Droplet-based pyrosequencing
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.
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 sample single stranded nucleic acid immobilized on a substrate;
(b) on the droplet microactuator, contacting the immobilized sample nucleic acid with one or more reagent droplets to yield a reaction 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;
(c) extending 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;
(d) on the droplet microactuator, transporting away from the nucleic acid immobilized on the substrate a droplet potentially including released PPi and contacting the transported droplet potentially including released PPi with one or more droplets comprising PPi-detection enzyme(s) to yield a detection droplet; and
(e) detecting and 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.
2. The method of claim 1 wherein the substrate comprises a substrate on a surface of the droplet microactuator.
3. The method of claim 1 wherein the substrate comprises one or more beads in a droplet on the droplet microactuator.
4. The method of claim 3 wherein one or more of the beads is magnetically responsive.
5. The method of claim 3 wherein one or more of the beads is not magnetically responsive.
6. The method of claim 1 wherein the substrate is not held in a well.
7. The method of claim 1 wherein the substrate is not held in place by stabilizing beads.
8. The method of claim 1 wherein the substrate is located in a droplet microactuator reservoir.
9. The method of claim 1 further comprising:
(f) contacting the immobilized sample nucleic acid having the extended double stranded portion with a series of different deoxynucleotides or dideoxynucleotides, in the presence of the polymerase, thereby incorporating the deoxynucleotide or dideoxynucleotide into the extended double stranded portion of the sample nucleic acid when the deoxynucleotide or dideoxynucleotide is complementary to a base immediately adjacent to the extended double stranded portion, and thereby releasing pyrophosphate (PPi) within the droplet; and
(g) identifying the deoxynucleotides or dideoxynucleotides incorporated.
10. The method of claim 1 wherein any yield of PPi is detected by a means comprising a luciferase-luciferin-based reaction.
11. The method of claim 10 wherein the luciferase-luciferin-based reaction is conducted in the presence of a photodetector.
12. The method of claim 11 wherein the photodetector comprises a photomultiplier device.
13. The method of claim 10 wherein the luciferase-luciferin-based reaction is conducted on the droplet microactuator.
14. The method of claim 1 wherein any yield of PPi is detected by a means comprising an enzymatic luminometric inorganic pyrophosphate detection assay.
15. The method of claim 14 wherein the enzymatic luminometric inorganic pyrophosphate detection assay is conducted in the presence of a photodetector.
16. The method of claim 14 wherein the luminometric inorganic pyrophosphate detection assay is conducted on the droplet microactuator.
17. The method of claim 1 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.
18. The method of claim 9 wherein step (b)(ii) is accomplished by transporting a droplet on the droplet microactuator into contact with the gold surface.
19. The method of claim 1 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.
20. The method of claim 9 wherein step (b)(i) is accomplished by contacting a droplet comprising thiolating reagents with a droplet comprising a nucleic acid sample on the droplet microactuator.
21. The method of claim 1 wherein the polymerase is exonuclease deficient.
22. The method of claim 1 wherein the detecting is conducted on the droplet microactuator.
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 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 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;
(c) extending 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
(d) transporting, using droplet operations mediated by the droplet microactuator, a droplet potentially including released PPi away from the one or more beads and contacting the transported droplet potentially including released PPi with one or more droplets comprising PPi-detection enzyme(s) to yield a detection droplet; and
(e) detecting and 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.