markers.

114. The method of claim 51, wherein the method is a method for determining the distance between two unit specific markers of the polymer, the identity of each unit specific marker being indicative of the identity of at least one unit of the polymer wherein the distance between two unit specific markers is the signature of said signals, wherein the labeled polymer is moved linearly relative to a station to produce a characteristic signal generated as each of the two unit specific markers passes by the station, and further comprising the step of determining the distance between the signals as an indication of the distance between the two unit specific markers.

52. A method for analyzing a polymer of linked units comprising:

labeling selected units of said polymer with light sensitive labels and passing said polymer through a channel;

irradiating said labeled units of said straightened polymer with electromagnetic radiation of a selected wavelength,

detecting sequentially electromagnetic radiation providing characteristic signals resulting from interaction with said labels or said units, and

analyzing said polymer based on said detected radiation.

53. The method of claim 52 wherein said irradiating is performed while said polymer passes through said nanochannel.

54. The method of claim 52 wherein said irradiating includes employing an optical waveguide.

55. The method of claim 52 wherein said passing includes passing said polymer through said nanochannel being between 1 and 500 Angstroms.

56. The method of claim 52 wherein said passing includes causing the polymer to pass nearly linearly through said nanochannel.

57. The method of claim 52 wherein said passing includes orienting said polymer in an electric field.

58. The method of claim 52 wherein said analyzing includes measuring the amount of time elapsed between detecting said characteristic signals, said amount of time elapsed being indicative of the proximity of two said labeled units.

59. The method of claim 52 wherein said light sensitive label includes fluorophore and said sequentially detected electromagnetic radiation includes fluorescent radiation emitted from said fluorophore.

60. The method of claim 59 wherein the polymer is a nucleic acid.

61. The method of claim 60 further including passing said units of said nucleic acid through a nanochannel.

62. The method of claim 61 wherein said passing includes passing said units of said nucleic acid through said nanochannel being between 1 and 500 Angstroms.

63. The method of claim 61 wherein said irradiating is performed while said units pass through said nanochannel.

64. The method of claim 59 wherein said irradiating includes employing an optical waveguide.

65. The method of claim 61 wherein said passing includes causing said units of said nucleic acid to pass nearly linearly through said nanochannel.

66. The method of claim 61 wherein said passing includes orienting said nucleic acid in an electric field.

67. The method of claim 60 wherein said analyzing includes measuring the amount of time elapsed between detecting two sequential characteristic signals, said amount of time elapsed being indicative of the proximity of two consecutive labeled units.

68. The method of claim 60 wherein said analyzing includes comparing said sequentially detected signals of said analyzed nucleic acid to a known pattern of signals characteristic of a known nucleic acid to determine relatedness of said analyzed nucleic acid to said known nucleic acid.

69. The method of claim 60 wherein only a portion of said units of said nucleic acid is labeled.

70. The method of claim 60 wherein said label includes at least three distinct fluorophores.

71. The method of claim 60 further comprising detecting the time elapsed between the sequential detection of said characteristic signals.

72. The method of claim 52 or 59 wherein the plurality of individual units of said polymer are labeled by at least two distinct labels attached to at least two distinct units, wherein the interaction between the units of the polymer and said at least two labels produces at least two said characteristic signals.

73. The method of claim 72 wherein a first of said labels interacts with said radiation to produce a first characteristic signal and a second of said labels interacts with said radiation to produce a second characteristic signal different from the first characteristic signal.

74. The method of claim 52 or 59 wherein said analyzing includes determining the distance between two unit specific labels of said polymer, the identity of each unit specific label being indicative of the identity of at least one unit of said polymer wherein the distance between two unit specific labels is the signature of said signals, wherein the labeled polymer is moved linearly relative to an interaction station to produce said characteristic signal generated as each of the two unit specific labels said irradiation at said station, and further comprising the step of determining the distance between the signals as an indication of the distance between the two unit specific labels.

75. The method of claim 52 or 59 wherein said analyzing includes sequencing said polymer by obtaining polymer dependent signals from a plurality of overlapping polymers, at least a portion of each of the polymers having a sequence of linked units identical to the other of the polymers, and comparing said polymer dependent signals to obtain a sequence of linked units which is identical in the plurality of polymers.

76. The method of claim 52 or 59 wherein said polymer is a nucleic acid and said labeling comprising

contacting a dividing cell with a nucleotide analog,

isolating from the cell nucleic acids that have incorporated the nucleotide analog, and

modifying the nucleic acid with incorporated nucleotide analog by labeling the incorporated nucleotide analog.

77. The method of claim 76 wherein said nucleotide analog is a brominated analog.

78. The method of claim 77 wherein said dividing cell is contacted with a nucleotide analog by growth arresting the cell in the cell division cycle, performing the contacting step, and allowing the cell to reenter the cell division cycle.

79. The method of claim 75 wherein the nucleic acids are isolated after said cells have reentered and completed said cell division cycle and before a second cell division cycle is completed.

115. The method of claim 52, wherein the channel is a nanochannel.

116. The method of claim 52, wherein the channel is a microchannel.

122. The method of claim 52, wherein the light sensitive labels are on a unit specific marker and wherein the polymer is labeled with the unit specific marker.

83. A method for analyzing a polymer of linked units comprising:

moving a plurality of individual units of a polymer of linked units with respect to a station; and

,

detecting sequentially signals arising from a detectable physical change in the polymer or the station as individual units pass the station to analyze a polymer, and wherein the signal is not an electromagnetic radiation signal.