Flowcell system for single molecule detection

• US 20080206764A1
• Filed: 10/26/2007
• Published: 08/28/2008
• Est. Priority Date: 06/07/2000
• Status: Abandoned Application

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience Labs (L'Oreal SA)
• Inventors: Reynolds, Bambi L., Draney, Dan, Bashford, Gregory R., Williams, John G.K., Chen, Jiyan, Narayanan, Nara, Sheaff, Pamela
• Application Number:
  US11/925,724
• Publication Number:
  US 20080206764A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2533/101 Primer extension
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

The present invention provides compounds, methods and systems for sequencing nucleic acid using single molecule detection. Using labeled NPs that exhibit charge-switching behavior, single-molecule DNA sequencing in a microchannel sorting system is realized. In operation, sequencing products are detected enabling real-time sequencing as successive detectable moieties flow through a detection channel. By electrically sorting charged molecules, the cleaved product molecules are detected in isolation without interference from unincorporated NPs and without illuminating the polymerase-DNA complex.

Claims

1-50. -50. (canceled)

51. A flowcell system for detecting a single molecule, said flowcell system comprising:

a substrate having a sample microchannel disposed therein;

a total internal reflection fluorescence microscope to view said sample microchannel; and

a detector for detecting said single molecule.

52. The flowcell system of claim 51, wherein a sample comprising single molecules flow in said sample microchannel.

53. The flowcell system of claim 52, wherein said sample comprises labeled NP probes for sequencing or genotyping a nucleic acid.

54. The flowcell system of claim 51, wherein said sample microchannel comprises a surface.

55. The flowcell system of claim 54, wherein said surface is a member selected from the group consisting of a support, a glass plate or slide, a well, a raised region, a dimple, a trench, a rod and an inner or an outer wall.

56. The flowcell system of claim 54, wherein a reactant is immobilized on said surface.

57. The flowcell system of claim 56, wherein said reactant is a template nucleic acid.

58. The flowcell system of claim 57, wherein said template nucleic acid further comprises a primer.

59. The flowcell system of claim 56, wherein said reactant is a DNA polymerase.

60. The flowcell system of claim 51, wherein said system further comprises up to four excitation lasers.

61. The flowcell system of claim 51, wherein said detector is selected from the group consisting of a microscope, a photon detector, a photodiode, a CCD, an ICCD, an optical fiber and a video chip.

62. The flowcell system of claim 61, wherein said detector is a CCD.

63. The flowcell system of claim 51, further comprising a data acquisition system with an image analysis algorithm.

64. The flowcell system of claim 51, wherein said flowcell system has a first energy field which is a member selected from the group consisting of an electric field, a thermal field, a magnetic field, an electromagnetic field, a photoelectric field, a light field, a mechanical field, a pressure field or combinations thereof.

65. The flowcell system of claim 51, wherein said flowcell system has a second energy field, which is a member selected from the group consisting of an electric field, a thermal field, a magnetic field, an electromagnetic field, a photoelectric field, a light field, a mechanical field, a pressure field or combinations thereof.

66. The flowcell system of claim 64, wherein said first energy field is a pressure field.

67. The flowcell system of claim 65, wherein said second energy field is a light field.

68. The flowcell system of claim 51, wherein said sample microchannel couples to a detector channel.

69. The flowcell system of claim 51, wherein said sample microchannel is about 0.5 μ

m to about 100 μ

m in depth.

70. The flowcell system of claim 51, wherein said flowcell is an array of flowcells.

71. The flowcell system of claim 51, wherein the cross-sectional geometry of said microchannel is selected from the group consisting of rectangular, oval, circular, triangular, and trapezoidal.

72. The flowcell system of claim 51, further comprising a member selected from the group consisting of a voltage probe, a conductivity electrode, a pH cell, a conductivity meter, a pH meter, a voltmeter, a flowrate monitor, a data acquisition system and a microcomputer.

73. A method for sequencing or genotyping a nucleic acid, said method comprising:

providing a target nucleic acid, a primer strand, a polymerase, and a plurality of NP probes each having a label;

mixing said target nucleic acid, said sequencing primer, said polymerase, said plurality of NP probes in a flowcell according to claim 51, under conditions permitting target dependent polymerization of said plurality NP probes each having a label, thereby providing a product; and

detecting each of said labels in said flowcell to provide a sequence of said target nucleic acid.

74. The method of claim 73, wherein the polymerization of said plurality NP probes produces a plurality of phosphate detectable moieties.

75. The method of claim 74, wherein said plurality of NP probes are incorporated on said primer strand hybridized to said target nucleic acid using said polymerase, thereby releasing a phosphate with a detectable moiety attached thereto.

76. The method of claim 73, wherein said flowcell is an array of flowcells.