Droplet-Based Pyrosequencing

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.

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.