Support bound probes and methods of analysis using the same

• US 6,403,320 B1
• Filed: 10/05/2000
• Issued: 06/11/2002
• Est. Priority Date: 06/07/1989
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Pirrung, Michael C., Fodor, Stephen P. A., Stryer, Lubert, Read, J. Leighton
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US09/684,377
• Time in Patent Office: 614 Days
• Field of Search: 435/6, 435/91.2, 435/7.1
• US Class Current: 506/9
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00434 Liquid crystal masks
• CPC Class Codes: B01J 2219/00436 Maskless processes
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/00475 Sheets
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00531 essentially square
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00621 : by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 : Covalent
• 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 2219/00648 : by the use of solid beads
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 : using computers
• CPC Class Codes: B01J 2219/00695 : Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00711 : Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B82Y 10/00 : Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07C 229/14 : to carbon atoms of carbon skeletons containing rings
• CPC Class Codes: C07C 229/16 : to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
• CPC Class Codes: C07D 263/44 : Two oxygen atoms
• CPC Class Codes: C07D 317/62 : with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
• CPC Class Codes: C07H 19/04 : Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/10 : with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: C07K 1/042 : characterised by the nature of the carrier
• CPC Class Codes: C07K 1/045 : using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 17/06 : attached to the carrier via a bridging agent
• CPC Class Codes: C07K 17/14 : Peptides being immobilised on, or in, an inorganic carrier
• CPC Class Codes: C07K 7/06 : having 5 to 11 amino acids
• CPC Class Codes: C12Q 1/6809 : Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 : characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 : for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 15/1433 : using image recognition
• 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 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6458 : Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G03F 7/00 : Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor using photoresist structures for special production processes, see the relevant places, e.g. B44C, H01L, e.g. H01L21/00, H05K
• CPC Class Codes: G03F 7/265 : Selective reaction with inorganic or organometallic reagents after image-wise exposure, e.g. silylation
• CPC Class Codes: G03F 7/38 : Treatment before imagewise removal, e.g. prebaking G03F7/265 takes precedence
• CPC Class Codes: G11C 13/0014 : comprising cells based on organic memory material
• CPC Class Codes: G11C 13/0019 : comprising bio-molecules

Abstract

The present invention provides methods and apparatus for sequencing, fingerprinting and mapping biological macromolecules, typically biological polymers. The methods make use of a plurality of sequence specific recognition reagents which can also be used for classification of biological samples, and to characterize their sources.

Claims

1. A method of analyzing a receptor bound to an array of nucleic acids, comprising:

providing an array of different nucleic acids immobilized on a substrate, the different nucleic acids occupying different localized areas on the substrate of less than 0.01 cm², at least some of the nucleic acids being bound to a fluorescently labeled receptor; and

detecting the localized areas of the array with a detector to detect light fluoresced from the labeled receptor bound to the nucleic acids in the localized areas, the detection step being one second or less, to detect which nucleic acids bind to the labeled receptor.

2. The method of claim 1, wherein the detector is a charge coupled device.

3. The method of claim 1, further comprising exposing the array to ultraviolet light.

4. The method of claim 1, wherein each location has an area of less than 10,000 square microns.

5. The method of claim 1, wherein each location has an area of less than 100 square microns.

6. The method of claim 1, wherein the array comprises at least 1000 probes.

7. The method of claim 1, wherein the combined area occupied by the at least 1000 nucleic acids is less than 1 cm².

8. The method of claim 1, wherein the array comprises at least 10⁶ nucleic acids per cm².

9. The method of claim 1, wherein the nucleic acids have lengths of 2-100 nucleotides.

10. The method of claim 1, wherein the labeled receptor is a labeled nucleic acid.

11. The method of claim 1, wherein the labeled receptor is a labeled polypeptide.

12. The method of claim 1, wherein the known locations are squares or rectangles.

13. The method of claim 1, wherein the method further comprises:

directing light from a light source at the surface of the substrate; and

storing a pattern of fluoresced light from the different localized areas in a data storage system.

