Droplet-based pyrosequencing

• US 20090280475A1
• Filed: 12/15/2006
• Published: 11/12/2009
• Est. Priority Date: 04/18/2006
• Status: Active Grant

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated), Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Pamula, Vamsee K., Pollack, Michael G., Fair, Richard B., Griffin, Peter, Allen, Dwayne J.
• Granted Patent: US 7,727,723 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1894 Cooling means; Cryo cooling
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 3/502792 for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

The present invention relates to a droplet microactuator and to systems, apparatuses and methods employing the droplet microactuator for executing various protocols using droplets. The invention includes a droplet microactuator or droplet microactuator system having one or more input reservoirs loaded with reagents for conducting sequencing protocols, such as the reagents for conducting a pyrosequencing protocol. The invention also includes a droplet microactuator or droplet microactuator system, having one or more input reservoirs loaded with a sample for conducting a pyrosequencing protocol.

Claims

1. A droplet microactuator comprising:

(a) one or more sequencing reagent droplets; and

(b) optionally, one or more nucleic acid sample droplets.

2. The droplet microactuator of claim 1 comprising the one or more nucleic acid sample droplets.

3. The droplet microactuator of claim 1 wherein one or more of the sequencing reagent droplets comprise reagents selected from the group consisting of primer, polymerase, dNTP, sulfurylase, APS and combinations of any of the foregoing reagents.

4. The droplet microactuator of claim 1 comprising one or more reservoirs comprising the one or more sequencing reagent droplets.

5. The droplet microactuator of claim 1 comprising one or more reservoirs comprising the nucleic acid sample droplet.

6. The droplet microactuator of claim 1 wherein the sequencing reagent droplets comprise:

(a) a priming droplet comprising a primer;

(b) a sequencing droplet comprising a polymerase and dNTP or a functionally equivalent analogue; and

(c) a detecting droplet reagents for the detection of ATP.

7. The droplet microactuator of claim 6 wherein the reagents for the detection of ATP comprise sulfurylase and APS.

8. The droplet microactuator of claim 1 comprising four sequencing droplets, each droplet comprising:

(a) a polymerase; and

(b) a dNTP for a different nucleotide base.

9. A system comprising a processor electronically coupled to the droplet microactuator of claim 1 and programmed to execute one or more sequencing protocols.

10. The system of claim 9 programmed to execute one or more pyrosequencing protocols.

11. A method of identifying a base at a target position in a sample nucleic acid, the method comprising:

(a) providing a droplet microactuator comprising a sample single stranded nucleic acid immobilized on a substrate;

(b) contacting the immobilized sample nucleic acid with one or more reagent droplets to yield a sample droplet comprising;

(i) an extension primer, which hybridizes to the sample nucleic acid to form a double-stranded portion of the sample nucleic acid;

(ii) a polymerase; and

(iii) a deoxynucleotide or dideoxynucleotide selected to incorporate at the target position and extend the double stranded portion of the sample nucleic acid and release pyrophosphate (PPi) within the droplet if it is complementary to a base immediately adjacent to the double stranded portion of the sample nucleic acid; and

(c) together with step (b) or separately from step (b), detecting the quantity of PPi released within the sample droplet wherein release of PPi is indicative of incorporation of deoxynucleotide or dideoxynucleotide and the identification of a base complementary thereto.

12. The method of claim 11 wherein the substrate comprises a substrate on a surface of the droplet microactuator.

13. The method of claim 11 wherein the substrate comprises one or more beads on a droplet microactuator.

14. The method of claim 13 wherein one or more of the beads is magnetically responsive.

15. The method of claim 13 wherein one or more of the beads is not magnetically responsive.

16. The method of claim 11 wherein the substrate is not held in a well.

17. The method of claim 11 wherein the substrate is not held in place by stabilizing beads.

18. The method of claim 11 wherein the substrate is located in a droplet microactuator reservoir.

19. The method of claim 11 further comprising repeating steps 11(b) (omitting 11(b)(i)) and 11(c) with a series of different deoxynucleotides or dideoxynucleotides, thereby extending the double stranded portion of the sample nucleic acid and identifying the deoxynucleotides or dideoxynucleotides incorporated.

20. The method of claim 11 wherein step 11(c) comprises contacting the sample droplet with one or droplets comprising PPi-detection enzyme(s).

21. The method of claim 11 wherein step 11(c) comprises detecting any yield of PPi enzymatically in the same step as the polymerase reaction.

22. The method of claim 11 wherein step 11(c) comprises detecting any yield of PPi in a separate step from the polymerase reaction.

23. The method of claim 11 wherein the sample droplet is transported away from the immobilized sample nucleic acid prior to detection of any yield of PPi.

24. The method of claim 11 wherein any yield of PPi is detected by a means comprising a luciferase-luciferin-based reaction.

25. The method of claim 11 wherein any yield of PPi is detected by a means comprising an enzymatic luminometric inorganic pyrophosphate detection assay.

26. The method of claim 24 wherein the luciferase-luciferin-based reaction is conducted in the presence of a photodetector.

27. The method of claim 25 wherein the luciferase-luciferin-based reaction is conducted in the presence of a photodetector.

28. The method of claim 26 wherein the photodetector comprises a photomultiplier device.

29. The method of claim 11 wherein:

(a) the substrate of 11(a) comprises a gold surface; and

(b) the sample nucleic acid sample is immobilized by;

(i) thiolating the nucleic acid sample; and

(ii) depositing the thiolated nucleic acid on the gold surface.

30. The method of claim 11 wherein:

(a) the substrate of 11(a) comprises a glass surface; and

(b) the sample nucleic acid sample is immobilized by;

(i) silanating all or a portion of the glass surface to provide a silanated surface; and

(ii) chemically coupling a single-stranded nucleic acid to the silanated surface.

31. The method of claim 29 wherein step 29(b)(i) is accomplished by contacting a droplet comprising thiolating reagents with a droplet comprising a nucleic acid sample on the droplet microactuator.

32. The method of claim 29 wherein step 29(b)(ii) is accomplished by transporting a droplet on the droplet microactuator into contact with the gold surface.

33. The method of claim 11 wherein the polymerase is exonuclease deficient.

34. A method of identifying a base at a target position in a single-stranded sample nucleic acid, the method comprising:

(a) providing a surface comprising immobilized single-stranded sample nucleic acid;

(b) priming the immobilized single-stranded sample nucleic acid to yield a double stranded segment adjacent to a target nucleic acid;

(c) extending the double stranded segment using a series of nucleotides, each of which will release a detectable molecule or signal if it is complementary to the base adjacent to the target nucleic acid; and

(d) detecting the detectable molecule or signal to determine when a complementary nucleotide is incorporated.

35. The method of claim 34 wherein the nucleic acid comprises DNA.

36. The method of claim 34 wherein the detectable molecule comprises pyrophosphate (PPi)

37. The method of claim 34 wherein step 34(c) comprises a polymerase reaction in a droplet.

38. The method of claim 34 wherein:

(a) the detectable molecule comprises pyrophosphate (PPi);

(b) step 34(c) comprises a polymerase reaction in a droplet; and

(c) step 34(d) comprises detecting any yield of PPi enzymatically in the same step as the polymerase reaction.