Method for isolation of independent, parallel chemical micro-reactions using a porous filter
First Claim
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1. A CMRA comprising:
- (a) a microreactor element comprising an array of open microchannels or open microwells, the longitudinal axes of said microchannels or microwells arranged in a substantially parallel manner; and
(b) a porous filter element in contact with the microreactor element to form a bottom to the microchannels or microwells, thereby defining a series of reaction chambers, wherein the porous filter element comprises a permselective membrane that blocks the passage of nucleic acids, proteins and beads there across, but permits the passage of low molecular weight solutes, organic solvents and water there across.
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Abstract
The present invention relates to the field of fluid dynamics. More specifically, this invention relates to methods and apparatus for conducting densely packed, independent chemical reactions in parallel in a substantially two-dimensional array. Accordingly, this invention also focuses on the use of this array for applications such as DNA sequencing, most preferably pyrosequencing, and DNA amplification.
243 Citations
106 Claims
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1. A CMRA comprising:
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(a) a microreactor element comprising an array of open microchannels or open microwells, the longitudinal axes of said microchannels or microwells arranged in a substantially parallel manner; and
(b) a porous filter element in contact with the microreactor element to form a bottom to the microchannels or microwells, thereby defining a series of reaction chambers, wherein the porous filter element comprises a permselective membrane that blocks the passage of nucleic acids, proteins and beads there across, but permits the passage of low molecular weight solutes, organic solvents and water there across. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 19, 90, 91, 92, 93, 94, 95, 96)
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- 9. A UMRA comprising a porous filter element against which molecules are concentrated by concentration polarization wherein discrete reaction chambers are formed in discrete locations on the surface of or within the porous filter element by depositing reactant molecules at discrete sites on or within the porous filter element.
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14. A UMRA comprising:
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(a) a porous membrane with discrete reaction sites formed by depositing mobile solid supports having said reactant molecules thereon, on the surface of, or within the porous membrane;
(b) a nucleic acid template immobilized to a solid support; and
(c) optionally, at least one immobilized enzyme. - View Dependent Claims (15, 16, 17, 18, 20, 21)
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22. An array comprising:
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(a) a first porous membrane with a plurality of discrete reaction sites disposed thereon, and/or within, wherein each reaction site has immobilized template adhered to the surface; and
(b) a second porous membrane with at least one enzyme located on the surface of, and/or within, the membrane, wherein the second porous membrane is in direct contact with the first porous membrane. - View Dependent Claims (23, 24, 25, 26, 27, 28)
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- 29. A CMRA comprising an array of open microchannels or microwells attached to a porous filter or membrane.
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36. An apparatus for determining the nucleic acid sequence in a template nucleic acid polymer, comprising:
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(a) a CMRA or UMRA;
(b) nucleic acid delivery means for introducing template nucleic acid polymers to the discrete reaction sites;
(c) nucleic acid delivery means to deliver reagents to the reaction sites to create a polymerization environment in which the nucleic acid polymers will act as template polymers for the synthesis of complementary nucleic acid polymers when nucleotides are added;
(d) convective flow delivery means to immobilize reagents to the porous membrane;
(e) detection means for detecting the formation of inorganic pyrophosphate enzymatically; and
(f) data processing means to determine the identity of each nucleotide in the complementary polymers and thus the sequence of the template polymers. - View Dependent Claims (37, 38, 39, 40, 41, 42, 43, 44, 45, 46)
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47. An apparatus for processing a plurality of analytes, the apparatus comprising:
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(a) a CMRA or an UMRA;
(b) fluid means for delivering processing reagents from one or more reservoirs to the flow chamber so that the analytes disposed therein are exposed to the reagents; and
(c) detection means for detecting a sequence of optical signals from each of the reaction sites, each optical signal of the sequence being indicative of an interaction between a processing reagent and the analyte disposed in the reaction site, wherein the detection means is in communication with the reaction site. - View Dependent Claims (48, 49, 50, 51, 52, 53, 54, 55, 58, 59, 60, 61, 62, 63, 64)
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56. An apparatus for determining the base sequence of a plurality of nucleotides on an array, the apparatus comprising:
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(a) a CMRA or UMRA;
(b) reagent delivery means for adding an activated nucleotide 5′
-triphosphate precursor of one known nitrogenous base to a reaction mixture to each reaction site, 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 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 in each reaction chamber from the sequence of incorporation of said nucleoside precursors. - View Dependent Claims (57)
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65. An apparatus for determining the nucleic acid sequence in a template nucleic acid polymer, comprising:
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(a) a CMRA or UMRA;
(b) nucleic acid delivery means for introducing a template nucleic acid polymers onto the reaction sites;
(c) nucleic acid delivery means to deliver reagents to the reaction chambers to create polymerization environment in which the nucleic acid polymers will act as a template polymers for the synthesis of complementary nucleic acid polymers when nucleotides are added;
(d) reagent delivery means for successively providing to the polymerization environment a series of feedstocks, each feedstock comprising a nucleotide selected from among the nucleotides from which the complementary nucleic acid polymer will be formed, such that if the nucleotide in the feedstock is complementary to the next nucleotide in the template polymer to be sequenced said nucleotide will be incorporated into the complementary polymer and inorganic pyrophosphate will be released;
(e) detection means for detecting the formation of inorganic pyrophosphate enzymatically; and
(f) data processing means to determine the identity of each nucleotide in the complementary polymers and thus the sequence of the template polymers. - View Dependent Claims (66, 67, 68, 69, 70, 71, 72, 73)
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74. A system for sequencing a nucleic acid comprising the following components:
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(a) a CMRA or UMRA;
(b) at least one enzyme immobilized on a solid support;
(c) means for flowing reagents over said porous membrane;
(d) means for detection; and
(e) means for determining the sequence of the nucleic acid. - View Dependent Claims (75, 76, 77, 78, 79, 80, 81)
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82. A system for sequencing a nucleic acid comprising the following components:
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(a) a CMRA or UMRA (b) at least one enzyme immobilized on a solid support;
(c) means for flowing reagents over said porous membrane;
(d) means for enzymatic detection; and
(e) means for determining the sequence of the nucleic acid. - View Dependent Claims (83, 84, 85, 86, 87, 88, 89)
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97. A method of determining the base sequence of nucleotides in an array format, the method comprising the steps of:
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(a) adding an activated nucleoside 5′
-triphopsphate precursor of one known nitrogenous base composition to a plurality of reaction sites localized on a CMRA or UMRA, wherein the reaction site is comprised of a template-directed nucleotide polymerase and a heterogenous population of single stranded templates hybridized to complementary oligonucleotide primer strands 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 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;
(b) 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
(c) sequentially repeating steps (a) and (b), wherein each sequential repetition adds and detects the incorporation of one type of activated nucleoside 5′
-triphosphate precursor of known nitrogenous base composition;
(d) determining the base sequence of the unpaired nucleotide residues of the template from the sequence of incorporation of said nucleoside precursors. - View Dependent Claims (98, 99, 100, 101)
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102. A method of determining the base sequence of a plurality of nucleotides on an array, said method comprising:
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(a) providing a plurality of sample DNA'"'"'s, each disposed within a plurality of reaction sites on a CMRA or UMRA;
(b) detecting the light level emitted from a plurality of reaction sites on respective proportional of an optically sensitive device;
(c) 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 regions;
(d) determining a light intensity for each of said discrete regions from the corresponding electrical signal;
(e) recording the variations of said electrical signals with time. - View Dependent Claims (103, 104, 105, 106)
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Specification