14. The method of claim 1, wherein the method further comprises translating the detector relative to the substrate.

15. The method of claim 1, further comprising analyzing the stored pattern of fluoresced light to determine which nucleic acids bind to the receptor.

16. The method of claim 1, wherein the array comprises more than 10 different nucleic acids.

17. The method of claim 1, wherein the array comprises more than 100 different nucleic acids.

18. The method of claim 1, wherein the array comprises more than 10,000 different nucleic acids.

19. The method of claim 1, wherein the array comprises more than 100,000 different nucleic acids.

20. The method of claim 1, wherein the array comprises more than 1000 different nucleic acids at a density of more than 1000 nucleic acids per cm².

21. The method of claim 1, wherein the array comprises more than 10,000 different nucleic acids at a density of more than 10,000 nucleic acids per cm².

22. The method of claim 1, wherein the array comprises more than 1,000,000 different nucleic acids at a density of more than 1,000,000 nucleic acids per cm².

23. The method of claim 1, further comprising:

illuminating the array with a device comprising;

a point light source to generate an excitation light;

an objective lens to focus the point light source at the surface of the substrate, whereby the fluorescently marked localized areas emit a fluoresced light in response to the excitation light;

a dichroic mirror to reflect light having a wavelength of the excitation light and passing light having a wavelength of the fluoresced light;

a photomultiplier and photon counter to detect the fluoresced light;

moving the substrate relative to the excitation light with an x-y translation stage; and

recording the fluoresced light as a function of a position on the surface of the substrate from which the fluoresced light was emitted with an appropriately programmed computer.

24. The method of claim 1, further comprising washing the surface of the substrate to remove unbound labeled receptor.

52. A method in accordance with claim 1 further comprising collecting and analyzing data from the detection step using a computer system.

53. A method in accordance with claim 52 wherein the computer system comprises an IBM PC or AT compatible computer.

54. A method in accordance with claim 1 further comprising translating the array using an X/Y translation table.

55. A method in accordance with claim 54 wherein the X/Y translation table is controlled by a digital computer.

56. A method in accordance with claim 1 wherein the detecting step uses a photomultiplier tube.

57. A method in accordance with claim 1 wherein the detecting step uses a pre-amplifier.

58. A method in accordance with claim 1 further comprising displaying data that has been acquired using a video display.

59. A method in accordance with claim 1 further comprising detecting and displaying the output of the detection using a photomultiplier tube, a pre-amplifier, a computer, and a video display.

60. A method in accordance with claim 1 wherein the detecting step involves counting the number of photons per unit area.

61. A method in accordance with claim 1 wherein the detecting step includes acquiring data every 1 to 100 μ

m.

62. A method in accordance with claim 1 wherein the detection step includes acquiring data with a data collection diameter of about 0.8 to 10 μ

m.

63. A method in accordance with claim 1 further comprising exposing the array to laser light.

64. A method in accordance with claim 1 further comprising exposing the array to laser light having a wavelength of greater than 520 nm.

65. A method in accordance with claim 1 further comprising translating the array with an X/Y translator under the control of a computer;

detecting and displaying the output of the detection using a photomultiplier tube, a pre-amplifier, and a computer; and

collecting and analyzing data from the detection step using a computer system.

100. A method in accordance with claim 23 wherein the receptor is a nucleic acid.

113. A method in accordance with claim 1 wherein the sequences of the nucleic acids are known.

123. An apparatus in accordance with claim 1 wherein the density of the different nucleic acid sequences on the array is more than 300/cm².

124. An apparatus in accordance with claim 1 wherein the density of the different nucleic acid sequences on the array is more than 300/cm².

25. An apparatus for analyzing ligand-receptor binding, comprising:

a substrate that comprises more than 1000 different ligands of known sequence collectively occupying less than 1 cm², the different ligands occupying different localized areas within the 1 cm²; and

a detector capable of detecting light from fluorescently marked localized areas on the substrate.

