Thermoelectric method of sequencing nucleic acids
First Claim
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1. A method of sequencing a nucleic acid comprising:
- (a) exposing a primer-hybridized nucleic acid template to a liquid flowing under conditions of laminar flow, wherein the primer comprises a 3′
end, and(b) while the liquid continues flowing under conditions of laminar flow, measuring heat generated or a temperature difference, if any, associated with incorporation of a known deoxynucleoside triphosphate into the 3′
end of the primer, and either(1) upon measuring heat generated or a temperature difference, identifying the nucleotide of the template that is complementary to the known deoxynucleoside triphosphate incorporated into the primer, or(2) upon failing to measure heat generated or a temperature difference, eliminating the possibility that the nucleotide that is complementary to the known deoxynucleoside triphosphate is present in the template at the 3′
end of the primer,wherein ambient temperature fluctuations are not controlled.
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Abstract
The present invention relates to a novel thermoelectric method for determining the sequence of nucleotides on a nucleic acid molecule through use of a thermopile and/or sequencing reagents flowing under the conditions of laminar flow. The methods disclosed herein involve the measurement of the heat generated by a deoxynucleotide incorporation event that can be accomplished without the need to control the temperature of any of a thermopile'"'"'s junctions.
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Citations
31 Claims
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1. A method of sequencing a nucleic acid comprising:
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(a) exposing a primer-hybridized nucleic acid template to a liquid flowing under conditions of laminar flow, wherein the primer comprises a 3′
end, and(b) while the liquid continues flowing under conditions of laminar flow, measuring heat generated or a temperature difference, if any, associated with incorporation of a known deoxynucleoside triphosphate into the 3′
end of the primer, and either(1) upon measuring heat generated or a temperature difference, identifying the nucleotide of the template that is complementary to the known deoxynucleoside triphosphate incorporated into the primer, or (2) upon failing to measure heat generated or a temperature difference, eliminating the possibility that the nucleotide that is complementary to the known deoxynucleoside triphosphate is present in the template at the 3′
end of the primer,wherein ambient temperature fluctuations are not controlled. - View Dependent Claims (2, 19)
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3. A method of sequencing a nucleic acid comprising:
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(a) exposing a primer-hybridized nucleic acid template to a liquid comprising a polymerase and a known deoxynucleoside triphosphate; and (b) measuring heat generated or difference in temperature, if any, as detected by a thermopile comprising at least one measuring junction and at least one reference junction, wherein each reference junction is not maintained at a constant temperature, and either (1) upon measuring heat generated or a temperature difference, identifying the nucleotide of the template that is complementary to the known deoxynucleoside triphosphate incorporated into the primer, or (2) upon failing to measure heat generated or a temperature difference, eliminating the possibility that the nucleotide that is complementary to the known deoxynucleoside triphosphate is present in the template at the 3′
end of the primer. - View Dependent Claims (4, 22, 23, 24)
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5. A method of sequencing a nucleic acid comprising:
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(a) obtaining a primer-hybridized nucleic acid template, wherein the primer comprises a 3′
end;(b) exposing the template to a liquid flowing under conditions of laminar flow, wherein the liquid comprises a polymerase and a known deoxynucleoside triphosphate; (c) while the liquid continues flowing under conditions of laminar flow, measuring heat generated or difference in temperature, if any, as detected by at least one thin-film thermopile comprising at least one first junction and at least one second junction, wherein each first junction is a measuring junction and each second junction is a reference junction, wherein each reference junction is not maintained at a constant temperature, and wherein the heat generated or the difference in temperature between the first and second junctions indicates incorporation of the known deoxynucleoside triphosphate into the 3′
end of the primer; and
either(d) upon measuring heat or a difference in temperature, identifying the nucleotide of the template that is complementary to the known deoxynucleoside triphosphate incorporated into the primer, or (e) upon failing to measure heat or a difference in temperature, eliminating the possibility that the nucleotide that is complementary to the known deoxynucleoside triphosphate is present in the template at the 3′
end of the primer. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 25, 26, 27)
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20. A method of sequencing a nucleic acid comprising:
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(a) exposing a primer-hybridized nucleic acid template to a liquid flowing under conditions of laminar flow, wherein the primer comprises a 3′
end, and(b) while the liquid continues flowing under conditions of laminar flow, measuring heat generated or a temperature difference, if any, associated with incorporation of a known deoxynucleoside triphosphate into the 3′
end of the primer, and either(1) upon measuring heat generated or a temperature difference, identifying the nucleotide of the template that is complementary to the known deoxynucleoside triphosphate incorporated into the primer, or (2) upon failing to measure heat generated or a temperature difference, eliminating the possibility that the nucleotide that is complementary to the known deoxynucleoside triphosphate is present in the template at the 3′
end of the primer,wherein the method is employed in the absence of a reaction chamber of a finite volume, and wherein ambient temperature fluctuations are not controlled. - View Dependent Claims (28, 29)
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21. A method of sequencing a nucleic acid comprising:
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(a) exposing a primer-hybridized nucleic acid template to a liquid flowing under conditions of laminar flow, wherein the primer comprises a 3′
end, and(b) while the liquid continues flowing under conditions of laminar flow, measuring heat generated or a temperature difference, if any, associated with incorporation of a known deoxynucleoside triphosphate into the 3′
end of the primer, and either(1) upon measuring heat generated or a temperature difference, identifying the nucleotide of the template that is complementary to the known deoxynucleoside triphosphate incorporated into the primer, or (2) upon failing to measure heat generated or a temperature difference, eliminating the possibility that the nucleotide that is complementary to the known deoxynucleoside triphosphate is present in the template at the 3′
end of the primer,wherein the method is employed in the absence of a reaction chamber of a finite volume, wherein ambient temperature fluctuations are not controlled, and wherein a reference junction temperature of a thermopile is not maintained. - View Dependent Claims (30, 31)
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Specification