WO2014106167A1

WO2014106167A1 - Digital microfluidic gene synthesis and error correction

Info

Publication number: WO2014106167A1
Application number: PCT/US2013/078263
Authority: WO
Inventors: Michael G. Pollack
Original assignee: Advanced Liquid Logic, Inc.
Priority date: 2012-12-31
Filing date: 2013-12-30
Publication date: 2014-07-03

Abstract

The invention provides methods for gene synthesis and error correction on a droplet actuator. In one embodiment, the invention provides a method for performing polymerase-mediated gene synthesis on a droplet actuator. The polymerase-mediated gene synthesis method uses oligonucleotide hybridization and PCR cycling to generate a pool of synthesized DNA strands, which may be subsequently enriched for the correct gene sequence.

Description

Digital Microfluidic Gene Synthesis and Error Correction

Related Applications

In addition to the patent applications cited herein, each of which is incorporated herein by reference, this patent application is related to and claims priority to U.S. Provisional Patent Application No. 61/747,605, filed on December 31, 2012, entitled "Digital Microfluidic Gene Synthesis and Error Correction", the entire disclosure of which is incorporated herein by reference.

Field of the Present Disclosure

The present disclosure relates to methods for gene synthesis and error correction on a droplet actuator. Specifically, the invention relates to a method for performing polymerase-mediated gene synthesis on a droplet actuator.

Background

A droplet actuator typically includes one or more substrates configured to form a surface or gap for conducting droplet operations. The one or more substrates establish a droplet operations surface or gap for conducting droplet operations and may also include electrodes arranged to conduct the droplet operations. The droplet operations substrate or the gap between the substrates may be coated or filled with a filler fluid that is immiscible with the liquid that forms the droplets.

Droplet actuators are used to conduct a variety of molecular protocols, such as amplification of nucleic acids (e.g., polymerase chain reaction (PCR)). In one application, PCR techniques are used in the synthesis of synthetic genes or long DNA biomolecules. In one example, a synthetic gene sequence is assembled using a set of oligonucleotide "building blocks." The oligonucleotide building blocks are hybridized in solution to form longer sequences and numerous cycles of PCR are performed to produce progressively longer and more complete DNA strands. However, the oligonucleotide building blocks used to construct the synthetic gene sequence often contain errors (e.g., deletion errors). If the oligonucleotide sequences contain errors, then the resulting pool of synthetic gene sequences will also contain errors. Typically, correct nucleotide sequences in a pool of synthesized DNA strands are separated from incorrect nucleotide sequences by gene cloning and sequencing, which are time-consuming and labor- intensive processes. Therefore, there is a need for new approaches for separating correct nucleotide sequences from incorrect nucleotide sequences in a pool of synthesized DNA strands.

Brief Description of the Present Disclosure

In one embodiment, the present disclosure provides a method for performing gene synthesis and error correction on a droplet actuator, the method comprising: a) performing a gene synthesis protocol on a droplet actuator to produce a pool of synthesized DNA strands, wherein the pool of synthesized DNA strands comprises both correct nucleotide sequences and incorrect nucleotide sequences; and b) performing an error correction protocol on the pool of synthesized DNA strands on the droplet-actuator, comprising selectively enriching DNA strands comprising the correct nucleotide sequences.

In one embodiment, the gene synthesis protocol comprises: a) transferring a droplet comprising synthetic oligonucleotide sequences designed for a DNA molecule of interest to a sample preparation reservoir of a droplet actuator; b) hybridizing the synthetic oligonucleotide sequences to generate hybridized oligonucleotides; c) filling-in gaps in the hybridized oligonucleotides using DNA polymerase and PCR cycling; and d) amplifying the assembled nucleotide sequences using amplification primers, thereby generating the pool of synthesized DNA strands. The pool of synthesized DNA strands may be generated off-actuator, particularly using phosphoramidite synthesis chemistry. A droplet comprising PCR reagents and the DNA polymerase may be combined with a droplet comprising the hybridized oligonucleotides using droplet operations, thereby generating a DNA assembly droplet, particularly wherein the PCR cycling comprises cyclically transporting the DNA assembly droplet between different temperature zones on the droplet actuator using droplet operations, more particularly wherein the DNA assembly droplet is PCR cycled until full-length DNA strands are obtained. One of the amplification primers may comprise a biotinylated primer, particularly wherein the biotinylated primer comprises a terminal oligonucleotide sequence used to design the DNA molecule of interest.

In another embodiment, a droplet including PCR reagents may be combined with the DNA assembly droplet using droplet operations, thereby generating a DNA amplification droplet. Amplification may be performed in a flow- through format wherein the amplification droplets are cyclically transported between different temperature zones using droplet operations, particularly wherein the different temperature zones comprise a zone of about 95°C and a zone of about 55°C. Excess amplification primers may be removed from the DNA amplification droplet by combining a droplet comprising a wash buffer and magnetically responsive beads with the amplification droplet using droplet operations, thereby generating a DNA capture droplet, particularly wherein the DNA capture droplet is transported into the presence of a magnet using droplet operations and performing a merge-and-split wash protocol to remove unbound material, thereby generating a washed DNA capture droplet. In another embodiment, the washed DNA capture droplet may be transported into a thermal zone using droplet operations to promote release of amplified DNA from the magnetically responsive beads, particularly wherein the thermal zone heats the washed DNA capture droplet to a temperature of about 65°C.

In another embodiment, the eluted amplified DNA may be transported away from the magnetically responsive beads, thereby generating an eluted amplified DNA droplet comprising biotinylated PCR amplicons. A droplet comprising streptavidin-coated magnetically responsive beads may be merged with the eluted amplified DNA droplet using droplet operations, thereby generating an amplified DNA/bead-comprising droplet. The amplified DNA/bead-comprising droplet may be transferred into a thermal zone using droplet operations for a period of time sufficient to promote formation of biotin-streptavidin complexes, particularly wherein the thermal zone heats the amplified DNA/bead-comprising droplet to a temperature of about 65°C. The biotinylated PCR amplicons may be immobilized on the streptavidin-coated magnetically responsive beads through formation of the biotin-streptavidin complexes.

In another embodiment, selectively enriching DNA strands comprising the correct nucleotide sequences may comprise priming DNA template strands in the pool of synthesized DNA strands and sequentially exposing primed DNA template strands to a nucleotide mix, wherein the nucleotide mix includes a nucleotide which is complementary to the next unpaired base of the correct nucleotide sequence and terminated versions of the other three nucleotides, particularly wherein the terminated versions of the other three nucleotides comprise dideoxy nucleotides.

In another embodiment, the error correction protocol comprises: a) providing a pool of biotinylated amplified DNA strands immobilized on magnetically responsive beads, thereby generating bead-bound amplified DNA strands; b) removing non-biotinylated DNA strands from the biotinylated amplified DNA strands; c) synthesizing the correct nucleotide sequences using prepared single stranded (ss) DNA templates immobilized on magnetically responsive beads, thereby generating full-length synthesized DNA strands complementary to the prepared ssDNA templates; d) recovering the DNA strands complementary to the prepared ssDNA templates from the magnetically responsive beads; and e) selectively enriching DNA strands comprising the correct nucleotide sequences using PCR amplification.

In another embodiment, the error correction protocol may further comprise hybridizing a sequencing primer to the bead-bound amplified DNA strands. The non-biotinylated DNA strands may be removed from the bead-bound amplified DNA strands by alkali denaturation, particularly wherein removing the non-biotinylated DNA strands from the bead-bound amplified DNA strands by alkali denaturation comprises: a) washing a droplet comprising the bead-bound amplified DNA strands with a first reagent droplet that comprises a denaturation solution, thereby generating a washed droplet comprising bead- bound amplified DNA strands; b) merging the washed droplet comprising bead-bound amplified DNA strands with a second reagent droplet and incubating for a period of time sufficient to denature DNA, thereby generating a ssDNA/bead-comprising droplet; and c) transporting the ssDNA/bead- comprising droplet into a magnetic field of a magnet using droplet operations and performing at least one bead washing protocol on the ssDNA /bead- comprising droplet. The washing step may comprise a merge-and-split protocol using droplet operations, particularly wherein the washed droplet comprises ead-bound amplified DNA strands and the second reagent droplet are incubated at ambient temperature. The at least one bead washing protocol may comprise a first bead washing protocol, wherein the first bead washing protocol may comprise an exchange of denaturation solution with a wash buffer in the ssDNA/bead-comprising droplet. Further, a second bead washing protocol may comprise an exchange of the wash buffer with an annealing buffer in the ssDNA/bead-comprising droplet. Removing non-biotinylated DNA strands from the biotinylated amplified DNA strands may comprise heat denaturation, particularly wherein heat denaturation comprises transporting the washed droplet comprising bead-bound amplified DNA strands to a thermal zone on the droplet actuator, more particularly wherein the thermal zone is about 95°C.

In another embodiment, the method further comprises combining the ssDNA/bead-comprising droplet with a droplet comprising primers, thereby generating an ssDNA template droplet. The ssDNA template droplet may be incubated at an annealing temperature for a period of time sufficient for annealing of primers to ssDNA, particularly at a temperature of about 80°C for a period of time of about two minutes. In another embodiment, the method may further comprise removing excess unbound primers from the ssDNA template droplet using a bead washing protocol, particularly wherein removing excess unbound primers from the ssDNA template droplet using the bead washing protocol comprises washing the ssDNA template droplet two times using polymerization buffer droplets. In another embodiment, synthesizing the correct nucleotide sequence comprises: a) combining the ssDNA template droplet with a reagent droplet, thereby generating a DNA synthesis droplet; b) incubating the DNA synthesis droplet for a period of time sufficient for incorporation of the nucleotides; c) transporting the DNA synthesis droplet into a magnetic field of a magnet using droplet operations; and d) washing the DNA synthesis droplet to remove unincorporated nucleotides. The reagent droplet may comprise the nucleotide which is complementary to the next unpaired base of the correct nucleotide sequence, the terminated versions of the other three nucleotides, a DNA polymerase, and a polymerization buffer. Washing the DNA synthesis droplet may comprise addition and removal of a polymerization buffer while retaining substantially all beads having a bound ssDNA template thereon in the droplet, particularly wherein unincorporated nucleotides are removed by enzymatic degradation.

In another embodiment, recovering the DNA strands complementary to the prepared ssDNA templates from the magnetically responsive beads comprises: a) transporting the DNA synthesis droplet to a thermal zone using droplet operations, wherein the DNA in the DNA synthesis droplet is denatured by heating, thereby producing single-stranded complementary DNA (cDNA) strands; and b) transporting the single-stranded cDNA strands away from the beads using droplet operations, thereby generating a cDNA droplet comprising a pool of single-stranded cDNA comprising full-length correct DNA strands and terminated incorrect DNA strands.

