WO2008024319A2 - Microfluidic devices for nucleic acid assembly - Google Patents

Microfluidic devices for nucleic acid assembly Download PDF

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Publication number
WO2008024319A2
WO2008024319A2 PCT/US2007/018437 US2007018437W WO2008024319A2 WO 2008024319 A2 WO2008024319 A2 WO 2008024319A2 US 2007018437 W US2007018437 W US 2007018437W WO 2008024319 A2 WO2008024319 A2 WO 2008024319A2
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Prior art keywords
assembly
nucleic acid
embodiments
reaction
microfluidic
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PCT/US2007/018437
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French (fr)
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WO2008024319A8 (en
WO2008024319A3 (en
Inventor
Craig Douglas Muir
Christopher J. Emig
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Codon Devices, Inc.
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Priority to US83896006P priority Critical
Priority to US60/838,960 priority
Priority to US60/878,332 priority
Priority to US87833206P priority
Priority to US87833006P priority
Priority to US60/878,330 priority
Application filed by Codon Devices, Inc. filed Critical Codon Devices, Inc.
Publication of WO2008024319A2 publication Critical patent/WO2008024319A2/en
Publication of WO2008024319A3 publication Critical patent/WO2008024319A3/en
Publication of WO2008024319A8 publication Critical patent/WO2008024319A8/en

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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00279Features relating to reactor vessels
    • B01J2219/00281Individual reactor vessels
    • B01J2219/00286Reactor vessels with top and bottom openings
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00279Features relating to reactor vessels
    • B01J2219/00306Reactor vessels in a multiple arrangement
    • B01J2219/00313Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315Microtiter plates
    • B01J2219/00317Microwell devices, i.e. having large numbers of wells
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00351Means for dispensing and evacuation of reagents
    • B01J2219/00423Means for dispensing and evacuation of reagents using filtration, e.g. through porous frits
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00585Parallel processes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/0059Sequential processes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718Type of compounds synthesised
    • B01J2219/0072Organic compounds
    • B01J2219/00722Nucleotides
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781Aspects relating to microreactors
    • B01J2219/00905Separation
    • B01J2219/00912Separation by electrophoresis
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0809Geometry, shape and general structure rectangular shaped
    • B01L2300/0816Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0832Geometry, shape and general structure cylindrical, tube shaped
    • B01L2300/0838Capillaries
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0421Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic electrophoretic flow
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0475Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00Heating or cooling apparatus; Heat insulating devices
    • B01L7/52Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples

Abstract

Certain aspects of the present invention provide devices and methods for assembling nucleic acid molecules on micro fluidic devices, e.g. via extension and/or ligation reactions. The invention also provides systems comprising one or more acoustic liquid handlers, one or more microfluidic devices, and/or one or more robotic liquid handlers for automating a nucleic acid assembly procedure. The microdevice may comprise the following components: a sipper, or means for transferring reagents (310); a main channel (315); a spitter, or means for transferring reagents (320), sample wells for storage of reagents or buffers (330, 335, 340), separation or concentration channels (350, 355) and sample or reaction wells (360, 365) e.g. a thermocycler.

Description

MICROFLUIDIC DEVICES FOR NUCLEIC ACID ASSEMBLY

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) from U.S. provisional application serial numbers 60/838,960 filed August 20, 2006, 60/878,332 filed December 31, 2006, and 60/878,330 filed December 31, 2006, the entire contents of each of which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

Methods and compositions of the invention relate to nucleic acid library design and assembly, and particularly to the design and assembly of nucleic acid libraries that express polypeptides.

BACKGROUND

Microfluidic devices and methods have been developed and used for a variety of analytical applications, including high throughput screening for drug discovery, gene expression analysis, and disease diagnosis.

