Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
First Claim
1. A method of DNA sequencing comprising the steps of:
- (a) providing a template system comprising at least one nucleic acid molecule of unknown sequence hybridized to a primer oligonucleotide in the presence of a DNA polymerase with reduced exonuclease activity;
(b) contacting the template system with a single type of deoxyribonucleotide under conditions which allow extension of the primer by incorporation of at least one deoxyribonucleotide to the 3′
end of the primer to form an extended primer;
(c) observing the template system in order to detect whether extension of the primer has occurred;
(d) detecting the number of deoxyribonucleotides incorporated into the primer;
(e) removing unincorporated deoxyribonucleotide; and
(f) repeating steps (a) through (e) to determine the nucleotide sequence of the nucleic acid molecule.
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Reexamination
Accused Products
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
30 Claims
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1. A method of DNA sequencing comprising the steps of:
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(a) providing a template system comprising at least one nucleic acid molecule of unknown sequence hybridized to a primer oligonucleotide in the presence of a DNA polymerase with reduced exonuclease activity; (b) contacting the template system with a single type of deoxyribonucleotide under conditions which allow extension of the primer by incorporation of at least one deoxyribonucleotide to the 3′
end of the primer to form an extended primer;(c) observing the template system in order to detect whether extension of the primer has occurred; (d) detecting the number of deoxyribonucleotides incorporated into the primer; (e) removing unincorporated deoxyribonucleotide; and (f) repeating steps (a) through (e) to determine the nucleotide sequence of the nucleic acid molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method of DNA sequencing comprising the steps of:
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(a) providing a template system comprising at least one nucleic acid molecule of unknown sequence hybridized to a primer oligonucleotide in the presence of a exonuclease deficient DNA polymerase; (b) contacting the template system with a single type of deoxyribonucleotide under conditions which allow extension of the primer by incorporation of at least one deoxyribonucleotide to the 3′
end of the primer to form an extended primer;(c) observing the template system in order to detect whether extension of the primer has occurred thereby identifying the deoxyribonucleotide added to the 3′
end of the primer;(d) detecting the number of deoxyribonucleotides incorporated into the primer; (e) removing unincorporated deoxyribonucleotide; (f) contacting the template system with a mixture including an exonuclease proficient DNA polymerase, an exonuclease deficient DNA polymerase and the identified deoxyribonucleotide of step (b); (g) removing the mixture of step (f); and (h) repeating steps (a) through (g) to determine the nucleotide sequence of the nucleic acid molecule. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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Specification