26. The apparatus of claim 25, wherein the ligands are nucleic acids.

27. The apparatus of claim 25, wherein the ligands are polypeptides.

28. The apparatus of claim 25, wherein the ligands have known sequences.

82. An apparatus in accordance with claim 25 further comprising an X/Y translation table.

83. An apparatus in accordance with claim 25 wherein the X/Y translation table is controlled by a digital computer.

84. An apparatus in accordance with claim 25 further comprising a photomultiplier tube.

85. An apparatus in accordance with claim 25 further comprising a pre-amplifier.

86. An apparatus in accordance with claim 25 further comprising a video display.

87. An apparatus in accordance with claim 25 further comprising a photomultiplier tube, a pre-amplifier, a computer, and a video display.

101. An apparatus in accordance with claim 25 wherein the receptor is a nucleic acid.

103. A method in accordance with claim 25 wherein the receptor is a peptide.

29. An apparatus for analyzing ligand-receptor binding, comprising:

a substrate that comprises at least 100 different ligands, collectively occupying less than 1 cm², the different ligands occupying different localized areas; and

a detector to detect a label bound to said ligands at said localized areas.

30. The apparatus of claim 29, wherein the different ligands are nucleic acids.

31. The apparatus of claim 29, wherein the different ligands have known sequences.

32. The apparatus of claim 29, wherein the different ligands are peptides.

33. The apparatus of claim 29, wherein the marker is a fluorescent marker.

34. The apparatus of claim 29, wherein the marker is a radiolabelled marker.

88. An apparatus in accordance with claim 29 further comprising a laser.

89. An apparatus in accordance with claim 29 further comprising an X/Y translator under the control of a computer;

a photomultiplier tube, a pre-amplifier, and a computer; and

a computer system.

90. An apparatus in accordance with claim 29 further comprising a computer system.

91. An apparatus in accordance with claim 90 wherein the computer system comprises an IBM PC or AT compatible computer.

94. An apparatus in accordance with claim 29 further comprising a photomultiplier tube.

95. An apparatus in accordance with claim 29 further comprising a pre-amplifier.

96. An apparatus in accordance with claim 29 further comprising a video display.

97. An apparatus in accordance with claim 29 further comprising a photomultiplier tube, a pre-amplifier, a computer, and a video display.

98. An apparatus in accordance with claim 29 further comprising a laser.

99. An apparatus in accordance with claim 29 further comprising an X/Y translator under the control of a computer;

a photomultiplier tube, a pre-amplifier, and a computer; and

a computer system.

102. An apparatus in accordance with claim 29 wherein the receptor is a nucleic acid.

105. A method in accordance with claim 29 wherein the receptor is a peptide.

126. An apparatus in accordance with claim 29 wherein the density of the different nucleic acid sequences on the array is more than 300/cm².

35. An apparatus for detecting binding of nucleic acids;

comprising;

(a) a substrate having a surface comprising more than 10 different nucleic acids, having known sequences, at localized areas on the surface of the substrate, each of the localized areas having an area of 10<sup>−

2</sup> cm² or less, at least one of the nucleic acids being bound to a labeled receptor;

(b) an excitation light source;

(c) a detector capable of receiving a signal from fluorescently marked localized areas on the substrate;

(d) a translator to move the substrate relative to the detector; and

(e) a data collection system adapted to receive input from the detector.

36. The apparatus of claim 35, wherein the nucleic acids have known sequences.

48. The apparatus of claim 35, wherein the ten different nucleic acids have lengths up to 100 nucleotides.

118. An apparatus in accordance with claim 35 wherein the density of the different nucleic acid sequences on the array is more than 1000/cm².

125. An apparatus in accordance with claim 35 wherein the density of the different nucleic acid sequences on the array is more than 300/cm².

37. A method of analyzing a receptor bound to an array of polymers, comprising:

providing an array of different polymers immobilized on a substrate, the different polymers occupying different localized areas on the substrate having an area of less than 0.01 cm², at least some of the nucleic acids being bound to a labeled receptor; and

detecting the localized areas of the array with a detector, each localized area being scanned for one second or less, to detect which polymers bind to the labeled receptor.

38. The method of claim 37, wherein the different polymers are different nucleic acids.

39. The method of claim 37, wherein the receptor is a nucleic acid.

40. The method of claim 37, wherein the different polymers are peptides.

66. A method in accordance with claim 37 further comprising collecting and analyzing data from the detection step using a computer system.

