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Kafe 바로가기국가/구분 | United States(US) Patent 등록 |
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국제특허분류(IPC7판) |
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출원번호 | US-0941403 (2015-11-13) |
등록번호 | US-9540689 (2017-01-10) |
발명자 / 주소 |
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출원인 / 주소 |
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인용정보 | 피인용 횟수 : 0 인용 특허 : 350 |
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. A method for sequencing DNA, comprising: a) providing a primer/template system comprising a template sequence hybridized to a primer oligonucleotide and a DNA polymerase that lacks 5′ to 3′ exonuclease activity;b) contacting the primer/template system with a single type of deoxyribonucleotide und
1. A method for sequencing DNA, comprising: a) providing a primer/template system comprising a template sequence hybridized to a primer oligonucleotide and a DNA polymerase that lacks 5′ to 3′ exonuclease activity;b) contacting the primer/template system with a single type of deoxyribonucleotide under conditions that produce a detectable signal when the DNA polymerase incorporates a deoxyribonucleotide onto the 3′ end of the primer oligonucleotide, wherein the single type of deoxyribonucleotide is an unlabeled and unblocked deoxyribonucleotide, and wherein the contacting occurs in a reaction chamber;c) converting, with a device, the detectable signal into an electrical signal based on an electrical potential generated across the device by the detectable signal, wherein the converting occurs in the reaction chamber, and wherein the amplitude of the electrical signal corresponds to the number of nucleotides incorporated onto the 3′ end of the primer oligonucleotide by the DNA polymerase; andd) detecting the electrical signal generated by the detectable signal produced in the reaction chamber. 2. The method of claim 1, further comprising (e) measuring the electrical signal with a voltmeter. 3. The method of claim 1, further comprising (e) flushing the reaction chamber with a dNTP free buffer. 4. The method of claim 3, further comprising repeating steps (b) through (e). 5. The method of claim 3, further comprising repeating steps (b) through (e) until the complete nucleotide sequence of the template sequence is determined. 6. The method of claim 1, wherein step (b) further comprises: introducing into, and evacuating from, the reaction chamber at least one reagent selected from the group consisting of: buffers, electrolytes, DNA template, DNA primer, deoxyribonucleotides, and polymerase enzymes. 7. The method of claim 1, wherein the deoxyribonucleotide is not chemically modified. 8. The method of claim 1, wherein the reaction chamber further includes a solid support. 9. The method of claim 8, wherein the template sequence is tethered to the solid support. 10. The method of claim 9, wherein the template sequence comprises a linker moiety for tethering to the solid support. 11. The method of claim 1, wherein the electrical signal provides real-time detection of incorporation of the deoxyribonucleotide monophosphate onto the primer strand by the DNA polymerase.
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