Patent ID: 7727723

Claim:
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.