Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12Q-001/68
C12P-019/34
C07H-021/02
출원번호
US-0969872
(2010-12-16)
등록번호
US-9096898
(2015-08-04)
발명자
/ 주소
Williams, Peter
Taylor, Thomas J.
Williams, Daniel J. B.
Gould, Ian
Hayes, Mark A.
출원인 / 주소
LIFE TECHNOLOGIES CORPORATION
인용정보
피인용 횟수 :
12인용 특허 :
348
초록▼
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.
대표청구항▼
1. A method of analyzing a nucleic acid molecule, the method comprising: (a) providing a template/primer duplex;(b) contacting the duplex with a single type of unlabeled and unblocked deoxyribonucleotide in the presence of a polymerase under conditions that allow extension of the primer by incorpora
1. A method of analyzing a nucleic acid molecule, the method comprising: (a) providing a template/primer duplex;(b) contacting the duplex with a single type of unlabeled and unblocked deoxyribonucleotide in the presence of a polymerase under conditions that allow extension of the primer by incorporation of at least one deoxyribonucleotide to the 3′ end of the primer, wherein the contacting occurs in a reaction cell; and(c) non-optically detecting in the reaction cell whether extension of the primer has occurred. 2. The method according to claim 1, further comprising detecting the number of deoxyribonucleotides incorporated into the primer. 3. The method according to claim 2, further comprising removing unincorporated deoxyribonucleotide. 4. The method according to claim 3, further comprising repeating steps (b) and (c) at least once. 5. The method according to claim 4, wherein the repeating step is conducted with a different nucleotide than previously introduced to the duplex. 6. The method according to claim 1, wherein the duplex is constrained within a reaction cell. 7. The method according to claim 1, wherein the duplex is immobilized to a solid support. 8. A method of analyzing a nucleic acid molecule, the method comprising: (a) providing a template/primer duplex;(b) contacting the duplex with a single type of unlabeled and unblocked deoxyribonucleotide in the presence of a polymerase under conditions that allow extension of the primer by incorporation of at least one deoxyribonucleotide to the 3′ end of the primer, wherein contacting occurs in a reaction cell; and(c) detecting a physical or chemical change within the reaction cell that is associated with incorporation of the deoxyribonucleotide into the duplex, thereby determining whether extension of the primer has occurred. 9. The method according to claim 8, further comprising detecting the number of deoxyribonucleotides incorporated into the primer. 10. The method according to claim 9, further comprising removing unincorporated deoxyribonucleotide. 11. The method according to claim 10, further comprising repeating steps (b) and (c) at least once. 12. The method according to claim 11, wherein the repeating step is conducted with a different nucleotide than previously introduced to the duplex. 13. The method according to claim 8, wherein the duplex is constrained within a reaction cell. 14. The method according to claim 13, wherein a localized region of the reaction cell detects the physical or chemical change associated with incorporation of the deoxyribonucleotide into the duplex. 15. The method according to claim 8, wherein the duplex is immobilized to a solid support. 16. A method of analyzing a nucleic acid molecule, the method comprising: (a) providing a template/primer duplex;(b) contacting the duplex with a single type of unlabeled and unblocked deoxyribonucleotide in the presence of a polymerase under conditions that allow extension of the primer by incorporation of at least one deoxyribonucleotide to the 3′ end of the primer, wherein contacting occurs in a reaction cell; and(c) non-optically detecting within the reaction cell a reaction by-product associated with incorporation of the deoxyribonucleotide into the duplex, thereby determining whether extension of the primer has occurred. 17. The method according to claim 16, further comprising detecting the number of deoxyribonucleotides incorporated into the primer. 18. The method according to claim 17, further comprising removing unincorporated deoxyribonucleotide. 19. The method according to claim 18, further comprising repeating steps (b) and (c) at least once. 20. The method according to claim 19, wherein the repeating step is conducted with a different nucleotide than previously introduced to the duplex. 21. The method according to claim 16, wherein the duplex is constrained within a reaction cell. 22. The method according to claim 21, wherein a localized region of the reaction cell detects the reaction by-product associated with incorporation of the deoxyribonucleotide into the duplex. 23. The method according to claim 16, wherein the duplex is immobilized to a solid support. 24. A method of analyzing a nucleic acid molecule, the method comprising: (a) providing a template/primer duplex;(b) contacting the duplex with a single type of unlabeled and unblocked deoxyribonucleotide in the presence of a polymerase under conditions that allow extension of the primer by incorporation of at least one deoxyribonucleotide to the 3′ end of the primer, wherein contacting occurs in a reaction cell; and(c) directly detecting in the reaction cell whether extension of the primer has occurred. 25. The method according to claim 24, further comprising detecting the number of deoxyribonucleotides incorporated into the primer. 26. The method according to claim 25, further comprising removing unincorporated deoxyribonucleotide. 27. The method according to claim 26, further comprising repeating steps (b) and (c) at least once. 28. The method according to claim 27, wherein the repeating step is conducted with a different nucleotide than previously introduced to the duplex. 29. The method according to claim 24, wherein the duplex is constrained within a reaction cell. 30. The method according to claim 24, wherein the duplex is immobilized to a solid support. 31. The method according to claim 1, wherein the step of non-optically detecting in the reaction cell comprises: converting the non-optical signal into an electrical signal in the reaction cell; andmeasuring said electrical signal with a voltmeter. 32. The method according to claim 8, wherein the step of detecting the physical or chemical change within in the reaction cell comprises: converting the physical or chemical change within the reaction chamber into an electrical signal in the reaction cell; andmeasuring said electrical signal with a voltmeter. 33. The method according to claim 16, wherein the step of non-optically detecting in the reaction cell comprises: converting the non-optical signal into an electrical signal in the reaction cell; andmeasuring said electrical signal with a voltmeter. 34. The method according to claim 24, wherein the step of non-optically detecting in the reaction cell comprises: producing a non-optical signal when a DNA polymerase enzyme incorporates said unlabeled and unblocked deoxyribonucleotide monophosphate onto the 3′ end of the primer strand;converting the non-optical signal into an electrical signal in the reaction cell; andmeasuring said electrical signal with a voltmeter.
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이 특허에 인용된 특허 (348)
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