SUMMARY OF THE INVENTION

Aspects of the invention relate to apparatuses for preparing and/or assembling macromolecules. Aspects of the invention provide microfluidic devices and methods for processing nucleic acid assembly reactions and assembling nucleic acids. In some embodiments, the invention provides microfluidic devices that are configured for preparing and/or assembling nucleic acids. Aspects of the invention also relate to microfluidic methods and devices for analyzing nucleic acid assembly reactions. According to the invention, certain microfluidic device configurations may be useful to synthesize, isolate, purify, and/or concentrate one or more reagents and/or intermediates during a nucleic acid assembly procedure. Certain isolated and/or concentrated reagents and/or intermediates may be combined and processed more rapidly and reproducibly to increase the throughput rate of the assembly. In some embodiments, the throughput rate of an assembly reaction may be increased by including a size selection step to isolate one or more intermediates during the assembly procedure. In certain embodiments, the throughput rate of an assembly reaction may be increased by including a step for concentrating certain reagents and/or intermediates during the assembly. In some embodiments, the concentration and/or purity of one or more intermediates may be normalized during an assembly to increase the throughput rate and/or decrease the error rate. In some embodiments, an acoustic liquid handler may be used to prepare mixtures of reagents or substrates (e.g., oligonucleotides) for one or more reactions (e.g., assembly reactions using a thermocycler). In some embodiments, an acoustic liquid handler may be used to prepare mixtures of reagents or substrates for access by a microfluidic system. In some embodiments, acoustic liquid delivery techniques may be used to introduce samples and/or reagents onto a microfluidic substrate, transfer samples and/or reagents from a first station or sample well to a second station or sample well on a microfluidic substrate, transfer samples and/or reagents from a first microfluidic substrate to a second microfluidic substrate, and/or remove samples and/or reagents from a microfluidic substrate (e.g., after assembly of a target nucleic acid product or intermediate). Accordingly, microfluidic devices of the invention may include reaction chambers, size separation stations, and/or other components that are provided in a preparative format. For example, the volumes and sizes of the microfluidic channels on a device may be adapted for preparative purposes as described herein. In some embodiments, the architecture of a microfluidic device may be adapted for assembly. For example, the architecture may include one or more connecting channels for transferring a size selected product (e.g., nucleic acid) from a first assembly station (e.g., temperature controlled well — for example, a thermocycling well) to a second assembly station. In some embodiments, the first and second assembly stations are integrated on the same microfluidic substrate (chip). In some embodiments, the first and second assembly stations are the same when the product of a first assembly reaction produced in an assembly station is recirculated to the same assembly station for further assembly after size selection in a size selection station. In some embodiments, an assembly process is recirculated through the same assembly station for a plurality of cycles (e.g., 5-10, 10-20, 20-30 or more cycles) required to assemble a target nucleic acid as described herein. However, it should be appreciated that a microfluidic substrate may include a plurality of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30 or more) integrated of assembly stations and one or more size separation stations. In some embodiments a single size separation station is used to process the products from each different assembly station. The connecting channels on the substrate may be designed for this procedure. In some embodiments, different size separation stations are used to process the reaction products from different assembly stations. In certain embodiments, each assembly station is connected to a unique size separation station that is in turn connected to the next assembly station. However, in other embodiments, a smaller number of size selection stations are used. In some embodiments, size selection stations are adapted to separate different size ranges (e.g., about 50 to 100 nucleotide long fragments, about 100 to about 500 nucleotide long fragments, about 500 to about 1,000 nucleotide long fragments, about 1,000 to about 2,000 nucleotide long fragments, about 2,000 to about 4,000 nucleotide long fragments, etc., or any subset or other configuration of different lengths). In some embodiments, a single microfluidic substrate may integrate a single assembly station with a plurality of size separation stations adapted for different size selections. Assembly products may be recirculated to the same assembly station after each round of size selection. However, different size selection stations may be used as the assembled product increases in size. It should be appreciated that any give size selection station may be used for a few (e.g., 2, 3, or 4) progressively larger sizes of consecutively assembled intermediate products depending on the assembly strategy (e.g., depending on whether it is a linear assembly strategy or an exponential assembly strategy as described herein).

Certain microfluidic device configurations may be adapted to perform isolation and/or concentration operations within an integrated (e.g., automated) assembly procedure. Microfluidic devices may be configured to isolate and/or concentrate a plurality of assembly reactions rapidly and efficiently. In some embodiments, a plurality of reactions may be processed in parallel. Accordingly, aspects of the invention are useful to increase the rate, yield, and/or precision of nucleic acid assembly. This can decrease the cost and/or delivery time for manufacturing a nucleic acid product.

