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 reduce 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 having a fluorescent moiety to the 3′
end of the primer to form an extended primer;
(c) detecting whether extension of the primer has occurred by detecting a fluorescent signal emitted by the fluorescent moiety, and further comprising destroying the fluorescent signal without removal of the fluorescent moiety;
(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.
316 Citations
21 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 reduce 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 having a fluorescent moiety to the 3′
end of the primer to form an extended primer;
(c) detecting whether extension of the primer has occurred by detecting a fluorescent signal emitted by the fluorescent moiety, and further comprising destroying the fluorescent signal without removal of the fluorescent moiety;
(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)
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4. 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 by detecting a change in the concentration of unincorporated deoxyribonucleotide;
(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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5. 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 having the capability of generating heat to the 3′
end of the primer to form an extended primer;
p1 (d) detecting whether extension of the prior has occurred by detecting the heat generated by incorporating the deoxyribonucleotides having the capability to generate heat;
(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 (6, 7)
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8. A method of DNA sequencing comprising the steps of:
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(a) providing a template system comprising a buffer and 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 which generates heat that is absorbed by the buffer to the 3′
end of the primer to form an extended primer;
(c) detecting whether extension of the primer has occurred by measuring the refractive index of the buffer;
(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 (11)
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9. 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 by detecting the concentration of pyrophosphate release by addition of the deoxyribonucleotide to the 3′
end of the primer where the concentration of pyrophosphate is detected by hydrolyzing the pyrophosphate and measuring heat generated by hydrolysis of the pyrophosphate;
(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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10. 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 wherein the DNA polymerase is a T4 DNA polymerase with a substitution of amino acid residue Asp112 by Ala and Glu114 by Ala;
(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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12. 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 an 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 having a fluorescent moiety to the 3′
end of the primer to form an extended primer;
(c) detecting whether extension of the primer has occurred by detecting a fluorescent signal emitted by the fluorescent moiety and destroying the fluorescent signal without removal of the fluorescent moiety 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 (13, 14)
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15. 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 an 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 capable of generating heat to the 3′
end of the primer to form an extended primer;
(c) detecting whether extension of the primer has occurred by detecting heat generated by incorporating the at least one deoxyribonucleotide 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, and exonuclease deficient DNA polymerase and the identified deoxyribonucleotide of step (b);
(g) removing the mixture of step (f); and
(b) repeating steps (a) through (g) to determine the nucleotide sequence of the nucleic acid molecule. - View Dependent Claims (16, 17)
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18. A method of DNA sequencing comprising the steps of:
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(a) providing a template system comprising a buffer and at least one nucleic acid molecule of unknown sequence hybridized to a primer oligonucleotide in the presence of an 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 capable of generating heat which is absorbed by the buffer to the 3′
end of the primer to form an extended primer;
(c) detecting whether extension of the primer has occurred by measuring the refractive index of the buffer 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 (20)
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19. 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 an 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 by detecting the concentration of pyrophosphate released by incorporation of a deoxyribonucleotide to the 3′
end of the primer where the concentration of pyrophosphate is detected by hydrolyzing the pyrophosphate and measuring the heat generated by hydrolysis of the pyrophosphate 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.
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21. 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 an exonuclease deficient DNA polymerase wherein the exonuclease deficient DNA polymerase is a T4 DNA polymerase with a substitution of amino acid residue Asp112 by Ala and Glu114 by Ala;
(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.
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Specification