Droplet-Based Pyrosequencing
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.
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;
(ii) splitting the second droplet to yield a third droplet comprising 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.
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 third 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 third 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 third droplet comprises reacting ATP to produce photons.
11 . The method of claim 1 wherein assaying the third droplet comprises conducting a luciferase-mediated reaction.
12 . The method of claim 1 wherein assaying the third droplet comprises quantifying ATP.
13 . The method of claim 1 wherein assaying the third droplet comprises quantifying PPi.
14 . The method of claim 1 wherein assaying the third droplet comprises detecting PPi by an enzymatic luminometric inorganic pyrophosphate detection assay.
15 . The method of claim 1 wherein the method comprises transporting the third 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 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 droplets comprising detection enzyme(s) to yield a detection droplet; and
(e) quantifying PPi released within the reaction droplet, wherein release of PPi is indicative of incorporation of deoxynucleotide or dideoxynucleotide and the identification of a base complementary thereto.