Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

• US 9,096,898 B2
• Filed: 12/16/2010
• Issued: 08/04/2015
• Est. Priority Date: 05/01/1998
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Williams, Peter, Taylor, Thomas J., Williams, Daniel J. B., Gould, Ian, Hayes, Mark A.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US12/969,872
• Publication Number:
  US 20110183321A1
• Time in Patent Office: 1,692 Days
• Field of Search: 435/6.1, 435/91.1, 435/91.2, 435/6, 536/23.1
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2563/103 luminescence
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/6863 Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

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.

Claims

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.

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; and

measuring said electrical signal with a voltmeter.

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.

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; and

measuring said electrical signal with a voltmeter.

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.

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; and

measuring said electrical signal with a voltmeter.

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.

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; and

measuring said electrical signal with a voltmeter.