Methods of amplifying and sequencing nucleic acids
First Claim
Patent Images
1. A method for sequencing nucleic acids comprising:
- (a) fragmenting large template nucleic acid molecules to generate a plurality of fragmented nucleic acids;
(b) delivering the fragmented nucleic acids into aqueous microreactors in a water-in-oil emulsion such that a plurality of aqueous microreactors comprise a single copy of a fragmented nucleic acid, a single bead capable of binding to the fragmented nucleic acid, and amplification reaction solution containing reagents necessary to perform nucleic acid amplification;
(c) amplifying the fragmented nucleic acids in the microreactors to form amplified copies of said nucleic acids and binding the amplified copies to beads in the microreactors;
(d) delivering the beads to an array of at least 10,000 reaction chambers on a planar surface, wherein a plurality of the reaction chambers comprise no more than a single bead; and
(e) performing a sequencing reaction simultaneously on a plurality of the reaction chambers.
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Abstract
An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.
418 Citations
84 Claims
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1. A method for sequencing nucleic acids comprising:
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(a) fragmenting large template nucleic acid molecules to generate a plurality of fragmented nucleic acids; (b) delivering the fragmented nucleic acids into aqueous microreactors in a water-in-oil emulsion such that a plurality of aqueous microreactors comprise a single copy of a fragmented nucleic acid, a single bead capable of binding to the fragmented nucleic acid, and amplification reaction solution containing reagents necessary to perform nucleic acid amplification; (c) amplifying the fragmented nucleic acids in the microreactors to form amplified copies of said nucleic acids and binding the amplified copies to beads in the microreactors; (d) delivering the beads to an array of at least 10,000 reaction chambers on a planar surface, wherein a plurality of the reaction chambers comprise no more than a single bead; and (e) performing a sequencing reaction simultaneously on a plurality of the reaction chambers. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 73, 74, 75)
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10. An array comprising a planar surface with a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 20 to 100 μ
- m and each cavity has a width in at least one dimension of between 20 μ
m and 70 μ
m, and wherein there are at least 10,000 reaction chambers. - View Dependent Claims (11, 12, 13, 14, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
- m and each cavity has a width in at least one dimension of between 20 μ
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15. An array comprising a planar top surface and a planar bottom surface wherein the planar top surface has at least 10,000 cavities thereon, each cavity forming an analyte reaction chamber, the planar bottom surface is optically conductive such that optical signals from the reaction chambers can be detected through the bottom planar surface, wherein the distance between the top surface and the bottom surface is no greater than 5 mm, wherein the reaction chambers have a center to center spacing of between 20 to 100 μ
- m and each chamber having a width in at least one dimension of between 20 μ
m and 70 μ
m. - View Dependent Claims (16)
- m and each chamber having a width in at least one dimension of between 20 μ
- 26. An array means for carrying out separate parallel common reactions in an aqueous environment, wherein the array means comprises a substrate comprising at least 10,000 discrete reaction chambers containing a starting material that is capable of reacting with a reagent, each of the reaction chambers being dimensioned such that when one or more fluids containing at least one reagent is delivered into each reaction chamber, the diffusion time for the reagent to diffuse out of the well exceeds the time required for the starting material to react with the reagent to form a product.
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40. A method for delivering a bioactive agent to an array, comprising dispersing over the array a plurality of mobile solid supports, each mobile solid support having at least one reagent immobilized thereon, wherein the reagent is suitable for use in a nucleic acid sequencing reaction, where the array comprises a planar surface with a plurality of reaction chambers disposed thereon, wherein the reaction chambers have a center to center spacing of between 20 to 100 μ
- m and each reaction chamber has a width in at least one dimension of between 20 μ
m and 70 μ
m.
- m and each reaction chamber has a width in at least one dimension of between 20 μ
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41. An apparatus for simultaneously monitoring an array of reaction chambers for light indicating that a reaction is taking place at a particular site, the apparatus comprising:
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(a) an array of reaction chambers formed from a planar substrate comprising a plurality of cavitated surfaces, each cavitated surface forming a reaction chamber adapted to contain analytes, and wherein the reaction chambers have a center to center spacing of between 20 to 100 μ
m, each reaction chamber having a volume of between 10 to 150 pL, the array comprising more than 10,000 discrete reaction chambers;(b) an optically sensitive device arranged so that in use the light from a particular reaction chamber will impinge upon a particular predetermined region of said optically sensitive device; (c) means for determining the light level impinging upon each of said predetermined regions and (d) means to record the variation of said light level with time for each of said reaction chamber.