In one aspect, the invention provides microfluidic devices for isolating nucleic acid intermediates during a polymerase-mediated assembly procedure. Certain polymerase-mediated assembly steps may generate a mixture of nucleic acids that include nucleic acids having a predicted size of interest, but also include a variety of incorrectly assembled nucleic acids. According to aspects of the invention, a microfluidic device may be used to purify nucleic acids or nucleic acid intermediates having a predetermined size corresponding to the expected size of an assembled product or intermediate. An assembly reaction mixture may be processed on a microfluidic substrate through a size separation or selection station arranged to segregate or otherwise group subject molecules based on size. In some embodiments, the assembly reaction mixture is introduced to the microfluidic substrate (e.g., using a collection port or sipper) from a reaction chamber on a different substrate (e.g., a multi-well plate). In some embodiments, the assembly reaction mixture or one or more of its components are prepared through the action of an acoustic liquid handler (e.g., an acoustic droplet ejector). For example, an acoustic liquid handler may be used to prepare precise mixtures of reaction components in a receptacle (e.g., a well of a multi-well plate) that can be accessed by a microfluidic device (e.g., by a sipper of a microfluidic device), hi some embodiments, acoustic liquid delivery technology may be used to directly deposit one or more reagents or a reaction mixture directly onto a microfluidic substrate. In some embodiments, the reaction mixture may be generated on the microfluidic substrate in an appropriate reaction well (e.g., in a thermocycling station on the substrate). Regardless of the source, the reaction mixture may be moved through a channel connected to the size separation station where nucleic acids of a target size or size range may be isolated. The size-selected nucleic acids may be retained in a compartment on a substrate for further in-chip processing. In some embodiments, the size-selected nucleic acids may be moved through a channel to a subsequent station on the same microfluidic substrate for further processing. In some embodiments, reaction mixtures and/or size- selected nucleic acids may be transferred to or from a size separation station using an acoustic liquid handling technique (e.g., via the action of an acoustic droplet ejector). In some embodiments, the size-selected nucleic acids are removed from the substrate (e.g., using a distribution port or spitter) for use in further assembly steps, hi some embodiments, further assembly steps are performed on the same substrate using a plurality of connected operational stations that are configured for performing subsequent mixing, reacting, concentration, and/or separation functions of the assembly procedure. hi some embodiments, acoustic liquid handling technology (e.g., using an acoustic droplet ejector, or using acoustic liquid handling components integrated into a microfluidic device) may be used to transfer nucleic acids and/or other reagents or products from one position to another on a micro fluidic substrate (e.g., between sample wells, reagent wells, and/or reaction stations). Accordingly, in some embodiments, one or more sample wells, reagent wells, reaction stations, and/or other wells on a microfluidic substrate are not connected via channels, but are configured to support transfer using acoustic liquid handling techniques.

It should be appreciated that aspects of the invention may involve isolating a plurality of intermediates that are then combined in a subsequent assembly step. In some embodiments, the concentration of each of the plurality of intermediates is normalized or otherwise modified to improve the subsequent assembly reaction. For example, certain intermediates may be assembled less efficiently than others in a first round of assembly. Accordingly, the concentration of each isolated intermediate may be adjusted to approximately the same level when they are combined for a further round of assembly. However, in some embodiments, the concentration of different intermediates may be set at different levels. For example, certain intermediates may be provided at higher concentrations than others if it is helpful for an assembly or other reaction. In some embodiments, the concentrations of one or more substrates or intermediates may be adjusted dynamically during an assembly process. For example, concentrations of different nucleic acids may be monitored continuously throughout the assembly procedure or after one or more predetermined assembly or isolation steps. The relative concentrations of different nucleic acids may be adjusted (e.g., normalized) at any stage during the assembly procedure resulting in a dynamic adjustment of different nucleic acid concentrations in response to measurements of nucleic acid levels during the assembly procedure. For example, dynamic normalization may include monitoring reaction products after one or more steps of the assembly process and re-normalizing the concentrations of one or more of the intermediate products from one or more steps prior to combining them for a subsequent step (e.g., by increasing or reducing the amount more of one or more nucleic acid samples that is added to a subsequent step and/or by increasing or reducing nucleic acid sample or reaction volumes). Dynamic adjustments may be automated. In another aspect, the invention provides microfluidic devices for removing error- containing nucleic acids during an assembly procedure. In some embodiments, an error correction step may be performed by mixing assembled nucleic acids and a MutS protein (or other mismatch recognition protein or other affinity mismatch reagent such as an aptamer) under conditions where the protein binds to heteroduplexes formed by error containing nucleic acids hybridized to error-free nucleic acids. The mismatch recognition agent may be free in solution or immobilized on a separating bead, surface, or other support. An error removal reaction mixture may be size selected on a micro fluidic device to isolate the unbound nucleic acids from the larger complexes of protein bound nucleic acids. In some embodiments, an error-removal reaction mixture is introduced to a microfluidic substrate (e.g., using a sipper) from a reaction chamber on a different substrate (e.g., a multi-well plate). In some embodiments, the error-removal reaction mixture or one or more of its components are introduced using acoustic liquid handling techniques (e.g., through the action an acoustic droplet ejector). In some embodiments, the reaction mixture may be generated on the microfluidic substrate in an appropriate reaction well (e.g., in a thermocycling station on the substrate). Regardless of the source, the reaction mixture may be moved through a channel connected to the size separation station where unbound nucleic acids may be isolated based on size or other sortable characteristic. The size-selected nucleic acids may be retained in a compartment on a substrate for further in-chip processing. In some embodiments, the size-selected nucleic acids may be moved through a channel to a subsequent station on the same microfluidic substrate for further processing. In some embodiments, reaction mixtures and/or size-selected nucleic acids may be transferred to or from a size separation station using an acoustic liquid handling technique (e.g., via the action of an acoustic droplet ejector). In some embodiments, the size-selected nucleic acids are removed from the substrate (e.g., using a spitter) for use in further assembly steps. In some embodiments, the size-selected nucleic acids are removed through the action of an acoustic liquid handler (e.g., an acoustic droplet ejector). Accordingly, in some embodiments, a size-separation station may be isolated from other stations and/or wells (e.g., not connected via a channel), but configure to support transfer of sample and/or size-selected product from and to other wells or stations using an acoustic liquid handler (e.g., an acoustic droplet ejector). In some embodiments, further assembly steps are performed on the same substrate using a plurality of connected operational stations that are configured for performing subsequent mixing, reacting, concentration, and/or separation functions of the assembly procedure. However, in certain embodiments, one or more of the further operational stations are isolated, but configured for liquid transfer using an acoustic liquid handler (e.g., an acoustic droplet ejector). According to aspects of the invention, size selection of unbound nucleic acids increases the yield of error-free nucleic acids or nucleic acid intermediates. It should be appreciated that aspects of the invention may involve isolating a plurality of error-free intermediates that are then combined in a subsequent assembly step. In some embodiments, the concentration of each of the plurality of intermediates is normalized or otherwise adjusted to improve the subsequent assembly reaction. In some embodiments, the concentration of one or more of the components may be dynamically adjusted (e.g., dynamically normalized) before, during or after any one of the steps of the assembly procedure in response to measured levels of nucleic acid reagents or products. In some embodiments, the adjustment may be automated.