In another embodiment, selectively enriching DNA strands comprising the correct nucleotide sequences using PCR amplification comprises: a) adding primers to the DNA template strands in the pool of synthesized DNA strands, thereby producing primed DNA template strands; and b) sequentially exposing primed DNA template strands to a nucleotide mix, wherein the nucleotide mix comprises a nucleotide which is complementary to the next unpaired base of the correct nucleotide sequence and terminated versions of the other three nucleotides.

In another embodiment, selectively enriching DNA strands comprising the correct nucleotide sequences using PCR amplification may comprise: a) adding primers to the DNA template strands in the pool of synthesized DNA strands, thereby producing primed DNA template strands; b) sequentially exposing primed DNA template strands to a nucleotide mix, wherein the nucleotide mix includes a reversibly-terminated nucleotide which is complementary to the next unpaired base of the correct nucleotide sequence and irreversibly-terminated versions of the other three nucleotides; and c) adding a deblocking agent to the primed DNA template strands to remove the terminal blocking groups from the incorporated correct nucleotides prior to the next synthesis cycle. PCR amplification may comprise: a) adding PCR primers targeted to the ends of the correct nucleotide sequence; b) combining a droplet comprising PCR reagents with the cDNA droplet, thereby generating a reaction droplet; and c) conducting PCR amplification on the reaction droplet. The PCR amplification may be performed in a flow-through format wherein for each cycle the reaction droplets are cyclically transported between different temperature zones within the droplet actuator using droplet operations. A size-fraction method may also be used to selectively enrich full- length correct DNA strands from shorter, prematurely terminated incorrect DNA strands. Further, a solid-phase capture method may also be used to selectively enrich or deplete either the correct nucleotide sequences or the incorrect nucleotide sequences.

Brief Description of the Drawings

Figure 1 illustrates a flow diagram of an example of a protocol for synthesis of a DNA molecule of interest;

Figure 2 illustrates a flow diagram of an example of an error correction protocol for selectively enriching correct nucleotide sequences from incorrect nucleotide sequences in a pool of synthesized DNA strands; and

Figure 3 illustrates a functional block diagram of an example of a microfluidics system that includes a droplet actuator.

Definitions

As used herein, the following terms have the meanings indicated.

"Activate," with reference to one or more electrodes, means affecting a change in the electrical state of the one or more electrodes which, in the presence of a droplet, results in a droplet operation. Activation of an electrode can be accomplished using alternating or direct current. Any suitable voltage may be used. For example, an electrode may be activated using a voltage which is greater than about 150 V, or greater than about 200 V, or greater than about 250 V, or from about 275 V to about 1000 V, or about 300 V. Where alternating current is used, any suitable frequency may be employed. For example, an electrode may be activated using alternating current having a frequency from about 1 Hz to about 10 MHz, or from about 10 Hz to about 60 Hz, or from about 20 Hz to about 40 Hz, or about 30 Hz.

"Amplify," "amplification," "nucleic acid amplification," or the like, refers to the production of multiple copies of a nucleic acid template (e.g., a template DNA molecule), or the production of multiple nucleic acid sequence copies that are complementary to the nucleic acid template (e.g., a template DNA molecule).

