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Kafe 바로가기국가/구분 | United States(US) Patent 등록 |
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국제특허분류(IPC7판) |
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출원번호 | US-0913433 (2013-06-08) |
등록번호 | US-9458500 (2016-10-04) |
발명자 / 주소 |
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출원인 / 주소 |
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인용정보 | 피인용 횟수 : 0 인용 특허 : 348 |
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 syst
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.
1. An apparatus for DNA sequencing comprising: a) at least one reaction chamber including a DNA primer/template system which produces a detectable reaction event when a DNA polymerase enzyme incorporates an unlabeled and unblocked deoxyribonucleotide monophosphate onto the 3′ end of the primer stran
1. An apparatus for DNA sequencing comprising: a) at least one reaction chamber including a DNA primer/template system which produces a detectable reaction event when a DNA polymerase enzyme incorporates an unlabeled and unblocked deoxyribonucleotide monophosphate onto the 3′ end of the primer strand;b) a component for introducing into, and evacuating from, said reaction chamber at least one reagent selected from the group consisting of: buffers, electrolytes, DNA template, DNA primer, unlabeled and unblocked deoxyribonucleotides, and polymerase enzymes;c) a system for converting said detectable reaction event into an electrical signal based on an electrical potential generated from said detectable reaction event in the at least one reaction chamber; andd) a voltmeter configured to measure said electrical signal in the at least one reaction chamber. 2. The apparatus of claim 1, wherein said at least one reaction chamber further comprises said deoxyribonucleotide monophosphate, and wherein said deoxyribonucleotide monophosphate is non-chemically modified. 3. The apparatus of claim 1, wherein said at least one reaction chamber further includes a solid support, wherein said primer/template system comprises a primer and a template, and wherein said template is tethered to said solid support. 4. The apparatus of claim 3, wherein said template comprises a linker moiety for tethering to said solid support. 5. The apparatus of claim 1, wherein said DNA polymerase lacks 5′ to 3′ exonuclease activity. 6. The apparatus of claim 1, wherein the amplitude of said electrical signal corresponds to the number of nucleotides added to said primer strand. 7. The apparatus of claim 1, wherein said apparatus provides real-time detection of incorporation of said deoxyribonucleotide monophosphate onto said primer strand. 8. A method of DNA sequencing comprising: a) providing in at least one reaction chamber a primer/template system comprising a template sequence hybridized to a primer oligonucleotide in the presence of a DNA polymerase;b) contacting said primer/template system with a single type of an unlabeled and unblocked deoxyribonucleotide under conditions which produces a detectable reaction event when said DNA polymerase incorporates a deoxyribonucleotide onto the 3′ end of said primer oligonucleotide;c) converting said detectable reaction event into an electrical signal based on an electrical potential generated from said detectable reaction event in the at least one reaction chamber; andd) measuring said electrical signal with a voltmeter in the at least one reaction chamber. 9. The method of 8, further comprising (e) flushing said reaction chamber with dNTP free buffer. 10. The method of claim 9, further comprising repeating steps (b) through (e). 11. The method of claim 9, further comprising repeating steps (b) through (e) until the complete nucleotide sequence of said template sequence is determined. 12. The method of claim 8, wherein said deoxyribonucleotide is non-chemically modified. 13. The method of claim 8, wherein said at least one reaction chamber further includes a solid support, and wherein said template sequence is tethered to said solid support. 14. The method of claim 13, wherein said template sequence comprises a linker moiety for tethering to said solid support. 15. The method of claim 8, wherein said DNA polymerase lacks 5′ to 3′ exonuclease activity. 16. The method of claim 8, wherein the amplitude of said electrical signal corresponds to the number of nucleotides added to said primer strand by said DNA polymerase. 17. The method of claim 8, wherein said electrical signal provides real-time detection of incorporation of said deoxyribonucleotide monophosphate onto said primer strand by said DNA polymerase. 18. The apparatus of claim 1, wherein the at least one reaction chamber comprises a plurality of reaction chambers formed on an array device. 19. The method of claim 8, wherein the at least one reaction chamber comprises a plurality of reaction chambers formed on an array device. 20. An apparatus for DNA sequencing, comprising: a) a reaction chamber including a polymerase and a DNA primer/template system that produces a non-optical signal when a DNA polymerase enzyme incorporates an unlabeled and unblocked deoxyribonucleotide monophosphate onto the 3′ end of the primer strand;b) a component for introducing into, and evacuating from, said reaction chamber a solution including a single type of unlabeled and unblocked deoxyribonucleotide;c) a system attached to a surface of the reaction chamber and configured to convert said detectable signal into an electrical signal; andd) a voltmeter configured to measure said electrical signal. 21. The apparatus of claim 20, wherein the reaction chamber further comprises the unlabeled and unblocked deoxyribonucleotide monophosphate. 22. The apparatus of claim 20, wherein the reaction chamber further includes a solid support, wherein said DNA primer/template system comprises a primer and a template, and wherein said template is tethered to said solid support. 23. The apparatus of claim 22, wherein said template comprises a linker moiety for tethering to said solid support. 24. The apparatus of claim 20, wherein said DNA polymerase lacks 5′ to 3′ exonuclease activity. 25. The apparatus of claim 20, wherein the amplitude of said electrical signal corresponds to the number of nucleotides added to said primer strand. 26. The apparatus of claim 20, wherein said apparatus provides real-time detection of incorporation of said deoxyribonucleotide monophosphate onto said primer strand. 27. The apparatus of claim 20 comprising a plurality of reaction chambers, wherein the plurality of reaction chambers is formed on an array device. 28. A method for DNA sequencing, comprising: a) providing in a reaction chamber a polymerase and a DNA primer/template system;b) contacting said DNA primer/template system with a single type of unlabeled and unblocked deoxyribonucleotide monophosphate under conditions which produces a non-optical signal when a DNA polymerase enzyme incorporates said unlabeled and unblocked deoxyribonucleotide monophosphate onto the 3′ end of the primer strand;c) converting with a system attached to a surface of the reaction chamber said non-optical signal into an electrical signal; andd) measuring said electrical signal with a voltmeter. 29. The method of claim 28, further comprising (e) flushing said reaction chamber with dNTP free buffer. 30. The method of claim 29, further comprising repeating steps (b) through (e). 31. The method of claim 29, further comprising repeating steps (b) through (e) until the complete nucleotide sequence of said template sequence is determined. 32. The method of claim 28, wherein said reaction chamber further includes a solid support, and wherein said template sequence is tethered to said solid support. 33. The method of claim 32, wherein said template sequence comprises a linker moiety for tethering to said solid support. 34. The method of claim 28, wherein said DNA polymerase lacks 5′ to 3′ exonuclease activity. 35. The method of claim 28, wherein the amplitude of said electrical signal corresponds to the number of nucleotides added to said primer strand by said DNA polymerase. 36. The method of claim 28, wherein said electrical signal provides real-time detection of incorporation of said deoxyribonucleotide monophosphate onto said primer strand by said DNA polymerase. 37. The method of claim 28, wherein said reaction chamber comprises a plurality of reaction chambers formed on an array device.
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