In a further aspect, the invention provides microfiuidic devices for concentrating, size separating and/or processing nucleic acid intermediates during a ligation-mediated assembly procedure. For example, during an iterative ligation assembly, a first plurality (e.g., two, three or more) of nucleic acid duplexes may be ligated in a reaction well (e.g., on a microfiuidic substrate or on a different substrate and then introduced to the microfiuidic substrate) and the reaction mixture may be moved to a first size separation or selection station on a microfiuidic substrate via a connecting channel. Nucleic acids of the size expected for a full length ligation product may be isolated in the size selection station. The size-selected ligation product then may be moved to a further reaction well where it is mixed with one or more additional duplexes for a second ligation reaction. This reaction mixture then may be moved through a connecting channel to the first or a second size selection station where the ligated product is isolated based on its size. This process may be repeated until a final target nucleic acid is assembled. It should be appreciated that different combinations of reaction steps may be performed on the microfiuidic substrate. In some embodiments, the microfluidic substrate also includes on or more stations for concentrating nucleic acid duplexes prior to a ligation reaction. These iterative ligation steps may be performed very rapidly and efficiently on a microfiuidic device of the invention. In some embodiments, one or more nucleic acids in a ligation-mediated assembly procedure may be transferred between wells and/or operational stations using an acoustic liquid handler (e.g., an acoustic droplet ejector) or other liquid handling system. Accordingly, some of the wells and/or operational stations may be configured for acoustic liquid transfer instead of being connected by a channel. In some embodiments, the concentration of one or more of the components may be dynamically adjusted (e.g., dynamically normalized) before, during or after any one of the steps of the ligation-mediated assembly procedure in response to measured levels of nucleic acid reagents or products. In some embodiments, the adjustment may be automated.

Accordingly, aspects of the invention provide microfluidic devices and methods for enhancing the assembly of target nucleic acids or intermediates thereof. In some embodiments, an assembled target nucleic acid may be amplified, sequenced or cloned after it is made. In some embodiments, a host cell may be transformed with the assembled target nucleic acid. The target nucleic acid may be integrated into the genome of the host cell. In some embodiments, the target nucleic acid may encode a polypeptide. The polypeptide may be expressed (e.g., under the control of an inducible promoter). The polypeptide may be isolated or purified. A cell transformed with an assembled nucleic acid may be stored, shipped, and/or propagated (e.g., grown in culture). One or more of these steps may be performed, at least in part, using an appropriate microfluidic device.

Methods and devices of the invention may involve small assembly reaction volumes. For example, reaction volumes of between about 5 nl and about 100 nl may be used. However, smaller or larger volumes may be used. In some embodiments, an acoustic liquid handler may be used for transferring volumes of less than lOOnl, less than 10 nl, for example less than 5 nl, or about 1 nl or less.

