Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
First Claim
1. A method of analyzing a nucleic acid molecule, the method comprising:
- (a) providing a template/primer duplex;
(b) contacting the duplex with a single type of unlabeled and unblocked deoxyribonucleotide in the presence of a polymerase under conditions that allow extension of the primer by incorporation of at least one deoxyribonucleotide to the 3′
end of the primer, wherein the contacting occurs in a reaction cell; and
(c) non-optically detecting in the reaction cell whether extension of the primer has occurred.
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Abstract
The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.
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Citations
34 Claims
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1. A method of analyzing a nucleic acid molecule, the method comprising:
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(a) providing a template/primer duplex; (b) contacting the duplex with a single type of unlabeled and unblocked deoxyribonucleotide in the presence of a polymerase under conditions that allow extension of the primer by incorporation of at least one deoxyribonucleotide to the 3′
end of the primer, wherein the contacting occurs in a reaction cell; and(c) non-optically detecting in the reaction cell whether extension of the primer has occurred. - View Dependent Claims (2, 3, 4, 5, 6, 7, 31)
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8. A method of analyzing a nucleic acid molecule, the method comprising:
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(a) providing a template/primer duplex; (b) contacting the duplex with a single type of unlabeled and unblocked deoxyribonucleotide in the presence of a polymerase under conditions that allow extension of the primer by incorporation of at least one deoxyribonucleotide to the 3′
end of the primer, wherein contacting occurs in a reaction cell; and(c) detecting a physical or chemical change within the reaction cell that is associated with incorporation of the deoxyribonucleotide into the duplex, thereby determining whether extension of the primer has occurred. - View Dependent Claims (9, 10, 11, 12, 13, 14, 15, 32)
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16. A method of analyzing a nucleic acid molecule, the method comprising:
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(a) providing a template/primer duplex; (b) contacting the duplex with a single type of unlabeled and unblocked deoxyribonucleotide in the presence of a polymerase under conditions that allow extension of the primer by incorporation of at least one deoxyribonucleotide to the 3′
end of the primer, wherein contacting occurs in a reaction cell; and(c) non-optically detecting within the reaction cell a reaction by-product associated with incorporation of the deoxyribonucleotide into the duplex, thereby determining whether extension of the primer has occurred. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 33)
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24. A method of analyzing a nucleic acid molecule, the method comprising:
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(a) providing a template/primer duplex; (b) contacting the duplex with a single type of unlabeled and unblocked deoxyribonucleotide in the presence of a polymerase under conditions that allow extension of the primer by incorporation of at least one deoxyribonucleotide to the 3′
end of the primer, wherein contacting occurs in a reaction cell; and(c) directly detecting in the reaction cell whether extension of the primer has occurred. - View Dependent Claims (25, 26, 27, 28, 29, 30, 34)
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Specification