Thermoelectric method of sequencing nucleic acids
First Claim
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1. A method of sequencing a nucleic acid, comprising:
- (a) providing an apparatus for sequencing a nucleic acid comprising;
(1) a flow channel;
(2) a substrate adapted to received an immobilized primer-hybridized nucleic acid template;
(3) a thin-film thermopile for heat or temperature measurement, wherein the thin-film thermopile comprises at least one measuring junction and at least one reference junction, wherein temperature control of the reference junction is not required;
(b) introducing a primer-hybridized nucleic acid template into the flow channel, wherein the primer comprises a 3′
end;
(c) introducing a liquid comprising a polymerase and a known deoxynucleoside triphosphate into the flow channel; and
(d) measuring heat generated or a temperature difference, if any, between the measuring junction and the reference junction, wherein temperature control of the reference junction is not required, and either;
(1) upon measuring the heat generated or difference in temperature, identifying the nucleotide of the template that is complementary to the known deoxynucleoside triphosphate incorporated into the 3′
end of the primer;
or(2) upon failing to measure heat generated 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.
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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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11 Claims
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1. A method of sequencing a nucleic acid, comprising:
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(a) providing an apparatus for sequencing a nucleic acid comprising; (1) a flow channel; (2) a substrate adapted to received an immobilized primer-hybridized nucleic acid template; (3) a thin-film thermopile for heat or temperature measurement, wherein the thin-film thermopile comprises at least one measuring junction and at least one reference junction, wherein temperature control of the reference junction is not required; (b) introducing a primer-hybridized nucleic acid template into the flow channel, wherein the primer comprises a 3′
end;(c) introducing a liquid comprising a polymerase and a known deoxynucleoside triphosphate into the flow channel; and (d) measuring heat generated or a temperature difference, if any, between the measuring junction and the reference junction, wherein temperature control of the reference junction is not required, and either; (1) upon measuring the heat generated or difference in temperature, identifying the nucleotide of the template that is complementary to the known deoxynucleoside triphosphate incorporated into the 3′
end of the primer;
or(2) upon failing to measure heat generated 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 (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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Specification