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42. An analytic sensor, comprising:
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(a) an array formed from a first bundle of optical fibers with a plurality of cavitated surfaces at one end thereof, each cavitated surface forming a reaction chamber adapted to contain analytes, and wherein the reaction chambers have a center to center spacing of between 20 to 100 μ
m, a width of 20 to 70 μ
m, the array comprising more than 10,000 discrete reaction chambers;(b) an enzymatic or fluorescent means for generating light in the reaction chambers; (c) light detection means comprising a light capture means and a second fiber optic bundle for transmitting light to the light detecting means, the second fiber optic bundle being in optical contact with the array, such that light generated in an individual reaction chamber is captured by a separate fiber or groups of separate fibers of the second fiber optic bundle for transmission to the light capture means. - View Dependent Claims (43, 44, 45, 46)
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47. A method for carrying out separate parallel common reactions in an aqueous environment, comprising:
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(a) delivering a fluid containing at least one reagent to an array, wherein the array comprises a substrate comprising at least 10,000 discrete reaction chambers, each reaction chamber adapted to contain analytes, and wherein the reaction chambers have a volume of between 10 to 150 pL and containing a starting material that is capable of reacting with the reagent, each of the reaction chambers being dimensioned such that when the fluid is delivered into each reaction chamber, the diffusion time for the reagent to diffuse out of the well exceeds the time required for the starting material to react with the reagent to form a product; and (b) washing the fluid from the array in the time period (i) after the starting material has reacted with the reagent to form a product in each reaction chamber but (ii) before the reagent delivered to any one reaction chamber has diffused out of that reaction chamber into any other reaction chamber. - View Dependent Claims (48, 49, 50, 51)
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52. A method for delivering nucleic acid sequencing enzymes to an array, said array having a planar surface with a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 20 to 100 μ
- m;
the method comprising dispersing over the array a plurality of mobile solid supports having one or more nucleic acid sequencing enzymes immobilized thereon, such that a plurality of the reaction chambers contain at least one mobile solid support. - View Dependent Claims (53)
- m;
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54. A method for delivering a plurality of nucleic acid templates to an array, said array having a planar surface with a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 20 to 100 μ
- m and the array having at least 10,000 reaction chambers;
the method comprising dispersing over the array a plurality of mobile solid supports, each mobile solid support having no more than a single species of nucleic acid template immobilized thereon, the dispersion causing no more than one mobile solid support to be disposed within any one reaction chamber. - View Dependent Claims (55, 56, 57, 58)
- m and the array having at least 10,000 reaction chambers;
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59. A method for sequencing a nucleic acid, the method comprising:
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(a) providing a plurality of single-stranded nucleic acid templates disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 20 to 100 μ
m and the planar surface has at least 10,000 reaction chambers;(b) performing a pyrophosphate based sequencing reaction simultaneously on all reaction chambers by annealing an effective amount of a sequencing primer to the nucleic acid templates and extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto the 3′
end of said sequencing primer, a sequencing reaction byproduct; and(c) identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid in each reaction chamber. - View Dependent Claims (60, 61)
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62. A method of determining the base sequence of a plurality of nucleotides on an array, the method comprising:
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(a) providing at least 10,000 DNA templates, each separately disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 20 to 100 μ
m, and a volume of between 10 to 150 pL;
wherein(b) adding an activated nucleotide 5′
-triphosphate precursor of one known nitrogenous base to a reaction mixture in each reaction chamber, each reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer strand at least one nucleotide residue shorter than the templates to form at least one unpaired nucleotide residue in each template at the 3′
-end of the primer strand, under reaction conditions which allow incorporation of the activated nucleoside 5′
-triphosphate precursor onto the 3′
-end of the primer strands, provided the nitrogenous base of the activated nucleoside 5′
-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the templates;(c) detecting whether or not the nucleoside 5′
-triphosphate precursor was incorporated into the primer strands in which incorporation of the nucleoside 5′
-triphosphate precursor indicates that the unpaired nucleotide residue of the template has a nitrogenous base composition that is complementary to that of the incorporated nucleoside 5′
-triphosphate precursor; and(d) sequentially repeating steps (b) and (c), wherein each sequential repetition adds and, detects the incorporation of one type of activated nucleoside 5′
-triphosphate precursor of known nitrogenous base composition; and(e) determining the base sequence of the unpaired nucleotide residues of the template in each reaction chamber from the sequence of incorporation of said nucleoside precursors.