Accordingly, aspects of the invention provide strategies for optimizing nucleic acid assembly by dynamically controlling (e.g., adjusting) reaction conditions during an assembly procedure. According to aspects of the invention, the efficiency and/or fidelity of a nucleic acid assembly reaction may be sensitive to minor changes, inconsistencies, errors, and/or other variations in the assembly reaction conditions (e.g., altered concentrations of buffer, salt, nucleic acid, or other reagents, altered temperature, altered pH, etc., or any combination thereof). Methods, devices, and systems for monitoring and/or modifying reaction conditions throughout the duration of a nucleic acid assembly procedure may be used to optimize the production of a target nucleic acid. In one aspect, the dynamics of one or more reaction conditions may be monitored during the progression of an assembly reaction. In some embodiments, reaction conditions may be monitored continuously during an assembly procedure (e.g., using continuous direct or indirect detection of one or more reagents and/or markers during an assembly reaction). In some embodiments, reaction conditions may be monitored at one or more steps during an assembly procedure (e.g., using direct or indirect detection of one or more reagents or markers in a reaction sample at one or more steps, for example after one or more assembly cycles). In another aspect, reaction conditions may be altered during the progression of an assembly procedure. In some embodiments, reaction conditions may be altered at any time by adding or removing reagents or solvents. In some embodiments, reaction conditions may be altered at predetermined steps during an assembly procedure. For example, reagent amounts or reaction or sample volumes may be adjusted (e.g., increased or decreased) after one or more assembly steps and prior to a subsequent assembly step. Accordingly, methods, systems and devices of the invention may provide dynamic feedback in response to detected changes in assembly reaction conditions in order to optimize the reaction conditions throughout a series of assembly cycles and/or for parallel assembly procedures. It should be appreciated that an assembly procedure may include a series of assembly steps in which a nucleic acid product is progressively assembled via a series of intermediate nucleic acids. In addition, one or more steps may be included to correct and/or remove error containing constructs from one or more of the intermediate products and/or the final product. One or more intermediate products and/or the final product also may be cloned into a vector prior to subsequent assembly or use. According to the invention, reaction conditions may be analyzed and/or adjusted after any one or more of the assembly, error correction/removal, and/or cloning steps described herein. In some embodiments, reaction conditions may be adjusted to stay within a narrow range of one or more predetermined conditions. For example, concentrations, temperatures, pH, and/or other reaction conditions may be dynamically monitored and adjusted to stay within +/- 0.01%, +/- 0.1%, +/- 1%, +/- 2%, +/- 5%, +/- 10%, +/- 15%, +/- 20%, +/- 25% of one or more predetermined reaction conditions. However, these ranges are not limiting, and reaction conditions may be maintained within narrower or broader ranges of one or more predetermined reaction conditions. In some embodiments, reaction conditions in sequential reaction steps may be dynamically monitored and adjusted to stay within +/- 0.01%, +/- 0.1%, +/- 1%, +/- 2%, +/- 5%, +/- 10%, +/- 15%, +/- 20%, +/- 25% of each other. However, these ranges are not limiting, and sequential reaction conditions may be maintained within narrower or broader ranges of each other. In certain embodiments, sequential reaction conditions may be dynamically normalized to be the same. In some embodiments, reaction conditions in parallel reactions may be dynamically monitored and adjusted to stay within +/- 0.01%, +/- 0.1%, +/- 1%, +/- 2%, +/- 5%, +/- 10%, +/- 15%, +/- 20%, +/- 25% of each other. However, these ranges are not limiting, and parallel reaction conditions may be maintained within narrower or broader ranges of each other. In certain embodiments, parallel reaction conditions may be dynamically normalized to be the same.