"Bead," with respect to beads on a droplet actuator, means any bead or particle that is capable of interacting with a droplet on or in proximity with a droplet actuator. Beads may be any of a wide variety of shapes, such as spherical, generally spherical, egg shaped, disc shaped, cubical, amorphous and other three dimensional shapes. The bead may, for example, be capable of being subjected to a droplet operation in a droplet on a droplet actuator or otherwise configured with respect to a droplet actuator in a manner which permits a droplet on the droplet actuator to be brought into contact with the bead on the droplet actuator and/or off the droplet actuator. Beads may be provided in a droplet, in a droplet operations gap, or on a droplet operations surface. Beads may be provided in a reservoir that is external to a droplet operations gap or situated apart from a droplet operations surface, and the reservoir may be associated with a flow path that permits a droplet including the beads to be brought into a droplet operations gap or into contact with a droplet operations surface. Beads may be manufactured using a wide variety of materials, including for example, resins, and polymers. The beads may be any suitable size, including for example, microbeads, microparticles, nanobeads and nanoparticles. In some cases, beads are magnetically responsive; in other cases beads are not significantly magnetically responsive. For magnetically responsive beads, the magnetically responsive material may constitute substantially all of a bead, a portion of a bead, or only one component of a bead. The remainder of the bead may include, among other things, polymeric material, coatings, and moieties which permit attachment of an assay reagent. Examples of suitable beads include flow cytometry microbeads, polystyrene microparticles and nanoparticles, functionalized polystyrene microparticles and nanoparticles, coated polystyrene microparticles and nanoparticles, silica microbeads, fluorescent microspheres and nanospheres, functionalized fluorescent microspheres and nanospheres, coated fluorescent microspheres and nanospheres, color dyed microparticles and nanoparticles, magnetic microparticles and nanoparticles, superparamagnetic microparticles and nanoparticles (e.g., DYNABEADS® particles, available from Invitrogen Group, Carlsbad, CA), fluorescent microparticles and nanoparticles, coated magnetic microparticles and nanoparticles, ferromagnetic microparticles and nanoparticles, coated ferromagnetic microparticles and nanoparticles, and those described in U.S. Patent Publication Nos. 20050260686, entitled "Multiplex flow assays preferably with magnetic particles as solid phase," published on November 24, 2005; 20030132538, entitled "Encapsulation of discrete quanta of fluorescent particles," published on July 17, 2003; 200501 18574, entitled "Multiplexed Analysis of Clinical Specimens Apparatus and Method," published on June 2, 2005; 20050277197. Entitled "Microparticles with Multiple Fluorescent Signals and Methods of Using Same," published on December 15, 2005; 20060159962, entitled "Magnetic Microspheres for use in Fluorescence-based Applications," published on July 20, 2006; the entire disclosures of which are incorporated herein by reference for their teaching concerning beads and magnetically responsive materials and beads. Beads may be pre-coupled with a biomolecule or other substance that is able to bind to and form a complex with a biomolecule. Beads may be pre- coupled with an antibody, protein or antigen, DNA/RNA probe or any other molecule with an affinity for a desired target. Examples of droplet actuator techniques for immobilizing magnetically responsive beads and/or non- magnetically responsive beads and/or conducting droplet operations protocols using beads are described in U.S. Patent Application No. 1 1/639,566, entitled "Droplet-Based Particle Sorting," filed on December 15, 2006; U.S. Patent Application No. 61/039, 183, entitled "Multiplexing Bead Detection in a Single Droplet," filed on March 25, 2008; U.S. Patent Application No. 61/047,789, entitled "Droplet Actuator Devices and Droplet Operations Using Beads," filed on April 25, 2008; U.S. Patent Application No. 61/086, 183, entitled "Droplet Actuator Devices and Methods for Manipulating Beads," filed on August 5, 2008; International Patent Application No. PCT/US2008/053545, entitled "Droplet Actuator Devices and Methods Employing Magnetic Beads," filed on February 11, 2008; International Patent Application No. PCT/US2008/058018, entitled "Bead-based Multiplexed Analytical Methods and Instrumentation," filed on March 24, 2008; International Patent Application No. PCT/US2008/058047, "Bead Sorting on a Droplet Actuator," filed on March 23, 2008; and International Patent Application No. PCT/US2006/047486, entitled "Droplet-based Biochemistry," filed on December 1 1, 2006; the entire disclosures of which are incorporated herein by reference. Bead characteristics may be employed in the multiplexing aspects of the invention. Examples of beads having characteristics suitable for multiplexing, as well as methods of detecting and analyzing signals emitted from such beads, may be found in U.S. Patent Publication No. 20080305481, entitled "Systems and Methods for Multiplex Analysis of PCR in Real Time," published on December 11, 2008; U.S. Patent Publication No. 20080151240, "Methods and Systems for Dynamic Range Expansion," published on June 26, 2008; U.S. Patent Publication No. 20070207513, entitled "Methods, Products, and Kits for Identifying an Analyte in a Sample," published on September 6, 2007; U.S. Patent Publication No. 20070064990, entitled "Methods and Systems for Image Data Processing," published on March 22, 2007; U.S. Patent Publication No. 20060159962, entitled "Magnetic Microspheres for use in Fluorescence-based Applications," published on July 20, 2006; U.S. Patent Publication No. 20050277197, entitled "Microparticles with Multiple Fluorescent Signals and Methods of Using Same," published on December 15, 2005; and U.S. Patent Publication No. 200501 18574, entitled "Multiplexed Analysis of Clinical Specimens Apparatus and Method," published on June 2, 2005. "Droplet" means a volume of liquid on a droplet actuator. Typically, a droplet is at least partially bounded by a filler fluid. For example, a droplet may be completely surrounded by a filler fluid or may be bounded by filler fluid and one or more surfaces of the droplet actuator. As another example, a droplet may be bounded by filler fluid, one or more surfaces of the droplet actuator, and/or the atmosphere. As yet another example, a droplet may be bounded by filler fluid and the atmosphere. Droplets may, for example, be aqueous or non-aqueous or may be mixtures or emulsions including aqueous and nonaqueous components. Droplets may take a wide variety of shapes; nonlimiting examples include generally disc shaped, slug shaped, truncated sphere, ellipsoid, spherical, partially compressed sphere, hemispherical, ovoid, cylindrical, combinations of such shapes, and various shapes formed during droplet operations, such as merging or splitting or formed as a result of contact of such shapes with one or more surfaces of a droplet actuator. For examples of droplet fluids that may be subjected to droplet operations using the approach of the invention, see International Patent Application No. PCT/US 06/47486, entitled, "Droplet-Based Biochemistry," filed on December 11, 2006. In various embodiments, a droplet may include a biological sample, such as whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, liquids containing multi- celled organisms, biological swabs and biological washes. Moreover, a droplet may include a reagent, such as water, deionized water, saline solutions, acidic solutions, basic solutions, detergent solutions and/or buffers. Other examples of droplet contents include reagents, such as a reagent for a biochemical protocol, such as a nucleic acid amplification protocol, an affinity-based assay protocol, an enzymatic assay protocol, a sequencing protocol, and/or a protocol for analyses of biological fluids. A droplet may include one or more beads. "Droplet Actuator" means a device for manipulating droplets. For examples of droplet actuators, see Pamula et al., U.S. Patent 6,911, 132, entitled "Apparatus for Manipulating Droplets by Electrowetting-Based Techniques," issued on June 28, 2005; Pamula et al, U.S. Patent Application No. 1 1/343,284, entitled "Apparatuses and Methods for Manipulating Droplets on a Printed Circuit Board," filed on filed on January 30, 2006; Pollack et al, International Patent Application No. PCT/US2006/047486, entitled "Droplet- Based Biochemistry," filed on December 1 1, 2006; Shenderov, U.S. Patents 6,773,566, entitled "Electrostatic Actuators for Microfluidics and Methods for Using Same," issued on August 10, 2004 and 6,565,727, entitled "Actuators for Microfluidics Without Moving Parts," issued on January 24, 2000; Kim and/or Shah et al, U.S. Patent Application Nos. 10/343,261, entitled "Electrowetting-driven Micropumping," filed on January 27, 2003, 1 1/275,668, entitled "Method and Apparatus for Promoting the Complete Transfer of Liquid Drops from a Nozzle," filed on January 23, 2006, 1 1/460, 188, entitled "Small Object Moving on Printed Circuit Board," filed on January 23, 2006, 12/465,935, entitled "Method for Using Magnetic Particles in Droplet Microfluidics," filed on May 14, 2009, and 12/513, 157, entitled "Method and Apparatus for Real-time Feedback Control of Electrical Manipulation of Droplets on Chip," filed on April 30, 2009; Velev, U.S. Patent 7,547,380, entitled "Droplet Transportation Devices and Methods Having a Fluid Surface," issued on June 16, 2009; Sterling et al, U.S. Patent 7, 163,612, entitled "Method, Apparatus and Article for Microfluidic Control via Electrowetting, for Chemical, Biochemical and Biological Assays and the Like," issued on January 16, 2007; Becker and Gascoyne et al, U.S. Patent Nos. 7,641,779, entitled "Method and Apparatus for Programmable fluidic Processing," issued on January 5, 2010, and 6,977,033, entitled "Method and Apparatus for Programmable fluidic Processing," issued on December 20, 2005; Deere et al, U.S. Patent 7,328,979, entitled "System for Manipulation of a Body of Fluid," issued on February 12, 2008; Yamakawa et al, U.S. Patent Pub. No. 20060039823, entitled "Chemical Analysis Apparatus," published on February 23, 2006; Wu, International Patent Pub. No. WO/2009/003184, entitled "Digital Microfluidics Based Apparatus for Heat- exchanging Chemical Processes," published on December 31, 2008; Fouillet et al., U.S. Patent Pub. No. 20090192044, entitled "Electrode Addressing Method," published on July 30, 2009; Fouillet et al, U.S. Patent 7,052,244, entitled "Device for Displacement of Small Liquid Volumes Along a Micro- catenary Line by Electrostatic Forces," issued on May 30, 2006; Marchand et al, U.S. Patent Pub. No. 20080124252, entitled "Droplet Microreactor," published on May 29, 2008; Adachi et al, U.S. Patent Pub. No. 20090321262, entitled "Liquid Transfer Device," published on December 31, 2009; Roux et al, U.S. Patent Pub. No. 20050179746, entitled "Device for Controlling the Displacement of a Drop Between two or Several Solid Substrates," published on August 18, 2005; Dhindsa et al, "Virtual Electrowetting Channels: Electronic Liquid Transport with Continuous Channel Functionality," Lab Chip, 10:832-836 (2010); the entire disclosures of which are incorporated herein by reference, along with their priority documents. Certain droplet actuators will include one or more substrates arranged with a droplet operations gap therebetween and electrodes associated with (e.g., layered on, attached to, and/or embedded in) the one or more substrates and arranged to conduct one or more droplet operations. For example, certain droplet actuators will include a base (or bottom) substrate, droplet operations electrodes associated with the substrate, one or more dielectric layers atop the substrate and/or electrodes, and optionally one or more hydrophobic layers atop the substrate, dielectric layers and/or the electrodes forming a droplet operations surface. A top substrate may also be provided, which is separated from the droplet operations surface by a gap, commonly referred to as a droplet operations gap. Various electrode arrangements on the top and/or bottom substrates are discussed in the above-referenced patents and applications and certain novel electrode arrangements are discussed in the description of the invention. During droplet operations it is preferred that droplets remain in continuous contact or frequent contact with a ground or reference electrode. A ground or reference electrode may be associated with the top substrate facing the gap, the bottom substrate facing the gap, in the gap. Where electrodes are provided on both substrates, electrical contacts for coupling the electrodes to a droplet actuator instrument for controlling or monitoring the electrodes may be associated with one or both plates. In some cases, electrodes on one substrate are electrically coupled to the other substrate so that only one substrate is in contact with the droplet actuator. In one embodiment, a conductive material (e.g., an epoxy, such as MASTER BOND™ Polymer System EP79, available from Master Bond, Inc., Hackensack, NJ) provides the electrical connection between electrodes on one substrate and electrical paths on the other substrates, e.g., a ground electrode on a top substrate may be coupled to an electrical path on a bottom substrate by such a conductive material. Where multiple substrates are used, a spacer may be provided between the substrates to determine the height of the gap therebetween and define dispensing reservoirs. The spacer height may, for example, be from about 5 μιη to about 600 μιη, or about 100 μιη to about 400 μιη, or about 200 μιη to about 350 μιη, or about 250 μιη to about 300 μιη, or about 275 μιη. The spacer may, for example, be formed of a layer of projections form the top or bottom substrates, and/or a material inserted between the top and bottom substrates. One or more openings may be provided in the one or more substrates for forming a fluid path through which liquid may be delivered into the droplet operations gap. The one or more openings may in some cases be aligned for interaction with one or more electrodes, e.g., aligned such that liquid flowed through the opening will come into sufficient proximity with one or more droplet operations electrodes to permit a droplet operation to be effected by the droplet operations electrodes using the liquid. The base (or bottom) and top substrates may in some cases be formed as one integral component. One or more reference electrodes may be provided on the base (or bottom) and/or top substrates and/or in the gap. Examples of reference electrode arrangements are provided in the above referenced patents and patent applications. In various embodiments, the manipulation of droplets by a droplet actuator may be electrode mediated, e.g., electrowetting-mediated or dielectrophoresis mediated or Coulombic force mediated. Examples of other techniques for controlling droplet operations that may be used in the droplet actuators of the invention include using devices that induce hydrodynamic fluidic pressure, such as those that operate on the basis of mechanical principles (e.g. external syringe pumps, pneumatic membrane pumps, vibrating membrane pumps, vacuum devices, centrifugal forces, piezoelectric/ultrasonic pumps and acoustic forces); electrical or magnetic principles (e.g. electroosmotic flow, electrokinetic pumps, ferrofluidic plugs, electrohydrodynamic pumps, attraction or repulsion using magnetic forces and magnetohydrodynamic pumps); thermodynamic principles (e.g. gas bubble generation/phase-change-induced volume expansion); other kinds of surface-wetting principles (e.g. electrowetting, and optoelectrowetting, as well as chemically, thermally, structurally and radioactively induced surface-tension gradients); gravity; surface tension (e.g., capillary action); electrostatic forces (e.g., electroosmotic flow); centrifugal flow (substrate disposed on a compact disc and rotated); magnetic forces (e.g., oscillating ions causes flow); magnetohydrodynamic forces; and vacuum or pressure differential. In certain embodiments, combinations of two or more of the foregoing techniques may be employed to conduct a droplet operation in a droplet actuator of the invention. Similarly, one or more of the foregoing may be used to deliver liquid into a droplet operations gap, e.g., from a reservoir in another device or from an external reservoir of the droplet actuator (e.g., a reservoir associated with a droplet actuator substrate and a flow path from the reservoir into the droplet operations gap). Droplet operations surfaces of certain droplet actuators of the invention may be made from hydrophobic materials or may be coated or treated to make them hydrophobic. For example, in some cases some portion or all of the droplet operations surfaces may be derivatized with low surface-energy materials or chemistries, e.g., by deposition or using in situ synthesis using compounds such as poly- or per- fluorinated compounds in solution or polymerizable monomers. Examples include TEFLON® AF (available from DuPont, Wilmington, DE), members of the cytop family of materials, coatings in the FLUOROPEL® family of hydrophobic and superhydrophobic coatings (available from Cytonix Corporation, Beltsville, MD), silane coatings, fluorosilane coatings, hydrophobic phosphonate derivatives (e.g.., those sold by Aculon, Inc), and NOVEC™ electronic coatings (available from 3M Company, St. Paul, MN), other fluorinated monomers for plasma-enhanced chemical vapor deposition (PECVD), and organosiloxane (e.g., SiOC) for PECVD. In some cases, the droplet operations surface may include a hydrophobic coating having a thickness ranging from about 10 nm to about 1,000 nm. Moreover, in some embodiments, the top substrate of the droplet actuator includes an electrically conducting organic polymer, which is then coated with a hydrophobic coating or otherwise treated to make the droplet operations surface hydrophobic. For example, the electrically conducting organic polymer that is deposited onto a plastic substrate may be poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS). Other examples of electrically conducting organic polymers and alternative conductive layers are described in Pollack et al, International Patent Application No. PCT/US2010/040705, entitled "Droplet Actuator Devices and Methods," the entire disclosure of which is incorporated herein by reference. One or both substrates may be fabricated using a printed circuit board (PCB), glass, indium tin oxide (ITO)- coated glass, and/or semiconductor materials as the substrate. When the substrate is ITO-coated glass, the ITO coating is preferably a thickness in the range of about 20 to about 200 nm, preferably about 50 to about 150 nm, or about 75 to about 125 nm, or about 100 nm. In some cases, the top and/or bottom substrate includes a PCB substrate that is coated with a dielectric, such as a polyimide dielectric, which may in some cases also be coated or otherwise treated to make the droplet operations surface hydrophobic. When the substrate includes a PCB, the following materials are examples of suitable materials: MITSUI™ BN-300 (available from MITSUI Chemicals America, Inc., San Jose CA); ARLON™ UN (available from Arlon, Inc, Santa Ana, CA).; NELCO® N4000-6 and N5000-30/32 (available from Park Electrochemical Corp., Melville, NY); ISOLA™ FR406 (available from Isola Group, Chandler, AZ), especially IS620; fluoropolymer family (suitable for fluorescence detection since it has low background fluorescence); polyimide family; polyester; polyethylene naphthalate; polycarbonate; polyetheretherketone; liquid crystal polymer; cyclo-olefin copolymer (COC); cyclo-olefin polymer (COP); aramid; THERMOUNT® nonwoven aramid reinforcement (available from DuPont, Wilmington, DE); NOMEX® brand fiber (available from DuPont, Wilmington, DE); and paper. Various materials are also suitable for use as the dielectric component of the substrate. Examples include: vapor deposited dielectric, such as PARYLENE™ C (especially on glass), PARYLENE™ N, and PARYLENE™ HT (for high temperature, ~300°C) (available from Parylene Coating Services, Inc., Katy, TX); TEFLON® AF coatings; cytop; soldermasks, such as liquid photoimageable soldermasks (e.g., on PCB) like ΤΑΓΥΟ™ PSR4000 series, TAIYO™ PSR and AUS series (available from Taiyo America, Inc. Carson City, NV) (good thermal characteristics for applications involving thermal control), and PROBIMER™ 8165 (good thermal characteristics for applications involving thermal control (available from Huntsman Advanced Materials Americas Inc., Los Angeles, CA); dry film soldermask, such as those in the VACREL® dry film soldermask line (available from DuPont, Wilmington, DE); film dielectrics, such as polyimide film (e.g., KAPTON® polyimide film, available from DuPont, Wilmington, DE), polyethylene, and fluoropolymers (e.g., FEP), polytetrafluoroethylene; polyester; polyethylene naphthalate; cyclo-olefin copolymer (COC); cyclo-olefin polymer (COP); any other PCB substrate material listed above; black matrix resin; polypropylene; and black flexible circuit materials, such as DuPont™ Pyralux® HXC and DuPont™ Kapton® MBC (available from DuPont, Wilmington, DE). Droplet transport voltage and frequency may be selected for performance with reagents used in specific assay protocols. Design parameters may be varied, e.g., number and placement of on-actuator reservoirs, number of independent electrode connections, size (volume) of different reservoirs, placement of magnets/bead washing zones, electrode size, inter-electrode pitch, and gap height (between top and bottom substrates) may be varied for use with specific reagents, protocols, droplet volumes, etc. In some cases, a substrate of the invention may derivatized with low surface-energy materials or chemistries, e.g., using deposition or in situ synthesis using poly- or per-fluorinated compounds in solution or polymerizable monomers. Examples include TEFLON® AF coatings and FLUOROPEL® coatings for dip or spray coating, other fluorinated monomers for plasma-enhanced chemical vapor deposition (PECVD), and organosiloxane (e.g., SiOC) for PECVD. Additionally, in some cases, some portion or all of the droplet operations surface may be coated with a substance for reducing background noise, such as background fluorescence from a PCB substrate. For example, the noise- reducing coating may include a black matrix resin, such as the black matrix resins available from Toray industries, Inc., Japan. Electrodes of a droplet actuator are typically controlled by a controller or a processor, which is itself provided as part of a system, which may include processing functions as well as data and software storage and input and output capabilities. Reagents may be provided on the droplet actuator in the droplet operations gap or in a reservoir fluidly coupled to the droplet operations gap. The reagents may be in liquid form, e.g., droplets, or they may be provided in a reconstitutable form in the droplet operations gap or in a reservoir fluidly coupled to the droplet operations gap. Reconstitutable reagents may typically be combined with liquids for reconstitution. An example of reconstitutable reagents suitable for use with the invention includes those described in Meathrel, et al, U.S. Patent 7,727,466, entitled "Disintegratable films for diagnostic devices," granted on June 1, 2010.

