THERMOELECTRIC METHOD OF SEQUENCING NUCLEIC ACIDS

• US 20090036317A1
• Filed: 07/31/2008
• Published: 02/05/2009
• Est. Priority Date: 07/31/2007
• Status: Active Grant

• Current Assignee: Eric Guilbeau
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: GUILBEAU, ERIC J.
• Granted Patent: US 8,043,814 B2
• Time in Patent Office: Days
• US Class Current: 506/6
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to a novel thermoelectric method for determining the sequence of nucleotides on a nucleic acid molecule through use of a thermopile and/or sequencing reagents flowing under the conditions of laminar flow. The methods disclosed herein involve the measurement of the heat generated by a deoxynucleotide incorporation event that can be accomplished without the need to control the temperature of any of a thermopile'"'"'s junctions.

Claims

1. A method of sequencing a nucleic acid comprising exposing a primer-hybridized nucleic acid template to a liquid flowing under conditions of laminar flow, wherein the primer comprises a 3′

end, and measuring heat generated or a temperature difference associated with incorporation of a known deoxynucleoside triphosphate into the 3′

end of the primer.

2. The method of claim 1, wherein the liquid comprises a polymerase and a known deoxynucleoside triphosphate.

3. The method of claim 1, further comprising obtaining the primer-hybridized nucleic acid template.

4. A method of sequencing a nucleic acid comprising:

(a) exposing a primer-hybridized nucleic acid template to a liquid comprising a polymerase and a known deoxynucleoside triphosphate; and

(b) measuring heat generated or difference in temperature, if any, as detected by a thermopile comprising at least one measuring junction and at least one reference junction.

5. The method of claim 4, wherein the thermopile is a thin-film thermopile or a bulk thermopile.

6. A method of sequencing a nucleic acid comprising:

(a) obtaining a primer-hybridized nucleic acid template, wherein the primer comprises a 3′

end;

(b) exposing the template to a liquid flowing under conditions of laminar flow, wherein the liquid comprises a polymerase and a known deoxynucleoside triphosphate;

(c) measuring heat generated or difference in temperature, if any, as detected by at least one thin-film thermopile comprising at least one first junction and at least one second junction, wherein the heat generated or the difference in temperature between the first and second junctions indicates incorporation of the known deoxynucleoside triphosphate into the 3′

end of the primer; and

either

(d) upon detecting heat or a difference in temperature, identifying the nucleotide of the template that is complementary to the known deoxynucleoside triphosphate incorporated into the primer, or

(e) upon failing to detect heat or a difference in temperature, eliminating the possibility that the nucleotide that is complementary to the known deoxynucleoside triphosphate is present in the template at the 3′

end of the primer.

7. The method of claim 6, wherein the primer-hybridized nucleic acid template is bound to a support.

8. The method of claim 7, wherein the support comprises a glass bead, a polymer bead, a glass microscope slide cover slip, a polymer microscope slide cover slip, a glass microscope slide, a polymer microscope slide, a DNA microarray fabrication glass support, a paramagnetic bead, a glass plate, or any combination thereof.

9. The method of claim 6, wherein the polymerase is selected from the group consisting of DNA polymerase I, DNA polymerase II, DNA polymerase III, Taq polymerase, T7 polymerase, T4 polymerase, Klenow polymerase and an exonuclease-deficient polymerase.

10. The method of claim 9, wherein the polymerase is an exonuclease-deficient polymerase.

11. The method of claim 6, wherein the known deoxynucleoside triphosphate is selected from the group consisting of dATP, dTTP, dGTP and dCTP, dATPα

S, and a tagged deoxynucleoside triphosphate.

12. The method of claim 6, wherein the first junction is a measuring junction and the second junction is a reference junction.

13. The method of claim 6, wherein the thin-film thermopile comprises at least 60 junction pairs.

14. The method of claim 13, wherein the thin-film thermopile comprises a material that provides an output of up to about 7.14 microvolts/milli-degree centigrade temperature difference.

15. The method of claim 6, wherein the thin-film thermopile comprises an electrically conductive metal.

16. The method of claim 6, wherein the thin-film thermopile is a bismuth/antimony thin thermopile or silicon/aluminum thin-film thermopile optionally doped with boron.

17. The method of claim 6, wherein the difference in temperature is between about 10⁻

4 and about 10<sup>−

5</sup> Kelvin.

18. The method of claim 6, wherein the liquid further comprises a pyrophosphatase.

19. The method of claim 18, wherein the difference in temperature is between about 10⁻

4 and about 10<sup>−

5</sup> Kelvin.