In one aspect, nucleic acid concentrations may be adjusted during assembly to optimize assembly efficiency and/or fidelity. An assembly procedure may include several parallel and/or sequential reaction steps in which a plurality of different nucleic acids are combined in order to be assembled (e.g., by extension or ligation as described herein) to generate a longer nucleic acid product to be used for further assembly, cloning, or other applications. In some embodiments, concentrations of different nucleic acids are assayed and/or adjusted prior to an initial assembly step (e.g., an initial multiplex oligonucleotide assembly step). In some embodiments, concentrations of different nucleic acids are assayed and/or adjusted after an initial assembly step. In some embodiments, concentrations of different nucleic acids are assayed and/or adjusted between sequential assembly steps. In some embodiments, concentrations of different nucleic acids are assayed and/or adjusted prior to one or more cloning steps. In some embodiments, concentrations of different nucleic acids are monitored and/or dynamically adjusted after one or more cloning steps, hi some embodiments, concentrations of different nucleic acids are monitored and/or dynamically adjusted prior to one or more error correction/error removal steps, m some embodiments, concentrations of different nucleic acids are monitored and/or dynamically adjusted after one or more error correction/error removal steps. It should be appreciated that an assembly reaction may include two or more sequential assembly steps in which nucleic acids assembled in parallel reactions in a first step are combined in a second step to be assembled together. Assembled product from the second step may be combined with additional nucleic acids for further assembly. Cloning and/or error correction/removal steps may be included in between two or more assembly steps. Dynamic monitoring and/or adjustment of nucleic acid concentrations may be applied at any one or more assembly, cloning, and/or error correction/removal steps. In some embodiments, nucleic acid concentrations may be monitored and/or dynamically adjusted prior to or after one or more assembly steps where a plurality of different nucleic acids are combined for further assembly. For example, nucleic acid concentrations may be monitored and/or dynamically adjusted prior to or after about 10%, about 20%, about 25%, about 50%, about 75%, about 80%, about 90%, or all assembly steps. In some embodiments, nucleic acid concentrations may be monitored and/or dynamically adjusted prior to or after one or more cloning steps where one or more different nucleic acids are combined with a vector for cloning and or selection. For example, nucleic acid concentrations may be monitored and/or dynamically adjusted prior to or after about 10%, about 20%, about 25%, about 50%, about 75%, about 80%, about 90%, or all cloning steps. It should be appreciated that the concentration of the vector nucleic acid also may be dynamically monitored and/or adjusted. hi any of the embodiments described herein, the concentrations of different nucleic acids may be adjusted to stay within a narrow range of one or more predetermined concentrations. For example, concentrations of different nucleic acids may be dynamically monitored and adjusted to stay within +/- 0.01%, +/- 0.1%, +/- 1%, +/- 2%, +/- 5%, +/- 10%, +/- 15%, +/- 20%, +/- 25% of one or more predetermined concentrations in serial or parallel reaction steps. However, these ranges are not limiting, and nucleic acid concentrations may be maintained within narrower or broader ranges of one or more predetermined reaction concentrations. It should appreciated that each different nucleic acid may be maintained at a different predetermined concentration based on one or more sequence characteristics (e.g., sequence specific features, or more general characteristics such as nucleic acid length, nucleic acid strandedness: double- stranded or single-stranded, GC content, the presence and/or length and/or GC content and/or sequence of single-stranded overhangs in a double-stranded nucleic acid, the degree of predicted secondary structures, the number and length of inverted and/or direct repeats, etc., or any combination thereof). However, in some embodiments, the concentration of each of a plurality of nucleic acids that are combined together in assembly and/or cloning reactions may be dynamically monitored and adjusted to stay within +/- 0.01%, +/- 0.1%, +/- 1%, +/- 2%, +/- 5%, +/- 10%, +/- 15%, +/- 20%, +/- 25% of each other. These ranges are not limiting, and nucleic acid concentrations may be maintained within narrower or broader ranges of each other. In certain embodiments, the concentrations of different nucleic acids may be dynamically normalized to be the same. Accordingly, in some embodiments, nucleic acid concentrations in parallel and/or sequential assembly steps may be dynamically monitored and adjusted to stay within +/- 0.01%, +/- 0.1%, +/- 1%, +/- 2%, +/- 5%, +/- 10%, +/- 15%, +/- 20%, +/- 25% of each other. In certain embodiments, nucleic acid concentrations in parallel or sequential assembly steps may be dynamically normalized to be the same.

Aspects of the invention may be automated to provide optimized reaction conditions for a series of assembly cycles and/or for parallel assembly steps. However, reaction conditions may be manually adjusted (e.g., through the actions of an operator) in response to detected changes in nucleic acid concentrations (e.g., in response to differences in relative concentrations of different nucleic acids that are combined in a single assembly and/or cloning step).

Aspects of the invention can be used in combination with one or more multiplex nucleic acid assembly techniques and/or cloning steps (e.g., one or more vector activation cycles described herein) in order to assemble a long nucleic acid product from small starting nucleic acids (e.g., from a plurality of oligonucleotides). For example, dynamic adjustment (e.g., dynamic normalization) of may be used in combination with any of the multiplex assembly reactions illustrated in the Figures or otherwise described herein. Dynamic adjustment (e.g., dynamic normalization) also may be used in combination with a concerted assembly (e.g., concerted ligation), one or more cycles of a vector-encoded trait activation technique, or any other cloning or assembly technique. In some embodiments, a plurality of assembly cycles can be performed in parallel and pairs of nucleic acid products from a first set of assembly cycles can be dynamically adjusted (e.g., dynamically normalized) and combined and assembled in a second set of assembly cycles. In turn, pairs of assembled nucleic acids from the second set of assembly cycles can be dynamically adjusted (e.g., dynamically normalized) and combined and assembled in a third set of assembly cycles. This process can be repeated one or more times until a final product is assembled to contain all of the starting nucleic acids from the first plurality of assembly cycles.

Design and assembly methods of the invention may be automated. Aspects of the invention can be implemented to provide reactive automation in a multiplex assembly procedure. Aspects of the invention also provide for intermediate quality control steps, for example, in an automated assembly procedure. Methods of the invention may reduce the cost and increase the speed and accuracy of nucleic acid assembly procedures, particularly automated assembly procedures.

Aspects of the invention relate to systems incorporating one or more acoustic liquid handlers, microfiuidic components, and/or robotic handlers as described herein. For example, a system may include an acoustic liquid handler for generating reaction mixtures, an optional temperature controlling device (e.g., a thermal cycler), a microfiuidic device for processing reaction mixtures (e.g., during and/or after one or more assembly reactions), and a robotic liquid handler for dispensing reaction products from the microfiuidic device into one or more suitable culture, storage, or transport containers. A system may include automated components adapted to connect the acoustic, temperature, microfiuidic, and/or robotic liquid handling components described herein.