"Droplet operation" means any manipulation of a droplet on a droplet actuator. A droplet operation may, for example, include: loading a droplet into the droplet actuator; dispensing one or more droplets from a source droplet; splitting, separating or dividing a droplet into two or more droplets; transporting a droplet from one location to another in any direction; merging or combining two or more droplets into a single droplet; diluting a droplet; mixing a droplet; agitating a droplet; deforming a droplet; retaining a droplet in position; incubating a droplet; heating a droplet; vaporizing a droplet; cooling a droplet; disposing of a droplet; transporting a droplet out of a droplet actuator; other droplet operations described herein; and/or any combination of the foregoing. The terms "merge," "merging," "combine," "combining" and the like are used to describe the creation of one droplet from two or more droplets. It should be understood that when such a term is used in reference to two or more droplets, any combination of droplet operations that are sufficient to result in the combination of the two or more droplets into one droplet may be used. For example, "merging droplet A with droplet B," can be achieved by transporting droplet A into contact with a stationary droplet B, transporting droplet B into contact with a stationary droplet A, or transporting droplets A and B into contact with each other. The terms "splitting," "separating" and "dividing" are not intended to imply any particular outcome with respect to volume of the resulting droplets (i.e., the volume of the resulting droplets can be the same or different) or number of resulting droplets (the number of resulting droplets may be 2, 3, 4, 5 or more). The term "mixing" refers to droplet operations which result in more homogenous distribution of one or more components within a droplet. Examples of "loading" droplet operations include microdialysis loading, pressure assisted loading, robotic loading, passive loading, and pipette loading. Droplet operations may be electrode- mediated. In some cases, droplet operations are further facilitated by the use of hydrophilic and/or hydrophobic regions on surfaces and/or by physical obstacles. For examples of droplet operations, see the patents and patent applications cited above under the definition of "droplet actuator." Impedance or capacitance sensing or imaging techniques may sometimes be used to determine or confirm the outcome of a droplet operation. Examples of such techniques are described in Sturmer et al, U.S. Patent Application Publication No. US20100194408, entitled "Capacitance Detection in a Droplet Actuator," published on Aug. 5, 2010, the entire disclosures of which are incorporated herein by reference. Generally speaking, the sensing or imaging techniques may be used to confirm the presence or absence of a droplet at a specific electrode. For example, the presence of a dispensed droplet at the destination electrode following a droplet dispensing operation confirms that the droplet dispensing operation was effective. Similarly, the presence of a droplet at a detection spot at an appropriate step in an assay protocol may confirm that a previous set of droplet operations has successfully produced a droplet for detection. Droplet transport time can be quite fast. For example, in various embodiments, transport of a droplet from one electrode to the next may exceed about 1 sec, or about 0.1 sec, or about 0.01 sec, or about 0.001 sec. In one embodiment, the electrode is operated in AC mode but is switched to DC mode for imaging. It is helpful for conducting droplet operations for the footprint area of droplet to be similar to electrowetting area; in other words, lx-, 2x- 3x-droplets are usefully controlled operated using 1, 2, and 3 electrodes, respectively. If the droplet footprint is greater than the number of electrodes available for conducting a droplet operation at a given time, the difference between the droplet size and the number of electrodes should typically not be greater than 1 ; in other words, a 2x droplet is usefully controlled using 1 electrode and a 3x droplet is usefully controlled using 2 electrodes. When droplets include beads, it is useful for droplet size to be equal to the number of electrodes controlling the droplet, e.g., transporting the droplet. "Filler fluid" means a fluid associated with a droplet operations substrate of a droplet actuator, which fluid is sufficiently immiscible with a droplet phase to render the droplet phase subject to electrode-mediated droplet operations. For example, the droplet operations gap of a droplet actuator is typically filled with a filler fluid. The filler fluid may, for example, be or include a low- viscosity oil, such as silicone oil or hexadecane filler fluid. The filler fluid may be or include a halogenated oil, such as a fluorinated or perfluorinated oil. The filler fluid may fill the entire gap of the droplet actuator or may coat one or more surfaces of the droplet actuator. Filler fluids may be conductive or non-conductive. Filler fluids may be selected to improve droplet operations and/or reduce loss of reagent or target substances from droplets, improve formation of microdroplets, reduce cross contamination between droplets, reduce contamination of droplet actuator surfaces, reduce degradation of droplet actuator materials, etc. For example, filler fluids may be selected for compatibility with droplet actuator materials. As an example, fluorinated filler fluids may be usefully employed with fluorinated surface coatings. Fluorinated filler fluids are useful to reduce loss of lipophilic compounds, such as umbelliferone substrates like 6-hexadecanoylamido-4-methylumbelliferone substrates (e.g., for use in Krabbe, Niemann-Pick, or other assays); other umbelliferone substrates are described in U.S. Patent Pub. No. 201 10118132, published on May 19, 2011, the entire disclosure of which is incorporated herein by reference. Examples of suitable fluorinated oils include those in the Galden line, such as Galden HT170 (bp = 170 °C, viscosity = 1.8 cSt, density = 1.77), Galden HT200 (bp = 200C, viscosity = 2.4 cSt, d = 1.79), Galden HT230 (bp = 230C, viscosity = 4.4 cSt, d = 1.82) (all from Solvay Solexis); those in the Novec line, such as Novec 7500 (bp = 128C, viscosity = 0.8 cSt, d = 1.61), Fluorinert FC-40 (bp = 155 °C, viscosity = 1.8 cSt, d = 1.85), Fluorinert FC-43 (bp = 174 °C, viscosity = 2.5 cSt, d = 1.86) (both from 3M). In general, selection of perfluorinated filler fluids is based on kinematic viscosity (< 7 cSt is preferred, but not required), and on boiling point (> 150

°C is preferred, but not required, for use in DNA/RNA-based applications (PCR, etc.)). Filler fluids may, for example, be doped with surfactants or other additives. For example, additives may be selected to improve droplet operations and/or reduce loss of reagent or target substances from droplets, formation of microdroplets, cross contamination between droplets, contamination of droplet actuator surfaces, degradation of droplet actuator materials, etc. Composition of the filler fluid, including surfactant doping, may be selected for performance with reagents used in the specific assay protocols and effective interaction or non-interaction with droplet actuator materials. Examples of filler fluids and filler fluid formulations suitable for use with the invention are provided in Srinivasan et al, International Patent Pub. Nos. WO/2010/027894, entitled "Droplet Actuators, Modified Fluids and Methods," published on March 11, 2010, and WO/2009/021 173, entitled "Use of Additives for Enhancing Droplet Operations," published on February 12, 2009; Sista et al, International Patent Pub. No. WO/2008/098236, entitled "Droplet Actuator Devices and Methods Employing Magnetic Beads," published on August 14, 2008; and Monroe et al, U.S. Patent Publication No. 20080283414, entitled "Electrowetting Devices," filed on May 17, 2007; the entire disclosures of which are incorporated herein by reference, as well as the other patents and patent applications cited herein. Fluorinated oils may in some cases be doped with fluorinated surfactants, e.g., Zonyl FSO-100 (Sigma-Aldrich) and/or others.

"Immobilize" with respect to magnetically responsive beads, means that the beads are substantially restrained in position in a droplet or in filler fluid on a droplet actuator. For example, in one embodiment, immobilized beads are sufficiently restrained in position in a droplet to permit execution of a droplet splitting operation, yielding one droplet with substantially all of the beads and one droplet substantially lacking in the beads.

"Magnetically responsive" means responsive to a magnetic field. "Magnetically responsive beads" include or are composed of magnetically responsive materials. Examples of magnetically responsive materials include paramagnetic materials, ferromagnetic materials, ferrimagnetic materials, and metamagnetic materials. Examples of suitable paramagnetic materials include iron, nickel, and cobalt, as well as metal oxides, such as Fe304, BaFel2019, CoO, NiO, Mn203, Cr203, and CoMnP. "Nucleic acid" as used herein means a polymeric compound comprising covalently linked subunits called nucleotides. A "nucleotide" is a molecule, or individual unit in a larger nucleic acid molecule, comprising a nucleoside (i.e., a compound comprising a purine or pyrimidine base linked to a sugar, usually ribose or deoxyribose) linked to a phosphate group.