67. A method in accordance with claim 66 wherein the computer system comprises an IBM PC or AT compatible computer.

68. A method in accordance with claim 37 further comprising translating the array using an X/Y translation table.

69. A method in accordance with claim 37 wherein the X/Y translation table is controlled by a digital computer.

70. A method in accordance with claim 37 wherein the detecting step uses a photomultiplier tube.

71. A method in accordance with claim 37 wherein the detecting step uses a pre-amplifier.

72. A method in accordance with claim 37 further comprising displaying data that has been acquired using a video display.

73. A method in accordance with claim 37 further comprising detecting and displaying the output of the detection using a photomultiplier tube, a pre-amplifier, a computer, and a video display.

74. A method in accordance with claim 37 wherein the detecting step involves counting the number of photons per unit area.

75. A method in accordance with claim 37 wherein the detecting step includes acquiring data every 1 to 100 μ

m.

76. A method in accordance with claim 37 wherein the detection step includes acquiring data with a data collection diameter of about 0.8 to 10 μ

m.

77. A method in accordance with claim 37 further comprising exposing the array to laser light.

78. A method in accordance with claim 37 further comprising exposing the array to laser light having a wavelength of greater than 520 nm.

79. A method in accordance with claim 37 further comprising translating the array with an X/Y translator under the control of a computer;

detecting and displaying the output of the detection using a photomultiplier tube, a pre-amplifier, and a computer; and

collecting and analyzing data from the detection step using a computer system.

80. An apparatus in accordance with claim 37 further comprising a computer system.

81. An apparatus in accordance with claim 80 wherein the computer system comprises an IBM PC or AT compatible computer.

92. An apparatus in accordance with claim 37 further comprising an X/Y translation table.

93. An apparatus in accordance with claim 92 wherein the X/Y translation table is controlled by a digital computer.

104. A method in accordance with claim 37 wherein the receptor is a peptide.

119. An apparatus in accordance with claim 37 wherein the density of the different nucleic acid sequences on the array is more than 1000/cm².

41. A method for screening comprising:

exposing a substrate having a ligand in a localized area to a marked receptor; and

detecting binding between the ligand and the marked receptor by scanning the substrate for 1 second or less in the localized area.

42. The method of claim 41, wherein the ligand is a nucleic acid.

43. The method of claim 41, wherein the marked receptor is a marked nucleic acid.

44. The method of claim 41, wherein the ligand is a polypeptide.

45. The method of claim 41, wherein the receptor is marked with a fluorescent label.

46. The method of claim 41, wherein the substrate has more than 1000 different ligands in 1000 different localized areas within an area less than 1 cm².

47. The method of claim 41, wherein the receptor is marked with a fluorescent marker.

114. An apparatus in accordance with claim 41 wherein the sequence of the ligand is known.

120. An apparatus in accordance with claim 41 wherein the density of the different nucleic acid sequences on the array is more than 1000/cm².

49. An apparatus for detection of fluorescent marked locations on a substrate, wherein the substrate comprises a plurality of different polymer sequences coupled to a surface of the substrate and wherein the plurality of different polymer sequences comprises a plurality of different nucleic acid sequences, each of the different polymer sequences being coupled in a different localized area of the surface at a density of more than 1000/cm², each of the localized areas having an area of 10⁻

3 cm² or less, the apparatus comprising;

(a) a light source constructed and arranged to direct light at a plurality of the localized areas;

(b) a detector constructed and arranged to detect light fluoresced from the plurality of the localized areas in response to the light source;

(c) a translator constructed and arranged to translate the substrate relative to the light source whereby the plurality of the localized areas are exposed to the light; and

(d) a data storage system constructed and arranged to store fluoresced light intensity as a function of location on the surface, the data storage system coupled to the translator and the detector.

