Droplet-based nucleic acid amplification method and apparatus

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.