"Polynucleotide" or "oligonucleotide" or "nucleic acid molecule" are used interchangeably herein to mean the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules" or simply "RNA") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules" or simply "DNA"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single-stranded or double-stranded form. Polynucleotides comprising RNA, DNA, or RNA/DNA hybrid sequences of any length are possible. Polynucleotides for use in the present invention may be naturally-occurring, synthetic, recombinant, generated ex vivo, or a combination thereof, and may also be purified utilizing any purification methods known in the art. Accordingly, the term "DNA" includes but is not limited to genomic DNA, plasmid DNA, synthetic DNA, semisynthetic DNA, complementary DNA ("cDNA"; DNA synthesized from a messenger RNA template), and recombinant DNA (DNA that has been artificially designed and therefore has undergone a molecular biological manipulation from its natural nucleotide sequence). A "gene" as used herein, refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.

"Polynucleotide fragment" as used herein means a polynucleotide of reduced length relative to a reference polynucleotide and comprising, over the common portion, a nucleotide sequence identical to that of the reference polynucleotide. Such a polynucleotide fragment may be, where appropriate, included in a larger polynucleotide of which it is a constituent. Such polynucleotide fragments comprise, or alternatively consist of, polynucleotides ranging in length from at least 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000 or 1500 consecutive nucleotides of a reference polynucleotide. Polynucleotide fragments include, for example, DNA fragments and R A fragments.

"Protocol" means a series of steps that includes, but is not limited to, droplet operations on one or more droplet microactuators and/or DNA synthesis or sequencing.

"Reservoir" means an enclosure or partial enclosure configured for holding, storing, or supplying liquid. A droplet actuator system of the invention may include on-cartridge reservoirs and/or off-cartridge reservoirs. On-cartridge reservoirs may be (1) on-actuator reservoirs, which are reservoirs in the droplet operations gap or on the droplet operations surface; (2) off-actuator reservoirs, which are reservoirs on the droplet actuator cartridge, but outside the droplet operations gap, and not in contact with the droplet operations surface; or (3) hybrid reservoirs which have on-actuator regions and off- actuator regions. An example of an off-actuator reservoir is a reservoir in the top substrate. An off-actuator reservoir is typically in fluid communication with an opening or flow path arranged for flowing liquid from the off-actuator reservoir into the droplet operations gap, such as into an on-actuator reservoir. An off-cartridge reservoir may be a reservoir that is not part of the droplet actuator cartridge at all, but which flows liquid to some portion of the droplet actuator cartridge. For example, an off-cartridge reservoir may be part of a system or docking station to which the droplet actuator cartridge is coupled during operation. Similarly, an off-cartridge reservoir may be a reagent storage container or syringe which is used to force fluid into an on-cartridge reservoir or into a droplet operations gap. A system using an off-cartridge reservoir will typically include a fluid passage means whereby liquid may be transferred from the off-cartridge reservoir into an on-cartridge reservoir or into a droplet operations gap. "Sequence identity" or "identity" in the context of nucleic acid sequences and as known in the art refers to the nucleic acid bases in two sequences that are the same when aligned for maximum correspondence over a specified comparison window. Thus, "percent sequence identity" refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference or template sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. Useful examples of percent sequence identities include, but are not limited to any integer percentage from 50% to 100%, in particular 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Sequence alignments and percent sequence identity calculations may be performed using methods and sequence analysis software known in the art, including but not limited to, the MegAlign™ program of the

LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.), multiple alignment using the Clustal method (Higgins and Sharp (1989) CABIOS. 5: 151-153) with the default parameters, including default parameters for pairwise alignments, the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.), BLASTP,

BLASTN, BLASTX (Altschul et al. (1990) J. Mol. Biol. 215:403-410, and DNASTAR (DNASTAR, Inc., Madison, Wis.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, the results of the analysis will be based on the default values of the program referenced, unless otherwise specified (i.e., any set of values or parameters which originally load with the software when first initialized).

"Transporting into the magnetic field of a magnet," "transporting towards a magnet," and the like, as used herein to refer to droplets and/or magnetically responsive beads within droplets, is intended to refer to transporting into a region of a magnetic field capable of substantially attracting magnetically responsive beads in the droplet. Similarly, "transporting away from a magnet or magnetic field," "transporting out of the magnetic field of a magnet," and the like, as used herein to refer to droplets and/or magnetically responsive beads within droplets, is intended to refer to transporting away from a region of a magnetic field capable of substantially attracting magnetically responsive beads in the droplet, whether or not the droplet or magnetically responsive beads is completely removed from the magnetic field. It will be appreciated that in any of such cases described herein, the droplet may be transported towards or away from the desired region of the magnetic field, and/or the desired region of the magnetic field may be moved towards or away from the droplet. Reference to an electrode, a droplet, or magnetically responsive beads being "within" or "in" a magnetic field, or the like, is intended to describe a situation in which the electrode is situated in a manner which permits the electrode to transport a droplet into and/or away from a desired region of a magnetic field, or the droplet or magnetically responsive beads is/are situated in a desired region of the magnetic field, in each case where the magnetic field in the desired region is capable of substantially attracting any magnetically responsive beads in the droplet. Similarly, reference to an electrode, a droplet, or magnetically responsive beads being "outside of or "away from" a magnetic field, and the like, is intended to describe a situation in which the electrode is situated in a manner which permits the electrode to transport a droplet away from a certain region of a magnetic field, or the droplet or magnetically responsive beads is/are situated away from a certain region of the magnetic field, in each case where the magnetic field in such region is not capable of substantially attracting any magnetically responsive beads in the droplet or in which any remaining attraction does not eliminate the effectiveness of droplet operations conducted in the region. In various aspects of the invention, a system, a droplet actuator, or another component of a system may include a magnet, such as one or more permanent magnets (e.g., a single cylindrical or bar magnet or an array of such magnets, such as a Halbach array) or an electromagnet or array of electromagnets, to form a magnetic field for interacting with magnetically responsive beads or other components on chip. Such interactions may, for example, include substantially immobilizing or restraining movement or flow of magnetically responsive beads during storage or in a droplet during a droplet operation or pulling magnetically responsive beads out of a droplet. "Washing" with respect to washing a bead means reducing the amount and/or concentration of one or more substances in contact with the bead or exposed to the bead from a droplet in contact with the bead. The reduction in the amount and/or concentration of the substance may be partial, substantially complete, or even complete. The substance may be any of a wide variety of substances; examples include target substances for further analysis, and unwanted substances, such as components of a sample, contaminants, and/or excess reagent. In some embodiments, a washing operation begins with a starting droplet in contact with a magnetically responsive bead, where the droplet includes an initial amount and initial concentration of a substance. The washing operation may proceed using a variety of droplet operations. The washing operation may yield a droplet including the magnetically responsive bead, where the droplet has a total amount and/or concentration of the substance which is less than the initial amount and/or concentration of the substance. Examples of suitable washing techniques are described in Pamula et al, U.S. Patent 7,439,014, entitled "Droplet-Based Surface Modification and Washing," granted on October 21, 2008, the entire disclosure of which is incorporated herein by reference.

The terms "top," "bottom," "over," "under," and "on" are used throughout the description with reference to the relative positions of components of the droplet actuator, such as relative positions of top and bottom substrates of the droplet actuator. It will be appreciated that the droplet actuator is functional regardless of its orientation in space.

When a liquid in any form (e.g., a droplet or a continuous body, whether moving or stationary) is described as being "on", "at", or "over" an electrode, array, matrix or surface, such liquid could be either in direct contact with the electrode/array/matrix/surface, or could be in contact with one or more layers or films that are interposed between the liquid and the electrode/array/matrix/surface. In one example, filler fluid can be considered as a film between such liquid and the electrode/array/matrix/surface.

When a droplet is described as being "on" or "loaded on" a droplet actuator, it should be understood that the droplet is arranged on the droplet actuator in a manner which facilitates using the droplet actuator to conduct one or more droplet operations on the droplet, the droplet is arranged on the droplet actuator in a manner which facilitates sensing of a property of or a signal from the droplet, and/or the droplet has been subjected to a droplet operation on the droplet actuator.

Description

The invention provides methods for gene synthesis and error correction on a droplet actuator. In one embodiment, the invention provides a method for performing polymerase-mediated gene synthesis on a droplet actuator. The polymerase-mediated gene synthesis method uses oligonucleotide hybridization and PCR cycling to generate a pool of synthesized DNA strands, which may be subsequently selectively enriched for the correct gene sequence.

Namely, the invention provides methods for selectively enriching correct nucleotide sequences from incorrect nucleotide sequences in a pool of synthesized DNA template strands. In one example, a primed DNA template strand is sequentially exposed to a nucleotide mix, wherein the nucleotide mix includes a nucleotide that is complementary to the next unpaired base of the correct nucleotide sequence and terminated versions of the other three nucleotides. In another example, a primed DNA template strand is sequentially exposed to a nucleotide mix, wherein the nucleotide mix includes a reversibly-terminated nucleotide that is complementary to the next unpaired base of the correct nucleotide sequence and irreversibly -terminated versions of the other three nucleotides.

"Correct DNA strand" or "intended DNA strand" as used herein means a DNA molecule comprising a correct nucleotide sequence. A "correct nucleotide sequence" as used herein is a nucleotide sequence having 100% sequence identity to the nucleotide sequence of a template DNA molecule or DNA molecule of interest. "Incorrect DNA strand" or "unintended DNA strand" as used herein means a DNA molecule comprising an incorrect nucleotide sequence. An "incorrect nucleotide sequence" as used herein is a nucleotide sequence having less than 100% sequence identity to the nucleotide sequence of a template DNA molecule or DNA molecule of interest. In one embodiment, amplification of DNA occurs using polymerase chain reaction ("PCR") cycling, which typically includes a heat denaturing step (wherein double stranded target UNA molecules are separated into two single- stranded target DNA molecules), an annealing step (wherein oligonucleotide primers complementary to the 3 ' boundaries of the target DNA molecules are annealed at low temperature), and a primer extension or elongation step (wherein DNA molecules are synthesized that are complementary to the single-stranded target DNA molecules via sequential nucleotide incorporation at the ends of the primers at an intermediate temperature). Typically, one set of these three consecutive steps is referred to as a "cycle."

7.1 Gene Synthesis and Error Correction Protocols

A common approach for construction of synthetic gene sequences or long DNA biomolecules is the polymerase-mediated synthesis method. Briefly, a collection of synthetic oligonucleotides are designed for a DNA molecule of interest such that the ends of each oligonucleotide overlap other oligonucleotides in the set. The oligonucleotides are hybridized in solution to form a connected chain. DNA polymerase is used to fill in the gaps in the hybridized chain and additional cycles of PCR are used to form progressively longer and more complete strands. At the end of this process, a pool of synthesized DNA strands that contain the desired nucleotide sequence has been constructed.