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63. A method of identifying the base in a target position in a DNA sequence of template DNA, wherein:
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(a) at least 10,000 separate DNA templates are separately disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 20 to 100 μ
m, said DNA being rendered single stranded either before or after being disposed in the reaction chambers,(b) an extension primer is provided which hybridizes to said immobilized single-stranded DNA at a position immediately adjacent to said target position; (c) said immobilized single-stranded DNA is subjected to a polymerase reaction in the presence of a predetermined deoxynucleotide or dideoxynucleotide, wherein if the predetermined deoxynucleotide or dideoxynucleotide is incorporated onto the 3′
end of said sequencing primer then a sequencing reaction byproduct is formed; and(d) identifying the sequencing reaction byproduct, thereby determining the nucleotide complementary to the base at said target position in each of the 10,000 DNA templates. - View Dependent Claims (64)
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65. An apparatus for analyzing a nucleic acid sequence, the apparatus comprising:
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(a) a reagent delivery cuvette, wherein the cuvette includes an array comprising a planar surface with a plurality of cavities thereon, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 20 to 100 μ
m, and there are in excess of 10,000 reaction chambers, and wherein the reagent delivery cuvette contains reagents for use in a sequencing reaction;(b) a reagent delivery means in communication with the reagent delivery cuvette; (c) an imaging system in communication with the reagent delivery chamber; and (d) a data collection system in communication with the imaging system.
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66. An apparatus for determining the base sequence of a plurality of nucleotides on an array, the apparatus comprising:
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(a) a reagent cuvette containing a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein there are in excess of 10,000 reaction chambers, each having a center to center spacing of between 20 to 100 μ
m and a volume of between 10 to 150 pL;(b) reagent delivery means for simultaneously adding to each reaction chamber an activated nucleotide 5′
-triphosphate precursor of one known nitrogenous base to a reaction mixture in each reaction chamber, each reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer strand at least one nucleotide residue shorter than the templates to form at least one unpaired nucleotide residue in each template at the 3′
-end of the primer strand, under reaction conditions which allow incorporation of the activated nucleoside 5′
-triphosphate precursor onto the 3′
-end of the primer strands, provided the nitrogenous base of the activated nucleoside 5′
-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the templates;(c) detection means for detecting in each reaction chamber whether or not the nucleoside 5′
-triphosphate precursor was incorporated into the primer strands in which incorporation of the nucleoside 5′
-triphosphate precursor indicates that the unpaired nucleotide residue of the template has a nitrogenous base composition that is complementary to that of the incorporated nucleoside 5′
-triphosphate precursor; and(d) means for sequentially repeating steps (b) and (c), wherein each sequential repetition adds and, detects the incorporation of one type of activated nucleoside 5′
-triphosphate precursor of known nitrogenous base composition; and(e) data processing means for determining the base sequence of the unpaired nucleotide residues of the template simultaneously in each reaction chamber from the sequence of incorporation of said nucleoside precursors.
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67. An apparatus for processing a plurality of analytes, the apparatus comprising:
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(a) a flow chamber having disposed therein a substrate comprising at least 50,000 cavitated surfaces on a fiber optic bundle, each cavitated surface forming a reaction chamber adapted to contain analytes, and wherein the reaction chambers have a center to center spacing of between 20 to 100 μ
m and a diameter of 20 to 70 μ
m;(b) fluid means for delivering processing reagents from one or more reservoirs to the flow chamber so that the analytes disposed in the reaction chambers are exposed to the reagents; and (c) detection means for simultaneously detecting a sequence of optical signals from each of the reaction chambers, each optical signal of the sequence being indicative of an interaction between a processing reagent and the analyte disposed in the reaction chamber, wherein the detection means is in communication with the cavitated surfaces. - View Dependent Claims (68, 69, 70, 71)
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72. A method for sequencing a nucleic acid, the method comprising:
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(a) providing a plurality of single-stranded nucleic acid templates in an array having at least 50,000 discrete reaction sites; (b) contacting the nucleic acid templates with reagents necessary to perform a pyrophosphate-based sequencing reaction coupled to light emission; (c) detecting the light emitted from a plurality of reaction sites on respective portions of an optically sensitive device; (d) converting the light impinging upon each of said portions of said optically sensitive device into an electrical signal which is distinguishable from the signals from all of said other reaction sites; (e) determining the sequence of the nucleic acid templates based on light emission for each of said discrete reaction sites from the corresponding electrical signal.
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76. A method for sequencing nucleic acids comprising:
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(a) fragmenting large template nucleic acid molecules to generate a plurality of fragmented nucleic acids; (b) attaching one strand of a plurality of the fragmented nucleic acids individually to beads to generate single stranded nucleic acids attached individually to beads; (c) delivering a population of the single stranded fragmented nucleic acids attached individually to beads to an array of at least 10,000 reaction chambers on a planar surface, wherein a plurality of the wells comprise no more than a one bead with on single stranded fragmented nucleic acid; (d) performing a sequencing reaction simultaneously on a plurality of the reaction chambers. - View Dependent Claims (77, 78, 79, 80, 81, 82, 83, 84)
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Specification