Droplet-based pyrosequencing

• US 9,243,282 B2
• Filed: 05/14/2012
• Issued: 01/26/2016
• Est. Priority Date: 04/18/2006
• Status: Expired due to Fees

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated), Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated), Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Pamula, Vamsee K., Fair, Richard B., Griffin, Peter B., Pollack, Michael G.
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US13/470,919
• Publication Number:
  US 20130164742A1
• Time in Patent Office: 1,352 Days
• US Class Current: 1/1
• 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 droplet-based pyrosequencing including a method of identifying a base at a target position in a sample nucleic acid. The method includes: (a) providing a droplet microactuator including a first droplet including a sample nucleic acid immobilized on a bead; and (b) on the droplet microactuator: (i) contacting the first droplet with one or more reagent droplets to yield a second droplet, wherein the one or more reagent droplets include reagents for extending a double stranded portion of the sample nucleic acid by incorporating a nucleotide at the target position; (ii) splitting the second droplet to yield a third droplet including the bead and a fourth droplet lacking the bead; and (iii) assaying the third droplet to determine whether the nucleotide was incorporated at the target position.

Claims

1. 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 first droplet comprising a sample nucleic acid immobilized on a bead; and

(b) on the droplet microactuator;

(i) contacting the first droplet with one or more reagent droplets to yield a second droplet, wherein the one or more reagent droplets comprise reagents for extending a double stranded portion of the sample nucleic acid by incorporating a nucleotide at the target position, wherein incorporation of a nucleotide complementary to the base at a target position produces a detectable signal;

(ii) splitting the second droplet to yield a third droplet comprising the bead and a fourth droplet lacking the bead; and

(iii) assaying the fourth droplet to determine whether the nucleotide was incorporated at the target position, wherein the assay comprises methods for detecting the detectable signal, further wherein detection of the detectable signal is indicative of incorporation of a nucleotide at a target position and identification of a base complementary thereto.

2. The method of claim 1 wherein one or more of the reagent droplets comprises an extension primer, which hybridizes to the sample nucleic acid to form the double-stranded portion of the sample nucleic acid.

3. The method of claim 1 wherein one or more of the reagent droplets comprises a polymerase.

4. The method of claim 1 wherein one or more of the reagent droplets comprises a deoxynucleotide or dideoxynucleotide selected to incorporate at the target position yielding the incorporated nucleotide.

5. The method of claim 1 wherein incorporating a nucleic acid at the target position comprises extending the double stranded portion of the sample nucleic acid and releasing pyrophosphate (PPi) within the droplet if the nucleotide is complementary to a base immediately adjacent to the double stranded portion of the sample nucleic acid.

6. The method of claim 1 wherein assaying the fourth droplet comprises contacting the third droplet with one or more droplets comprising PPi-detection enzyme(s) to yield a detection droplet.

7. The method of claim 1 wherein assaying the fourth droplet comprises quantifying PPi released within the second droplet.

8. The method of claim 1 wherein the bead is magnetically responsive.

9. The method of claim 1 wherein the bead is not magnetically responsive.

10. The method of claim 1 wherein assaying the fourth droplet comprises reacting ATP to produce photons.

11. The method of claim 1 wherein assaying the fourth droplet comprises conducting a luciferase-mediated reaction.

12. The method of claim 1 wherein assaying the fourth droplet comprises quantifying ATP.

13. The method of claim 1 wherein assaying the fourth droplet comprises quantifying PPi.

14. The method of claim 1 wherein assaying the fourth droplet comprises detecting PPi by an enzymatic luminometric inorganic pyrophosphate detection assay.

15. The method of claim 1 wherein the method comprises transporting the fourth droplet into the presence of a photodetector and conducting the assaying in the presence of the photodetector.

16. The method of claim 1 wherein:

(a) the bead is magnetically responsive; and

(b) splitting the second droplet to yield a third droplet comprises restraining the magnetically-responsive bead during an electrowetting-mediated droplet operation.

17. The method of claim 16 wherein the droplet is surrounded by an oil filler fluid.

18. The method of claim 17 wherein the droplet is compressed between two substrates of the droplet actuator.

19. The method of claim 17 wherein the filler fluid comprises a surfactant.

20. The method of claim 17 wherein the droplet microactuator comprises electrodes arranged for conducting droplet operations and the splitting is mediated by the electrodes.

21. The method of claim 17 wherein the splitting is electrowetting mediated.

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

(a) providing a droplet microactuator comprising sample single stranded nucleic acid immobilized on one or more beads in a bead-containing droplet on the droplet microactuator;

(b) contacting, using droplet operations mediated by the droplet microactuator, the bead-containing droplet with one or more reagent droplets to yield a reaction droplet;

(c) extending a double stranded portion of the sample nucleic acid and release pyrophosphate (PPi) within the reaction droplet;

(d) transporting, using droplet operations mediated by the droplet microactuator, a droplet away from the one or more beads and contacting the transported droplet potentially including released PPi with droplets comprising detection enzyme(s) to yield a detection droplet; and

(e) quantifying PPi released within the detection droplet, wherein release of PPi is indicative of incorporation of deoxynucleotide or dideoxynucleotide and the identification of a base complementary thereto.

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

(a) providing a droplet microactuator comprising electrodes and a first droplet comprising a sample nucleic acid immobilized on a bead; and

(b) on the droplet microactuator;

(i) contacting the first droplet with one or more reagent droplets to yield a second droplet, wherein the one or more reagent droplets comprise reagents for extending a double stranded portion of the sample nucleic acid by incorporating a nucleotide at the target position, wherein incorporation of a nucleotide complementary to the base at a target position produces a detectable signal;

(ii) splitting the second droplet to yield a third droplet comprising the bead and a fourth droplet lacking the bead, wherein the splitting is mediated by the electrodes; and

(iii) assaying the fourth droplet to determine whether the nucleotide was incorporated at the target position, wherein the assay comprises methods for detecting the detectable signal, further wherein detection of the detectable signal is indicative of incorporation of a nucleotide at a target position and identification of a base complementary thereto.

24. 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 first droplet comprising a sample nucleic acid immobilized on a bead; and

(b) on the droplet microactuator;

(i) contacting the first droplet with one or more reagent droplets to yield a second droplet, wherein the one or more reagent droplets comprise reagents for extending a double stranded portion of the sample nucleic acid by incorporating a nucleotide at the target position, wherein incorporation of a nucleotide complementary to the base at a target position produces a detectable signal;

(ii) splitting the second droplet to yield a third droplet comprising the bead and a fourth droplet lacking the bead, wherein the splitting is electrowetting-mediated; and

(iii) assaying the fourth droplet to determine whether the nucleotide was incorporated at the target position, wherein the assay comprises methods for detecting the detectable signal, further wherein detection of the detectable signal is indicative of incorporation of a nucleotide at a target position and identification of a base complementary thereto.