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) detecting 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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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.
149 Citations
42 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) detecting 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)
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13. 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) detecting 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 (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
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25. 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;
(b) contacting the template system with a single type of deoxyribonucleotide including a fluorescent moiety under conditions which allow extension of the primer by incorporation of a deoxyribonucleotide to the 3′
end of the primer to form an extended primer;
(c) removing unincorporated deoxyribonucleotide;
(d) detecting whether extension of the primer has occurred by removing the fluorescent moiety to a remote location and detecting the fluorescent moiety removed from the incorporated deoxyribonucleotide; and
(e) repeating steps (a) through (d) to determine the nucleotide sequence of the nucleic acid molecule.
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26. 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;
(b) contacting the template system with a single type of deoxyribonucleotide including a fluorescent moiety under conditions which allow extension of the primer by incorporation of a deoxyribonucleotide to the 3′
end of the primer to form an extended primer;
(c) removing unincorporated deoxyribonucleotide;
(d) detecting whether extension of the primer has occurred by removing the fluorescent moiety to a remote location and detecting the fluorescent moiety removed from the incorporated deoxyribonucleotide; and
(e) repeating steps (a) through (d) to determine the nucleotide sequence of the nucleic acid molecule.
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27. A test apparatus for DNA sequencing, said apparatus including one or more of a plurality of elements including:
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(a) a reaction chamber that incorporates a DNA template/primer system which produces a detectable signal when a DNA polymerase enzyme incorporates a deoxyribonucleotide monophosphate onto the 3′
end of the primer strand;
(b) a means for introducing into, and evacuating from, the reaction chamber a plurality of reagents including, but not limited to, buffers, electrolytes, DNA template, DNA primer, deoxyribonucleotides and chemically modified deoxyribonucleotides and polymerase enzymes;
(c) a means for amplifying said signal (d) a transduction element which transduces said signal into an electrical signal. - View Dependent Claims (28, 29, 34)
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- 30. The apparatus of claim 30 in which the signal is an increase in temperature of the contents of the reaction chamber
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35. A test apparatus for DNA sequencing, said apparatus including one or more of a plurality of elements including:
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(a) a reaction chamber that incorporates a DNA template/primer system in which deoxyribonucleotide monophosphate may be caused to react with the DNA template/primer system in the presence of a DNA polymerase enzyme (b) a means for introducing into, and evacuating from, the reaction chamber a plurality of reagents including, but not limited to, buffers, electrolytes, DNA template, DNA primer, deoxyribonucleotides and chemically modified deoxyribonucleotides and polymerase enzymes;
(c) a detection chamber in which one or more products of a one or more chemical reactions in the reaction chamber are caused to generate a detectable signal following a chemical reaction which incorporates one or more deoxyribonucleotides into the DNA template/primer system (d) a means for amplifying said signal (e) a transduction element which transduces said signal into an electrical signal. - View Dependent Claims (36, 37, 38, 39, 40, 41, 42)
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Specification