SYSTEM AND METHODS FOR NUCLEIC ACID SEQUENCING OF SINGLE MOLECULES BY POLYMERASE SYNTHESIS

• US 20100093068A1
• Filed: 10/24/2007
• Published: 04/15/2010
• Est. Priority Date: 12/14/1998
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience Labs (L'Oreal SA)
• Inventors: Williams, John G.K.
• Granted Patent: US 8,980,584 B2
• Time in Patent Office: Days
• US Class Current: 435/287.2
• CPC Class Codes: C07D 209/12 Radicals substituted by oxygen atoms
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 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: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention relates to improved methods for sequencing and genotyping nucleic acid in a single molecule configuration. The method involves single molecule detection of fluorescent labeled PPi moieties released from NTPs as a polymerase extension product is created.

Claims

1-52. -52. (canceled)

53. A flow chamber for sequencing a target nucleic acid molecule, said flow chamber comprising:

(a) a support; and

(b) an array of immobilized molecules, said immobilized molecule selected from the group consisting of a nucleic acid polymerizing enzyme and a target nucleic acid;

wherein said flow cell is configured to permit labeled nucleotide analogs that are not positioned on said support to move through said flow chamber; and

(c) each member of said array is so spatially resolved to be detectable as an individual molecule.

54. The flow chamber of claim 53, wherein said immobilized molecule is a nucleic acid polymerizing enzyme.

55. The flow chamber of claim 53, wherein said immobilized molecule is a target nucleic acid.

56. The flow chamber of claim 53, wherein said flow chamber is made of plastic or glass.

57. The flow chamber of claim 53, further comprising a detector capable of detecting a single molecule.

58. The flow chamber of claim 57, wherein said detector is selected from the group consisting of an optical reader, a high-efficiency photon detection system, a photodiode, a camera, a charge couple device, an intensified charge coupled device, a near-field scanning microscope, a far-field confocal microscope, a microscope that detects wide-field epi-illumination, and a total internal reflection fluorescence microscope.

59. The flow chamber of claim 53, wherein each member of said array is present as an individual molecule each located in a defined location.

60. The flow chamber of claim 53, wherein each member of said array is different.

61. The flow chamber of claim 53, wherein said array of molecules is addressable.

62. The flow chamber of claim 57, wherein said labeled nucleotide analogs are detected.

63. The flow chamber of claim 57, wherein said detecting comprises acquiring a sequence of images.

64. The flow chamber of claim 63, wherein said sequence of images is analyzed to track single molecules.

65. The flow chamber of claim 57, wherein said detecting is carried out by a mechanism that provides low background and enables the detection of single molecules.

66. The flow chamber of claim 57, wherein said detecting is carried out by an evanescent optical field.

67. The flow chamber of claim 57, wherein said detecting is carried out by a mechanism selected from the group consisting of fluorescence resonance energy transfer, an electron transfer mechanism, an excited-state lifetime mechanism and a ground-state complex quenching mechanism.

68. A flow chamber, said flow chamber comprising a plurality of channels, wherein each of said plurality of channels comprises:

(a) a support; and

(b) a single immobilized molecule, said immobilized molecule selected from the group consisting of a nucleic acid polymerizing enzyme and a target nucleic acid;

wherein said flow cell is configured to permit labeled nucleotide analogs that are not positioned on said support to move through said flow chamber; and

(c) each member of said array is so spatially resolved to be detectable as an individual molecule.

69. The flow chamber of claim 68, wherein said immobilized molecule is a nucleic acid polymerizing enzyme.

70. The flow chamber of claim 68, wherein said immobilized molecule is a target nucleic acid.

71. The flow chamber of claim 68, wherein said flow chamber is made of plastic or glass.

72. The flow chamber of claim 68, further comprising a detector capable of detecting a single molecule.

73. The flow chamber of claim 68, wherein said detector is selected from the group consisting of an optical reader, a high-efficiency photon detection system, a photodiode, a camera, a charge couple device, an intensified charge coupled device, a near-field scanning microscope, a far-field confocal microscope, a microscope that detects wide-field epi-illumination, and a total internal reflection fluorescence microscope.

74. The flow chamber of claim 68, wherein each member of said array is present as an individual molecule each located in a defined location.

75. The flow chamber of claim 68, wherein each member of said array is different.

76. The flow chamber of claim 68, wherein said array of molecules is addressable.

77. The flow chamber of claim 72, wherein said labeled nucleotide analogs are detected.

78. The flow chamber of claim 72, wherein said detecting comprises acquiring a sequence of images.

79. The flow chamber of claim 78, wherein the sequence of images is analyzed to track single molecules.

80. The flow chamber of claim 72, wherein said detecting is carried out by a mechanism that provides low background and enables the detection of single molecules.

81. The flow chamber of claim 72, wherein said detecting is carried out by an evanescent optical field.

82. The flow chamber of claim 72, wherein said detecting is carried out by a mechanism selected from the group consisting of fluorescence resonance energy transfer, an electron transfer mechanism, an excited-state lifetime mechanism and a ground-state complex quenching mechanism.