Aspects of the invention provide methods and compositions that can be used to efficiently assemble a target nucleic acid, particularly a long target nucleic acid. In some embodiments, a target nucleic acid may be amplified, sequenced or cloned after it is made. In some embodiments, a host cell may be transformed with the assembled target nucleic acid. The target nucleic acid may be integrated into the genome of the host cell. In some embodiments, the target nucleic acid may encode a polypeptide. The polypeptide may be expressed (e.g., under the control of an inducible promoter). The polypeptide may be isolated or purified. A cell transformed with an assembled nucleic acid may be stored, shipped, and/or propagated (e.g., grown in culture).

In another aspect, the invention provides methods of obtaining target nucleic acids by sending sequence information and delivery information to a remote site. The sequence may be analyzed at the remote site. The starting nucleic acids may be designed and/or produced at the remote site. The starting nucleic acids may be assembled in a reaction involving a microfiuidic device at the remote site. In some embodiments, the starting nucleic acids, an intermediate product in the assembly reaction, and/or the assembled target nucleic acid may be shipped to the delivery address that was provided.

Other aspects of the invention provide systems for designing starting nucleic acids and/or for assembling the starting nucleic acids to make a target nucleic acid using one or more micro fluidic processes. Other aspects of the invention relate to methods and devices for automating a multiplex oligonucleotide assembly procedure that involves one or more micro fluidic steps. Other aspects of the invention relate to methods and devices for automating a multiplex oligonucleotide assembly reaction that include one or more dynamic adjustment (e.g., dynamic normalization) methods of the invention. Yet further aspects of the invention relate to business methods of marketing one or more devices, methods, systems, and/or automated procedures that involve microfluidic devices for multiplex nucleic acid assembly.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. The claims provided below are hereby incorporated into this section by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. IA illustrates an embodiment of a microfluidic device comprising a sipper (110), a channel (115), a spitter (120), wells (130; 140) and a separation and/or concentration station (150);

FIG. IB illustrates embodiments of different acid assembly strategies; FIG. 2A illustrates an embodiment of a microfluidic device comprising a sipper (210), a central channel (215), a spitter (220), sample wells (230; 240), a separation station (250) and a reaction well (260); FIGS. 2B-2D illustrate different embodiments of a polymerase-based multiplex assembly reaction;

FIG. 3 A illustrates an embodiment of a microfluidic device comprising a sipper (310), a channel (315), a spitter (320), sample/reagent wells (330; 335; 340; 360; 365) and separation stations (350; 355); FIG. 3B illustrates an embodiment of a MutS error removal procedure; FIG. 4A illustrates an embodiment of a micro fluidic device comprising a sipper (410), a spitter (420), sample /reagent wells (430; 435; 440; 460; 465) and separation station (450);

FIGS. 4B-4D illustrate different embodiments of a ligase-based multiplex assembly reaction;

FIG. 5 A outlines an embodiment of a ligase-based assembly procedure that can be automated on a micro fluidic device;

FIG. 5B illustrates a non-limiting example of a recirculating geometry for automating a ligase-based assembly procedure on a microfluidic device; FIGS. 5C-5E illustrate embodiments of microfluidic stations used for different assembly steps (FIG. 5C shows a non-limiting example of an enzymatic reaction chamber, FIG. 5D shows a non-limiting example of a flow separation region, FIG. 5E shows a non-limiting example of an assembly product selection region);

FIGS. 6A-6B illustrate a non-limiting examples of a cascaded geometry for automating a ligase-based assembly procedure on a microfluidic device; and

FIG. 7 illustrates a non-limiting embodiment of a cross-sectional side view of a microfluidic interconnect for connecting two or more microfluidic substrates.

DETAILED DESCRIPTION OF THE INVENTION Aspects of the invention provide microfluidic devices for preparing and/or assembling macromolecules. Aspects of the invention may be useful for increasing the accuracy, yield, throughput, and/or cost efficiency of nucleic acid assembly reactions.

Aspects of the invention provide devices and methods for isolating and purifying one or more assembly intermediates or products. For example, assembly intermediates or products may be isolated or purified based on size using a microfluidic device that includes a size separation or selection station arranged to segregate or otherwise group subject molecules based on size (e.g., an electrophoretic station, a column, etc.). According to aspects of the invention, size-selected intermediates may increase the efficiency, accuracy, and/or yield of subsequent assembly steps. Size selection may be used at different stages in an assembly reaction as described in more detail herein. It should be appreciated that nucleic acids of interest may be separated, selected, or isolated based on other properties. For example, a nucleic acid of interest may be affinity purified. Accordingly, when an aspect of the invention is described herein in the context of a size selection or separation, it should be appreciated that one or more other separation or selection technique(s) or systems (e.g., affinity) may be substituted for the size separation or selection technique or system if the other technique(s) can be used to isolate the nucleic acid of interest.