The oligonucleotides used to construct synthetic genes are typically synthesized by automated machines using phosphoramidite synthesis chemistry. This synthesis process is prone to producing oligonucleotides that contain errors (e.g., deletion errors). As the length of the oligonucleotide sequences are increased, the probability of the oligonucleotides containing errors is also increased. Typically, relatively long oligonucleotide sequences (e.g., 40 to 50 nucleotides in length), which are assembled more efficiently, are used to construct synthetic gene sequences. Because the oligonucleotides used to construct a nucleotide sequence of interest may contain errors, the resulting pool of synthesized DNA strands may also contain errors. The invention provides a method for performing a polymerase-mediated synthesis protocol on a droplet actuator. On-bench protocols for each step of a polymerase-mediated protocol may be adapted and described as a discrete step-by-step, droplet-based protocol.

The invention also provides methods of selectively enriching correct nucleotide sequences from incorrect nucleotide sequences in a pool of synthesized DNA strands. Namely, the methods use a pool of synthesized DNA strands as templates for synthesis of the intended (i.e., the correct) nucleotide sequences. In one example, a primed DNA template strand is sequentially exposed to a nucleotide mix, wherein the nucleotide mix includes a nucleotide which is complementary to the next unpaired base of the correct nucleotide sequence and terminated versions of the other three nucleotides. In another example, a primed DNA template strand is sequentially exposed to a nucleotide mix, wherein the nucleotide mix includes a reversibly -terminated nucleotide which is complementary to the next unpaired base of the correct nucleotide sequence and irreversibly-terminated versions of the other three nucleotides.

Digital microfluidic protocol steps are performed in aqueous droplets within an oil-filled droplet operations gap of a droplet actuator. Samples and assay reagents are manipulated as discrete droplets upon an arrangement of electrodes (i.e., digital electrowetting). Sample droplets and reagent droplets for use in conducting the various protocol steps may be dispensed and/or combined according to appropriate assay protocols using droplet operations on a droplet actuator. Incubation and washing of assay droplets, including temperature adjustments as needed, may also be performed on a droplet actuator. Further, each of these processes may be conducted while the droplet is partially or completely surrounded by a filler fluid on the droplet actuator.

7.1.1 Gene Synthesis Protocol

Figure 1 illustrates a flow diagram of an example of a protocol 100 for synthesis of a DNA molecule of interest. Protocol 100 uses a set of synthetic oligonucleotide sequences designed for a DNA molecule of interest and PCR cycling to generate a pool of synthesized DNA strands. Protocol 100 includes, but is not limited to, the following steps.

At a step 110, a collection of synthetic oligonucleotide sequences designed for a DNA molecule of interest are transferred to a sample preparation reservoir of a droplet actuator. In one example, the oligonucleotide sequences used to construct a DNA molecule of interest are synthesized off-actuator using amidite synthesis chemistry. The sample preparation reservoir may be adjusted to a certain temperature that is suitable for hybridization of the synthetic oligonucleotide sequences.

At a step 115, gaps in the hybridized oligonucleotides are filled-in using DNA polymerase and PCR cycling. A droplet including DNA polymerase and PCR reagents (e.g., dNTPs, buffer) may be combined using droplet operations with a hybridized oligonucleotide droplet to yield a DNA assembly droplet. PCR cycling (e.g., 55 cycles) may, for example, be performed in a flow-through format where for each cycle the DNA assembly droplet is cyclically transported using droplet operations between different temperature zones (e.g., between a 95°C zone and a 55°C zone) within the oil filled droplet actuator. Cycling with DNA polymerase results in the formation of increasingly longer DNA fragments until full-length DNA strands are obtained.

At a step 120, the assembled DNA strands are amplified to generate a pool of synthesized DNA strands. To provide a platform for subsequent digital microfluidic processing steps, one of the amplification primers may be biotinylated. In one example, the biotinylated primer may be one of the terminal oligonucleotide sequences used to assemble the nucleotide sequence of interest. The biotinylated oligonucleotide provides a ready method for anchoring the DNA strand to magnetically responsive beads, such as streptavidin-coated magnetic beads. A droplet including PCR reagents (e.g., dNTPs, enzyme, primers) may be combined using droplet operations with a DNA assembly droplet to yield a DNA amplification droplet. PCR amplification may, for example, be performed in a flow-through format where for each cycle the reaction droplets are cyclically transported using droplet operations between different temperature zones (e.g., between a 95°C zone and a 55°C zone) within the oil filled droplet actuator. To remove excess biotinylated primers from the DNA amplification droplet, a droplet including wash buffer and magnetically responsive beads (e.g., Dynabeads DNA DIRECT from Dynal) may be combined using droplet operations with a DNA amplification droplet to yield a DNA capture droplet. The DNA capture droplet may be transported using droplet operations into the presence of a magnet and washed using a merge-and-split wash protocol to remove unbound material. The washed DNA capture droplet may be transported using droplet operations into a thermal zone to promote release of DNA from the beads, e.g., by heating to approximately 65°C. The eluted amplified DNA contained in the droplet surrounding the beads may then be transported away from the beads to yield an eluted amplified DNA droplet. A droplet including streptavidin-coated magnetically responsive beads may be merged with the eluted amplified DNA droplet, yielding an amplified DNA/ bead-containing droplet. The amplified DNA/bead-containing droplet may be transported using droplet operations into a thermal zone (e.g., about 65 °C) for a period of time sufficient to promote formation of biotin-streptavidin complexes. The biotinylated PCR amplicons are immobilized on the beads through formation of biotin-streptavidin complexes. The pool of synthesized DNA strands includes both correct nucleotide sequences and incorrect nucleotide sequences.

7.1 .2 Error Correction Protocol

Figure 2 illustrates a flow diagram of an example of an error correction protocol 200 for selectively enriching correct nucleotide sequences from incorrect nucleotide sequences in a pool of synthesized DNA strands. Error correction protocol 200 uses a pool of biotinylated DNA strands as templates for synthesizing the intended (i.e., the correct) nucleotide sequence. A primed DNA template strand is sequentially exposed to a nucleotide mix, wherein the nucleotide mix includes a nucleotide that is complementary to the next unpaired base of the correct nucleotide sequence and terminated versions of the other three nucleotides. The terminated versions of the other three nucleotides cannot be extended further in the DNA synthesis reaction. In one example, the terminated nucleotides may be dideoxy nucleotides. The un- terminated nucleotides are incorporated into the correct DNA strand because the order in which they are delivered matches the correct nucleotide sequence. Any template that does not match the correct nucleotide sequence will incorporate a terminated nucleotide, which prevents further extension of that strand even if it differs from the correct nucleotide sequence by only one base pair.

In one example, a pool of biotinylated DNA strands immobilized on magnetically responsive beads are prepared on a droplet actuator, as described with reference to Figure 1. In another example, a pool of biotinylated DNA strands immobilized on magnetically responsive beads are prepared on-bench and subsequently loaded onto a sample reservoir of a droplet actuator. Protocol 200 includes, but is not limited to, the following steps.

At a step 210, single-strand (ss) DNA templates for DNA synthesis are prepared from a pool of biotinylated DNA strands. In this step, the non- biotinylated strands are removed from the amplified DNA strands and a sequencing primer is hybridized to the bead-bound template strands. In one example, the non-biotinylated strands are removed by alkali denaturation. An amplified DNA/ bead-containing droplet is washed using a merge-and-split protocol with a reagent droplet that contains a denaturation solution (e.g., 0.5 M sodium hydroxide (NaOH)). After washing, the amplified DNA/bead- containing droplet is merged with a second reagent droplet and incubated at ambient temperature for a period of time sufficient to denature DNA. The amplified DNA/bead-containing droplet that now has ssDNA bound therein is transported using droplet operations into the magnetic field of a magnet. A first bead washing protocol is used to exchange the denaturation solution in the ssDNA/bead-containing droplet with a wash buffer. A second washing protocol is used to exchange the wash buffer in the ssDNA/bead-containing droplet with an annealing buffer.

At a step 215, the non-biotinylated strands are removed from the amplified DNA strands by heat denaturation. The amplified DNA/bead-containing droplet is transported using droplet operations to a thermal zone on the droplet actuator to denature the DNA, e.g., by heating to approximately 95°C. The amplified DNA/ bead-containing droplet that now has ssDNA bound therein is transported using droplet operations into the magnetic field of a magnet and washed using a merge-and-split wash protocol to remove unbound material. A second washing protocol is used to exchange the wash buffer in the ssDNA/bead-containing droplet with an annealing buffer.

The ssDNA/bead-containing droplet is combined using droplet operations with a primer droplet to yield an ssDNA template droplet. The ssDNA template droplet is incubated at an annealing temperature (e.g., about 80°C) for a period of time (e.g., about 2 minute) sufficient for annealing of primer to ssDNA. After the incubation period, a bead washing protocol is used to remove excess unbound primers from the ssDNA template droplet. In one example, the ssDNA template droplet is washed two times using polymerization buffer droplets. The ssDNA template droplet in polymerization buffer is ready for DNA synthesis.

At a step 220, the intended (i.e., correct) nucleotide sequence is synthesized using the prepared ssDNA templates immobilized on magnetically responsive beads. An example of a DNA synthesis protocol is as follows. An ssDNA template droplet may be combined with a reagent droplet that includes the correct complementary nucleotide, terminated versions (e.g., dideoxy nucleotides) of the other three nucleotides and DNA polymerase in polymerization buffer to yield a DNA synthesis droplet. After a period of time sufficient for incorporation of the nucleotides, the DNA synthesis droplet may be transported to a magnet and washed to remove unincorporated nucleotides. Washing may be accomplished by addition and removal of polymerization buffer while retaining substantially all beads (with bound template thereon) in the droplet. In another example, the unincorporated nucleotides may be removed by enzymatic degradation. The cycle is repeated multiple times with a user defined sequence of nucleotide additions to generate a full-length complementary strand.

At a step 225, the synthesized complementary DNA strands are removed from the magnetically responsive beads. The DNA synthesis droplet may be transported using droplet operations into a thermal zone on the droplet actuator to denature the DNA, e.g., by heating to approximately 95°C. The single-strand complementary DNA (cDNA) strands contained in the droplet surrounding the beads may then be transported away from the beads to yield a cDNA droplet. This pool of single-stranded cDNA strands includes full- length correct DNA strands and terminated incorrect DNA strands.

At a step 230, PCR amplification is used to selectively enrich correct nucleotide sequences. PCR primers targeted to the ends of the correct nucleotide sequence are used to ensure that the terminated strands are not amplified (terminated strands will have only one of the primer sequences). A droplet including PCR reagents (e.g., dNTPs, enzyme, primers) may be combined using droplet operations with a single-stranded cDNA droplet to yield a reaction droplet. PCR amplification may, for example, be performed in a flow-through format where for each cycle the reaction droplets are cyclically transported using droplet operations between different temperature zones (e.g., between a 95°C zone and a 55°C zone) within the oil filled droplet actuator. The number of PCR cycles is selected to generate a sufficiently large number of correct nucleotide sequences relative to the number of prematurely terminated incorrect DNA strands.

