Methods and apparatuses for analyzing polynucleotide sequences

• US 20050014175A1
• Filed: 05/14/2004
• Published: 01/20/2005
• Est. Priority Date: 06/28/1999
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Quake, Stephen R.
• Granted Patent: US 7,462,449 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2300/14 Means for pressure control
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0655 pinch valves
• 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: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: C12Q 1/68 involving 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 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: F04B 43/043 Micropumps
• CPC Class Codes: F15C 5/00 Manufacture of fluid circuit elements; Manufacture of assemblages of such elements integrated circuits
• CPC Class Codes: F16K 2099/0074 using photolithography, e.g. etching
• CPC Class Codes: F16K 2099/0076 using electrical discharge machining [EDM], milling or drilling
• CPC Class Codes: F16K 2099/0078 : using moulding or stamping
• CPC Class Codes: F16K 2099/008 : Multi-layer fabrications
• CPC Class Codes: F16K 2099/0084 : Chemistry or biology, e.g. "lab-on-a-chip" technology
• CPC Class Codes: F16K 2099/0094 : Micropumps
• CPC Class Codes: F16K 99/0001 : Microvalves microdevices B81B1/00; manufacture or treatment of devices or systems in or on a substrate B81C1/00; microfluidic structures B01L3/5027; micropumps F04B19/006
• CPC Class Codes: F16K 99/0015 : Diaphragm or membrane valves
• CPC Class Codes: F16K 99/0051 : using electrostatic means
• CPC Class Codes: F16K 99/0059 : actuated by a pilot fluid
• CPC Class Codes: G01N 33/549 : with antigen or antibody entrapped within the carrier

Abstract

Methods for high speed, high throughput analysis of polynucleotide sequences, and apparatuses with which to carry out the methods are provided in the invention.

Claims

1-54. -54. (Cancelled)

55. A method of deriving nucleotide sequence information from individual polynucleotide molecules, the method comprising the steps of:

locating the respective positions of a plurality of target nucleic acid template molecules in a field of immobilized polynucleotides;

exposing said field to a polymerase and at least one nucleotide comprising a detectable label under conditions that allow said nucleotide to be incorporated in a primer hybridized to said template molecule if said nucleotide is complementary to a nucleotide in said template immediately 5′

of said primer;

determining the position or positions of optical signal emanating from label attached to a nucleotide that has been incorporated in said primer; and

recording the identity and position of an incorporated nucleotide based upon said optical signal.

56. The method of claim 55, further comprising the step of compiling one or more nucleic acid sequences at one or more of said locations.

57. The method of claim 55, wherein said exposing step comprises inhibiting incorporation of more than one nucleotide on average per primer prior to said determining step.

58. The method of claim 55, wherein said locating step comprises randomly depositing said target nucleic acid template molecules onto said field.

59. The method of claim 55, wherein said field is contained on a surface coated with a polyelectrolyte multilayer.

60. The method of claim 55, wherein said target nucleic acid template molecules are fixed on said field by a chemical linkage.

61. The method of claim 60, wherein said chemical linkage is a biotin/avidin or a biotin/streptavidin linkage.

62. The method of claim 55, wherein said field is arrayed on a surface comprising glass or fused silica.

63. The method of claim 55, further comprising the step of washing said field in order to eliminate unincorporated nucleotides.

64. The method of claim 55, further comprising the step of neutralizing said label after said determining step.

65. A method for identifying one or more nucleotides in a nucleic acid target molecule, the method comprising the steps of:

(a) obtaining an array of support-bound target nucleic acids in which each of said support-bound nucleic acids is individually optically resolvable;

(b) exposing said array to a polymerase, a primer capable of hybridizing to at least some of said support-bound nucleic acids and at least one nucleotide comprising a detectable label under conditions that allow said nucleotide to be incorporated into said primer if said nucleotide is complementary to a nucleotide in said target immediately 5′

to said primer;

(c) washing said array in order to remove unincorporated nucleotides;

(d) determining the presence and position of label attached to an incorporated nucleotide;

(e) neutralizing any label observed in said determining step;

(f) repeating steps a, b, c, and d;

(g) identifying label present at any position in which presence of label has previously been determined in said determining step;

(h) repeating steps a, b, c, d, and e; and

(i) identifying a sequencing of nucleotides in said target nucleic acid based upon the sequence of labels detected at any given position in said array.

66. The method of claim 65, wherein said label is optically detectable.

67. The method of claim 66, wherein said label is a fluorescent label.

68. The method of claim 65, wherein said label present in an incorporated nucleotide, inhibits incorporation of a subsequent nucleotide at a position immediately 5′

of said incorporated nucleotide.

71. The method of claim 65, wherein said label is an acceptor fluorophore, and wherein said primer comprises a donor fluorophore label attached thereto.

72. The method of claim 65, wherein said label is an acceptor fluorophore and wherein said polymerase comprises a donor fluorophore attached thereto.

73. The method of claim 66, wherein said identifying step comprises visualizing said label using an optical microscope.

74. The method of claim 73, wherein said optical microscope comprises a total internal reflection objective.

69. The method of 68, wherein said inhibition is reversible.

70. The method of claim 69, further comprising the step of cleaving label present on an incorporated nucleotide after said first identifying step.