20. The method of claim 6, further comprising repeating steps (b)-(e) at least once.

27. The method of claim 6, wherein the thin-film thermopile comprises an electrically conductive metal.

21. An apparatus for sequencing a nucleic acid comprising:

(a) a flow channel adapted for laminar flow during use;

(b) a substrate adapted to receive an immobilized primer-hybridized nucleic acid template during use;

(c) a thin-film thermopile for temperature measurement during use.

22. The apparatus of claim 21, wherein a liquid flowing under conditions of laminar flow flows through the flow channel during use.

23. The apparatus of claim 22, wherein the liquid comprises a polymerase and a known deoxynucleoside triphosphate.

24. The apparatus of claim 23, wherein the liquid further comprises a pyrophosphatase.

25. The apparatus of claim 22, wherein pressure-driven or electrokinetic methods drive the flow of the liquid through the flow channel during use.

26. The apparatus of claim 21, wherein the substrate is positioned at one or more defined areas in the flow channel during use.

28. The apparatus of claim 21, wherein the thin-film thermopile is a bismuth/antimony thin-film thermopile or a silicon/aluminum thin-film thermopile optionally doped with boron.

29. The apparatus of claim 21, wherein the thin-film thermopile comprises at least one measuring junction and at least one reference junction.

30. The apparatus of claim 29, further comprising any one or more of the following:

(a) a signal detector;

(b) a signal amplifier;

(c) a signal storing device;

(d) a signal processor;

(e) a device for consecutively introducing a known deoxyribonucleotide triphosphate into the flow channel;

(f) a device for introducing pyrophosphatase into the flow channel;

(g) a device for introducing a rinse solution into the flow channel;

(h) a signal recorder; and

/or

(i) a signal display device,

wherein the signal correlates to the amount of heat generated or the temperature difference between the measuring and reference junctions.

33. A method of sequencing a nucleic acid comprising:

(a) introducing a primer-hybridized nucleic acid template into the flow channel of the apparatus of claim 21, wherein the primer comprises a 3′

end;

(b) introducing a liquid comprising a polymerase and a known deoxynucleoside triphosphate into the flow channel of the apparatus of claim 21; and

(c) subjecting the liquid to conditions of laminar flow.

34. The method of claim 33, wherein the thin-film thermopile of the apparatus of claim 21 comprises at least one measuring junction and at least one reference junction.

35. The method of claim 34, further comprising:

(d) measuring heat generated or a temperature difference between the measuring junction and the reference junction and either;

(e) upon detecting the heat generated or a difference in temperature, identifying the nucleotide of the template that is complementary to the known deoxynucleoside triphosphate incorporated into the 3′

end of the primer;

or

(f) upon failing to detect heat or a difference in temperature, eliminating the possibility that the nucleotide that is complementary to the known deoxynucleoside triphosphate is present in the template at the 3′

end of the primer.

36. The method of claim 35, further comprising repeating steps (b)-(f) at least once.

37. The method of claim 33, wherein the liquid further comprises a pyrophosphatase.

31. An apparatus for sequencing a nucleic acid comprising:

(a) a flow channel;

(b) a substrate adapted to receive an immobilized primer-hybridized nucleic acid template during use;

(c) a thin-film thermopile for heat or temperature measurement during use, wherein the thin-film thermopile comprises at least one measuring junction and at least one reference junction.

32. The apparatus of claim 31, wherein the flow channel is adapted for laminar flow, transitional flow, or turbulent flow during use.

38. A method of sequencing a nucleic acid comprising:

(a) introducing a primer-hybridized nucleic acid template into the flow channel of the apparatus of claim 31, wherein the primer comprises a 3′

end;

(b) introducing a liquid comprising a polymerase and a known deoxynucleoside triphosphate into the flow channel of the apparatus of claim 31;

(c) measuring heat generated or a temperature difference between the measuring junction and the reference junction and either;

(d) upon detecting the heat generated or difference in temperature, identifying the nucleotide of the template that is complementary to the known deoxynucleoside triphosphate incorporated into the 3′

end of the primer;

or

(e) upon failing to detect heat or a difference in temperature, eliminating the possibility that the nucleotide that is complementary to the known deoxynucleoside triphosphate is present in the template at the 3′

end of the primer.

39. The method of claim 38, wherein the liquid is subjected to the conditions of laminar flow.

40. The method of claim 38, wherein the liquid is subjected to conditions of transitional or turbulent flow.

41. The method of claim 38, further comprising repeating steps (b)-(e) at least once.