In another example, a size-fraction method may be used to selectively enrich full-length correct DNA strands from shorter, prematurely terminated incorrect DNA strands.

In yet another example, a solid-phase capture method may be used to selectively enrich or deplete either the correct nucleotide sequences or the incorrect nucleotide sequences. For example, sequences which are complementary to the 3 '-end of the correctly synthesized strand may be coupled to magnetically responsive beads and used to capture the correct nucleotide sequences from the pool by hybridization.

In another embodiment, a primed DNA template strand is sequentially exposed to a nucleotide mix, wherein the nucleotide mix includes a reversibly- terminated nucleotide which is complementary to the next unpaired base of the correct nucleotide sequence and irreversibly-terminated versions of the other three nucleotides. In this example, the digital microfluidic protocol is substantially the same as the protocol 200 described with reference to Figure 2, except that a deblocking reaction is incorporated in the DNA synthesis step to remove the terminal blocking group from the incorporated correct nucleotide prior to the next synthesis cycle.

7.2 Systems

Figure 3 illustrates a functional block diagram of an example of a micro fluidics system 300 that includes a droplet actuator 305. Digital microfluidic technology conducts droplet operations on discrete droplets in a droplet actuator, such as droplet actuator 305, by electrical control of their surface tension (electro wetting). The droplets may be sandwiched between two substrates of droplet actuator 305, a bottom substrate and a top substrate separated by a droplet operations gap. The bottom substrate may include an arrangement of electrically addressable electrodes. The top substrate may include a reference electrode plane made, for example, from conductive ink or indium tin oxide (ITO). The bottom substrate and the top substrate may be coated with a hydrophobic material. Droplet operations are conducted in the droplet operations gap. The space around the droplets (i.e., the gap between bottom and top substrates) may be filled with an immiscible inert fluid, such as silicone oil, to prevent evaporation of the droplets and to facilitate their transport within the device. Other droplet operations may be effected by varying the patterns of voltage activation; examples include merging, splitting, mixing, and dispensing of droplets.

Droplet actuator 305 may be designed to fit onto an instrument deck (not shown) of micro fluidics system 300. The instrument deck may hold droplet actuator 305 and house other droplet actuator features, such as, but not limited to, one or more magnets and one or more heating devices. For example, the instrument deck may house one or more magnets 310, which may be permanent magnets. Optionally, the instrument deck may house one or more electromagnets 315. Magnets 310 and/or electromagnets 315 are positioned in relation to droplet actuator 305 for immobilization of magnetically responsive beads. Optionally, the positions of magnets 310 and/or electromagnets 315 may be controlled by a motor 320. Additionally, the instrument deck may house one or more heating devices 325 for controlling the temperature within, for example, certain reaction and/or washing zones of droplet actuator 305. In one example, heating devices 325 may be heater bars that are positioned in relation to droplet actuator 305 for providing thermal control thereof.

A controller 330 of microfluidics system 300 is electrically coupled to various hardware components of the invention, such as droplet actuator 305, electromagnets 315, motor 320, and heating devices 325, as well as to a detector 335, an impedance sensing system 340, and any other input and/or output devices (not shown). Controller 330 controls the overall operation of microfluidics system 300. Controller 330 may, for example, be a general purpose computer, special purpose computer, personal computer, or other programmable data processing apparatus. Controller 330 serves to provide processing capabilities, such as storing, interpreting, and/or executing software instructions, as well as controlling the overall operation of the system. Controller 330 may be configured and programmed to control data and/or power aspects of these devices. For example, in one aspect, with respect to droplet actuator 305, controller 330 controls droplet manipulation by activating/ deactivating electrodes .

In one example, detector 335 may be an imaging system that is positioned in relation to droplet actuator 305. In one example, the imaging system may include one or more light-emitting diodes (LEDs) (i.e., an illumination source) and a digital image capture device, such as a charge-coupled device (CCD) camera.

Impedance sensing system 340 may be any circuitry for detecting impedance at a specific electrode of droplet actuator 305. In one example, impedance sensing system 340 may be an impedance spectrometer. Impedance sensing system 340 may be used to monitor the capacitive loading of any electrode, such as any droplet operations electrode, with or without a droplet thereon. For examples of suitable capacitance detection techniques, see Sturmer et al, International Patent Publication No. WO/2008/101 194, entitled "Capacitance Detection in a Droplet Actuator," published on Aug. 21, 2008; and Kale et al, International Patent Publication No. WO/2002/080822, entitled "System and Method for Dispensing Liquids," published on Oct. 17, 2002; the entire disclosures of which are incorporated herein by reference.

Droplet actuator 305 may include disruption device 345. Disruption device 345 may include any device that promotes disruption (lysis) of materials, such as tissues, cells and spores in a droplet actuator. Disruption device 345 may, for example, be a sonication mechanism, a heating mechanism, a mechanical shearing mechanism, a bead beating mechanism, physical features incorporated into the droplet actuator 305, an electric field generating mechanism, a thermal cycling mechanism, and any combinations thereof. Disruption device 345 may be controlled by controller 330.

It will be appreciated that various aspects of the invention may be embodied as a method, system, computer readable medium, and/or computer program product. Aspects of the invention may take the form of hardware embodiments, software embodiments (including firmware, resident software, micro-code, etc.), or embodiments combining software and hardware aspects that may all generally be referred to herein as a "circuit," "module" or "system." Furthermore, the methods of the invention may take the form of a computer program product on a computer-usable storage medium having computer-usable program code embodied in the medium.

Any suitable computer useable medium may be utilized for software aspects of the invention. The computer-usable or computer-readable medium may be, for example but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, device, or propagation medium. The computer readable medium may include transitory and/or non-transitory embodiments. More specific examples (a non- exhaustive list) of the computer-readable medium would include some or all of the following: an electrical connection having one or more wires, a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), an optical fiber, a portable compact disc read-only memory (CD-ROM), an optical storage device, a transmission medium such as those supporting the Internet or an intranet, or a magnetic storage device. Note that the computer-usable or computer-readable medium could even be paper or another suitable medium upon which the program is printed, as the program can be electronically captured, via, for instance, optical scanning of the paper or other medium, then compiled, interpreted, or otherwise processed in a suitable manner, if necessary, and then stored in a computer memory. In the context of this document, a computer-usable or computer-readable medium may be any medium that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device.

Program code for carrying out operations of the invention may be written in an object oriented programming language such as Java, Smalltalk, C++ or the like. However, the program code for carrying out operations of the invention may also be written in conventional procedural programming languages, such as the "C" programming language or similar programming languages. The program code may be executed by a processor, application specific integrated circuit (ASIC), or other component that executes the program code. The program code may be simply referred to as a software application that is stored in memory (such as the computer readable medium discussed above). The program code may cause the processor (or any processor-controlled device) to produce a graphical user interface ("GUI"). The graphical user interface may be visually produced on a display device, yet the graphical user interface may also have audible features. The program code, however, may operate in any processor-controlled device, such as a computer, server, personal digital assistant, phone, television, or any processor-controlled device utilizing the processor and/or a digital signal processor.

The program code may locally and/or remotely execute. The program code, for example, may be entirely or partially stored in local memory of the processor-controlled device. The program code, however, may also be at least partially remotely stored, accessed, and downloaded to the processor- controlled device. A user's computer, for example, may entirely execute the program code or only partly execute the program code. The program code may be a stand-alone software package that is at least partly on the user's computer and/or partly executed on a remote computer or entirely on a remote computer or server. In the latter scenario, the remote computer may be connected to the user's computer through a communications network.

The invention may be applied regardless of networking environment. The communications network may be a cable network operating in the radio- frequency domain and/or the Internet Protocol (IP) domain. The communications network, however, may also include a distributed computing network, such as the Internet (sometimes alternatively known as the "World Wide Web"), an intranet, a local-area network (LAN), and/or a wide-area network (WAN). The communications network may include coaxial cables, copper wires, fiber optic lines, and/or hybrid-coaxial lines. The communications network may even include wireless portions utilizing any portion of the electromagnetic spectrum and any signaling standard (such as the IEEE 802 family of standards, GSM/CDMA/TDMA or any cellular standard, and/or the ISM band). The communications network may even include powerline portions, in which signals are communicated via electrical wiring. The invention may be applied to any wireless/wireline communications network, regardless of physical componentry, physical configuration, or communications standard(s).

Certain aspects of invention are described with reference to various methods and method steps. It will be understood that each method step can be implemented by the program code and/or by machine instructions. The program code and/or the machine instructions may create means for implementing the functions/acts specified in the methods.

The program code may also be stored in a computer-readable memory that can direct the processor, computer, or other programmable data processing apparatus to function in a particular manner, such that the program code stored in the computer-readable memory produce or transform an article of manufacture including instruction means which implement various aspects of the method steps. The program code may also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed to produce a processor/computer implemented process such that the program code provides steps for implementing various functions/acts specified in the methods of the invention.

Concluding Remarks

The foregoing detailed description of embodiments refers to the accompanying drawings, which illustrate specific embodiments of the present disclosure. Other embodiments having different structures and operations do not depart from the scope of the present disclosure. The term "the present disclosure" or the like is used with reference to certain specific examples of the many alternative aspects or embodiments of the applicants' present disclosure set forth in this specification, and neither its use nor its absence is intended to limit the scope of the applicants' present disclosure or the scope of the claims. This specification is divided into sections for the convenience of the reader only. Headings should not be construed as limiting of the scope of the present disclosure. The definitions are intended as a part of the description of the present disclosure. It will be understood that various details of the present disclosure may be changed without departing from the scope of the present disclosure. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

The Claims We claim:

1. A method for performing gene synthesis and error correction on a droplet actuator, the method comprising: a. performing a gene synthesis protocol on a droplet actuator to produce a pool of synthesized DNA strands, wherein the pool of synthesized DNA strands comprises both correct nucleotide sequences and incorrect nucleotide sequences; and b. performing an error correction protocol on the pool of synthesized DNA strands on the droplet-actuator, comprising selectively enriching DNA strands comprising the correct nucleotide sequences.

2. The method of claim 2, wherein the gene synthesis protocol comprises: a. transferring a droplet comprising synthetic oligonucleotide sequences designed for a DNA molecule of interest to a sample preparation reservoir of a droplet actuator; b. hybridizing the synthetic oligonucleotide sequences to generate hybridized oligonucleotides; c. filling-in gaps in the hybridized oligonucleotides using DNA polymerase and PCR cycling; and d. amplifying the assembled nucleotide sequences using amplification primers, thereby generating the pool of synthesized DNA strands.

3. The method of claim 2, wherein the pool of synthesized DNA strands is generated off-actuator.

4. The method of any of claims 2 to 3, wherein the pool of synthesized DNA strands is generated using phosphoramidite synthesis chemistry.

