METHOD OF DETERMINING THE NUCLEOTIDE SEQUENCE OF OLIGONUCLEOTIDES AND DNA MOLECULES
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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
62 Claims
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1-32. -32. (canceled)
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33. 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 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; and(c) non-optically detecting whether extension of the primer has occurred. - View Dependent Claims (34, 35, 36, 37, 38, 39)
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40. 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 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; and(c) detecting a physical or chemical change associated with incorporation of the deoxyribonucleotide into the duplex, thereby determining whether extension of the primer has occurred. - View Dependent Claims (41, 42, 43, 44, 45, 46, 47)
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48. 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 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; and(c) non-optically detecting 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 (49, 50, 51, 52, 53, 54, 55)
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56. 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 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; and(c) directly detecting whether extension of the primer has occurred. - View Dependent Claims (57, 58, 59, 60, 61, 62)
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Specification