Patent ID: 7851184

Claim:
A method of amplifying a nucleic acid in a sample, the method comprising: (a) providing a system comprising: (i) one or more sample droplets potentially comprising a target nucleic acid and at least partially surrounded by a filler fluid comprising an oil; (ii) one or more amplification reagent droplets comprising reagents for amplifying the target nucleic acid; (iii) a droplet microactuator comprising a substrate comprising electrodes for conducting electrowetting-mediated droplet operations on the substrate; (iv) a processor capable of executing instructions and electronically coupled to and controlling the electrodes; (v) one or more temperature control means arranged for heating a region of the substrate such that a droplet on the substrate comprising the electrodes can be transported by the droplet operations into the region for heating; and (vi) a magnet arranged to immobilize magnetically responsive beads in a droplet on the substrate during a droplet operation mediated by the electrodes; (b) combining, by conducting the droplet operations on the substrate comprising the electrodes, the one or more amplification reagent droplets and the one or more sample droplets to yield an amplification-ready droplet; (c) thermal cycling the amplification-ready droplet sufficient to result in amplification of a target nucleic acid when present in the amplification-ready droplet thereby yielding an amplified droplet, the thermal cycling comprising transporting, by conducting the droplet operations on the substrate comprising the electrodes, the amplification-ready droplet into and out of proximity with the region for heating; (d) merging, by conducting the droplet operations on the substrate comprising the electrodes, the amplified droplet comprising amplified target nucleic acid with a droplet comprising one or more magnetically responsive beads having an affinity for the amplified nucleic acid yielding a merged droplet thereby immobilizing amplified nucleic acid on the one or more magnetically responsive beads; and (e) executing a droplet-based washing protocol to separate unbound components of the merged droplet from the one or more magnetically responsive beads, wherein the droplet-based washing protocol comprises: (i) merging, by conducting the droplet operations on the substrate comprising the electrodes, a wash droplet with the droplet comprising the one or more magnetically responsive beads to yield a merged droplet; (ii) immobilizing the one or more magnetically responsive beads in the merged droplet using the magnet; and (iii) separating, b conducting the substrate comprising the electrodes, a droplet comprising unbound components and lacking the immobilized beads from the merged droplet thereby carrying away the unbound components from the beads.