5. The method of any of claims 2 to 4, wherein a droplet comprising PCR reagents and the DNA polymerase is combined with a droplet comprising the hybridized oligonucleotides using droplet operations, thereby generating a DNA assembly droplet.

6. The method of claim 5, wherein the PCR cycling comprises cyclically transporting the DNA assembly droplet between different temperature zones on the droplet actuator using droplet operations.

7. The method of claim 6, wherein the DNA assembly droplet is PCR cycled until full-length DNA strands are obtained.

8. The method of any of claims 2 to 7, wherein one of the amplification primers comprises a biotinylated primer.

9. The method of claim 8, wherein the biotinylated primer comprises a terminal oligonucleotide sequence used to design the DNA molecule of interest.

10. The method of any of claims 8 to 9, wherein a droplet including PCR reagents is combined with the DNA assembly droplet using droplet operations, thereby generating a DNA amplification droplet.

11. The method of claim 10, wherein amplification is performed in a flow- through format wherein the amplification droplets are cyclically transported between different temperature zones using droplet operations.

12. The method of claim 1 1, wherein the different temperature zones comprise a zone of about 95°C and a zone of about 55°C.

13. The method of any of claims 10 to 12, wherein excess amplification primers are removed from the DNA amplification droplet by combining a droplet comprising a wash buffer and magnetically responsive beads with the amplification droplet using droplet operations, thereby generating a DNA capture droplet.

14. The method of claim 13, comprising transporting the DNA capture droplet into the presence of a magnet using droplet operations and performing a merge-and-split wash protocol to remove unbound material, thereby generating a washed DNA capture droplet.

15. The method claim 14, further comprising transporting the washed DNA capture droplet into a thermal zone using droplet operations to promote release of amplified DNA from the magnetically responsive beads.

16. The method of claim 15, wherein the thermal zone heats the washed DNA capture droplet to a temperature of about 65°C.

17. The method of any of claims 15 to 16, further comprising transporting eluted amplified DNA away from the magnetically responsive beads, thereby generating an eluted amplified DNA droplet comprising biotinylated PCR amplicons.

18. The method of claim 17, further comprising merging a droplet comprising streptavidin-coated magnetically responsive beads with the eluted amplified DNA droplet using droplet operations, thereby generating an amplified DNA/ bead-comprising droplet.

19. The method of claim 18, further comprising transporting the amplified DNA/bead-comprising droplet into a thermal zone using droplet operations for a period of time sufficient to promote formation of biotin-streptavidin complexes.

20. The method of claim 19, wherein the thermal zone heats the amplified DNA/bead-comprising droplet to a temperature of about 65°C.

21. The method of any of claims 19 to 20, wherein the biotinylated PCR amplicons are immobilized on the streptavidin-coated magnetically responsive beads through formation of the biotin-streptavidin complexes.

22. The method of any of claims 1 to 21, wherein selectively enriching DNA strands comprising the correct nucleotide sequences comprises priming DNA template strands in the pool of synthesized DNA strands and sequentially exposing primed DNA template strands to a nucleotide mix, wherein the nucleotide mix includes a nucleotide which is complementary to the next unpaired base of the correct nucleotide sequence and terminated versions of the other three nucleotides.

23. The method of claim 22, wherein the terminated versions of the other three nucleotides comprise dideoxy nucleotides.

24. The method of any of claims 1 to 21, wherein the error correction protocol comprises: a. providing a pool of biotinylated amplified DNA strands immobilized on magnetically responsive beads, thereby generating bead-bound amplified DNA strands; b. removing non-biotinylated DNA strands from the biotinylated amplified DNA strands; c. synthesizing the correct nucleotide sequences using prepared single stranded (ss) DNA templates immobilized on magnetically responsive beads, thereby generating full-length synthesized DNA strands complementary to the prepared ssDNA templates; d. recovering the DNA strands complementary to the prepared ssDNA templates from the magnetically responsive beads; and e. selectively enriching DNA strands comprising the correct nucleotide sequences using PCR amplification.

25. The method of claim 24, further comprising hybridizing a sequencing primer to the bead-bound amplified DNA strands.

26. The method of claim 24, wherein the non-biotinylated DNA strands are removed from the bead-bound amplified DNA strands by alkali denaturation.

27. The method of claim 26, wherein removing the non-biotinylated DNA strands from the bead-bound amplified DNA strands by alkali denaturation comprises: a. washing a droplet comprising the bead-bound amplified DNA strands with a first reagent droplet that comprises a denaturation solution, thereby generating a washed droplet comprising bead-bound amplified DNA strands; b. merging the washed droplet comprising bead-bound amplified DNA strands with a second reagent droplet and incubating for a period of time sufficient to denature DNA, thereby generating a ssDNA/bead- comprising droplet; and c. transporting the ssDNA/bead-comprising droplet into a magnetic field of a magnet using droplet operations and performing at least one bead washing protocol on the ssDNA /bead-comprising droplet.

28. The method of claim 27, wherein the washing step of 27a comprises a merge-and-split protocol using droplet operations.

29. The method of claim 28, wherein the washed droplet comprising bead- bound amplified DNA strands and the second reagent droplet are incubated at ambient temperature.

30. The method of any of claims 27 to 29, wherein the at least one bead washing protocol comprises a first bead washing protocol, wherein the first bead washing protocol comprises an exchange of denaturation solution with a wash buffer in the ssDNA/bead-comprising droplet.

31. The method of claim 30, wherein the at least one bead washing protocol further comprises a second bead washing protocol, wherein the second bead washing protocol comprises an exchange of the wash buffer with an annealing buffer in the ssDNA/bead-comprising droplet.

32. The method of any of claims 27 to 31, wherein removing non-biotinylated DNA strands from the biotinylated amplified DNA strands comprises heat denaturation.

33. The method of claim 32, wherein heat denaturation comprises transporting the washed droplet comprising bead-bound amplified DNA strands to a thermal zone on the droplet actuator.

34. The method of claim 33, wherein the thermal zone is about 95°C.

35. The method of any of claims 27 to 34, further comprising combining the ssDNA/bead-comprising droplet with a droplet comprising primers, thereby generating an ssDNA template droplet.

36. The method of claim 35, further wherein the ssDNA template droplet is incubated at an annealing temperature for a period of time sufficient for annealing of primers to ssDNA.

37. The method of claim 36, wherein the ssDNA template droplet is incubated at a temperature of about 80°C for a period of time of about two minutes.

38. The method of claim 37, further comprising removing excess unbound primers from the ssDNA template droplet using a bead washing protocol.

39. The method of claim 38, wherein removing excess unbound primers from the ssDNA template droplet using the bead washing protocol comprises washing the ssDNA template droplet two times using polymerization buffer droplets.

40. The method of any of claims 35 to 39, wherein synthesizing the correct nucleotide sequence comprises: a. combining the ssDNA template droplet with a reagent droplet, thereby generating a DNA synthesis droplet; b. incubating the DNA synthesis droplet for a period of time sufficient for incorporation of the nucleotides; c. transporting the DNA synthesis droplet into a magnetic field of a magnet using droplet operations; and d. washing the DNA synthesis droplet to remove unincorporated nucleotides.

41. The method of claim 40, wherein the reagent droplet comprises the nucleotide which is complementary to the next unpaired base of the correct nucleotide sequence, the terminated versions of the other three nucleotides, a DNA polymerase, and a polymerization buffer.

42. The method of claim 40, wherein washing the DNA synthesis droplet comprises addition and removal of a polymerization buffer while retaining substantially all beads having a bound ssDNA template thereon in the droplet.

43. The method of claim 42, wherein the unincorporated nucleotides are removed by enzymatic degradation.

44. The method of any of claims 40 to 43, wherein recovering the DNA strands complementary to the prepared ssDNA templates from the magnetically responsive beads comprises: a. transporting the DNA synthesis droplet to a thermal zone using droplet operations, wherein the DNA in the DNA synthesis droplet is denatured by heating, thereby producing single-stranded complementary DNA (cDNA) strands; and b. transporting the single-stranded cDNA strands away from the beads using droplet operations, thereby generating a cDNA droplet comprising a pool of single-stranded cDNA comprising full-length correct DNA strands and terminated incorrect DNA strands.

45. The method of claim 44, wherein selectively enriching DNA strands comprising the correct nucleotide sequences using PCR amplification comprises: a. adding primers to the DNA template strands in the pool of synthesized DNA strands, thereby producing primed DNA template strands; and b. sequentially exposing primed DNA template strands to a nucleotide mix, wherein the nucleotide mix comprises a nucleotide which is complementary to the next unpaired base of the correct nucleotide sequence and terminated versions of the other three nucleotides.

The method of claim 44, wherein selectively enriching DNA strands comprising the correct nucleotide sequences using PCR amplification comprises: a. adding primers to the DNA template strands in the pool of synthesized DNA strands, thereby producing primed DNA template strands; b. sequentially exposing primed DNA template strands to a nucleotide mix, wherein the nucleotide mix includes a reversibly -terminated nucleotide which is complementary to the next unpaired base of the correct nucleotide sequence and irreversibly -terminated versions of the other three nucleotides; and c. adding a deblocking agent to the primed DNA template strands to remove the terminal blocking groups from the incorporated correct nucleotides prior to the next synthesis cycle.

The method of any of claims 45 to 46, wherein the PCR amplification comprises: a. adding PCR primers targeted to the ends of the correct nucleotide sequence; b. combining a droplet comprising PCR reagents with the cDNA droplet, thereby generating a reaction droplet; and c. conducting PCR amplification on the reaction droplet.

The method of claim 47, wherein the PCR amplification is performed in a flow-through format wherein for each cycle the reaction droplets are cyclically transported between different temperature zones within the droplet actuator using droplet operations.

49. The method of claim 47, wherein a size-fraction method is used to selectively enrich full-length correct DNA strands from shorter, prematurely terminated incorrect DNA strands.

50. The method of claim 47 or 48, wherein a solid-phase capture method is used to selectively enrich or deplete either the correct nucleotide sequences or the incorrect nucleotide sequences.

51. The method of any of claims 1 to 50, wherein the droplet actuator comprises: a. a bottom substrate separated from a top substrate to form a droplet operations gap, wherein the droplet operations gap is filled with a filler fluid; and b. an electrode arrangement disposed on the bottom and/or top substrate for conducting droplet operations.

52. The method of 51, wherein the electrode arrangement comprises electrowetting electrodes.

53. A microfluidics system programmed to execute the method of any of claims 1 to 52 on a droplet actuator.

54. A storage medium comprising program code embodied in the medium for executing the method of any of claims 1 to 52 on a droplet actuator.

55. A microfluidics system comprising the droplet actuator test coupled to a processor, wherein the processor executes program code embodied in a storage medium for executing the method of any of claims 1 to 52 on the droplet actuator.