115. An apparatus in accordance with claim 49 wherein the sequences of the nucleic acids are known.

50. An apparatus for detection of fluorescent markers at a plurality of localized areas on a substrate, comprising:

(a) a light source constructed and arranged to direct light at the plurality of localized areas;

(b) a detector constructed and arranged to detect light fluoresced from the plurality of localized areas in response to the light source;

(c) a translator constructed and arranged to translate the substrate relative to the light source whereby the plurality of localized areas are exposed to the light; and

(d) a data storage system constructed and arranged to store fluoresced light intensity as a function of location on the surface, the data storage system coupled to the translator and the detector;

wherein the substrate comprises a plurality of different polymer sequences coupled to a surface of the substrate and wherein the plurality of different polymer sequences comprises a plurality of different nucleic acid sequences, each of the different polymer sequences being coupled in a different of the localized areas, each localized area having an area of 10<sup>−

2</sup> cm² or less.

116. An apparatus in accordance with claim 50 wherein the sequences of the nucleic acids are known.

121. An apparatus in accordance with claim 50 wherein the density of the different nucleic acid sequences on the array is more than 1000/cm².

51. An apparatus for detection of fluorescent markers at a plurality of localized areas on a substrate, comprising:

(a) a light source constructed and arranged to direct light at the plurality of localized areas, wherein the substrate comprises a plurality of different polymer sequences coupled to a surface of the substrate and wherein the plurality of different polymer sequences comprises a plurality of different nucleic acid sequences, each of the different polymer sequences being coupled in a different of the localized areas, each of the localized areas having an area of 10<sup>−

2</sup> cm² or less;

(b) a detector constructed and arranged to detect light fluoresced from the plurality of localized areas in response to the light source;

(c) a translator constructed and arranged to translate the substrate relative to the light source whereby the plurality of localized areas are exposed to the light; and

(d) a data storage system constructed and arranged to store fluoresced light intensity as a function of location on the surface, the data storage system coupled to the translator and the detector.

117. An apparatus in accordance with claim 51 wherein the sequences of the nucleic acids are known.

122. An apparatus in accordance with claim 51 wherein the density of the different nucleic acid sequences on the array is more than 1000/cm².

106. A method for detecting binding between a nucleic acid probe bound to a substrate and a nucleic acid target comprising:

providing an array of positionally distinguishable nucleic acids of known sequence bound to a substrate having a density of more than 300 different nucleic acids/cm²; and

detecting a double stranded nucleic acid.

107. A method in accordance with claim 106 wherein the density of the array is 1,000 different nucleic acids/cm².

108. A method in accordance with claim 107 wherein the density of the array is more than 10,000 different nucleic acids/cm².

109. A method of analyzing using an array of nucleic acids, comprising:

providing an array of different nucleic acids of known sequence immobilized on a substrate at a density more than 1,000 nucleic acids/cm², the different nucleic acids being positionally distinguishable and, at least some of the nucleic acids being bound to a fluorescently labeled receptor;

illuminating an area of the array greater than 10<sup>−

3</sup> cm² with an energy source; and

detecting fluoresced light from the known locations of the array with a detector, the detection step being one second or less, to detect which nucleic acids bind to the labeled receptor.

110. A method in accordance with claim 109 wherein the receptor is a nucleic acid.

111. A method in accordance with claim 109 wherein the receptor is a peptide.

112. A method in accordance with claim 109 wherein the density is more than 10,000 nucleic acids/cm².

127. A method of analyzing using an array of polymers, comprising:

providing an array of different polymers of known sequence immobilized on a substrate at a density more than 1,000 polymers/cm², the different polymers being positionally distinguishable and at least some of the polymers being bound to a fluorescently labeled target polymers;

illuminating an area of the array greater than 10<sup>−

3</sup> cm² with an energy source; and

detecting fluoresced light from the known locations of the array with a detector, the detection step being one second or less, to detect which polymers on the array bind to the labeled target polymers.

128. A method in accordance with claim 127 wherein the polymers are nucleic acids.

129. A method in accordance with claim 127 wherein the polymers are peptides.

130. A method in accordance with claim 128 the density of the different nucleic acids is more than 10,000/cm².

131. A method in accordance with claim 129 wherein the density of the different peptides is more than 10,000/cm².