Aspects of the invention also provide devices and methods for concentrating nucleic acid assembly reagents, intermediates, and/or products. For example, a microfluidic device may include a concentration station arranged to concentrate or otherwise select subject molecules. A concentration station may have one or more size barriers (e.g., a filter, a pore arrangement, a gel, a column), buffer or matrix barriers that can concentrate assembly reagents, intermediates, and/or products (e.g., via isotachophoresis), or other suitable concentration selectors. According to aspects of the invention, concentrated reagents or intermediates may increase the efficiency, accuracy, and/or yield of subsequent assembly steps. Concentration may be used at different stages in an assembly reaction as described in more detail herein.

Aspects of the invention also provide devices and methods for normalizing or otherwise adjusting the concentration of reaction intermediates during an assembly procedure. Aspects of the invention also provide devices and methods for the dynamic adjustment (e.g., dynamic normalization) of the concentrations of one or more of the components of the assembly procedure in response to detected levels of one or more of the different components during the assembly procedure.

Aspects of the invention may be performed on a variety of different microfluidic devices. However, certain microfluidic devices may be configured to optimize the reaction throughput. Particular configurations may be optimized for different assembly steps (e.g., polymerase-based assembly, ligation based assembly, error removal, or a combination thereof) as described herein. Microfluidic devices may include one or more operational stations for preparing, mixing, separating, isolating, assembling, modifying, dispensing, cloning, transforming, and/or storing, etc., one or more reagents, intermediates, or products associated with a nucleic acid assembly procedure. The invention also provides systems, system controllers, robotic components, software, hardware, etc., for manipulating and controlling microfluidic devices and integrating them into nucleic assembly procedures that may involve one or more automated steps. In some embodiments, systems of the invention include a micro fluidic device handler integrated with one or more instruments, devices and/or other equipment for sample analysis, processing, storage, etc. For example, a system of the invention may comprise a microfluidic device handler integrated with a nucleic acid synthesizer, nucleic acid sequencer, mass spectrometer, electron microscope, digital microphotogrammetry equipment, liquid handler, colony picker, or other device or equipment. In some embodiments, acoustic liquid handling technology may be incorporated into a system of the invention (e.g., as a stand-alone device that is incorporated into an assembly system via one or more robotic interfaces — for example, an automated and/or programmable robotic interface, or as an integral part of a microfluidic device so that volumes may be transferred from a first station on a microfluidic substrate to a second substrate on the same or a different microfluidic substrate).

A microfluidic device may include a sipper to aspirate a sample (e.g., a solution or suspension of a nucleic acid assembly reagent or intermediate) into a channel or reservoir on a substrate. Accordingly, the sipper may be in fluid communication with either a channel or a reservoir. The device also may include a separation station, a concentration station, or a combination thereof, in fluid connection with the sipper via one or more channels. A used herein, a functional station on a microfluidic substrate (e.g., a chip) is a location comprising one or more microfluidic structures that are adapted for a particular function. For example, a station may be a reservoir or a series of channels or any other structure or combination of structures. A functional station also may include one or more additional components for activating (e.g., heating, irradiating, etc.), detecting (e.g., visualizing, etc.), etc., or any combination thereof. A detector element may be included to detect reagents, intermediates, or products of interest (e.g., after separation or concentration). In some embodiments, the detector element may be integrated with a separation or isolation station. In some embodiments, the detector element may interact with materials in a channel at an outlet of the separation or concentration station. A sampling station may be included to remove material from the device. The sampling station may be downstream from the separation or concentration station. This allows, for example, an assembly intermediate or product of a predetermined size or a concentrated reagent, intermediate, or product to be sampled (e.g., removed from the device for further analysis or processing). In some embodiments, the sampling station may be a well that can be accessed with a liquid handling apparatus (e.g., a manual or automated pipettor). In certain embodiments, the sampling station may be a spitter. A microfluidic device may include additional or alternative operational stations (e.g., thermocyclers, reagent wells, sample wells, reaction wells, waste wells, etc.) in fluid communication with each other according to an architecture that may be adapted for one or more nucleic acid assembly procedures. In some embodiments, the sipper and spitter may be the same component and the channel architecture and fluid flow determine when it is used as a sipper and when it is used as a spitter. In some embodiments, sippers and spitters may be separate and a single microfluidic substrate may include one or more (e.g., two or more, for example, 2, 3, 4, 5, 6, 7, 8, 9 ,10 or more) sippers and/or spitters. However, in some embodiments, an assembly reaction of the invention may be performed using a basic or standard (e.g., commercially available) microfluidic device, for example, a device having operational stations and an architecture designed for analytical applications. It should be appreciated that a microfluidic device may include two or more of any particular operational station (including sippers and/or spitters). Two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 to 20, 20 to 50, 50 to 100, or more) identical or different operational stations may be configured in series, in parallel, or a combination thereof. In some embodiments, devices may be used and/or designed to perform a plurality of parallel assembly reactions. Assembly reagents, intermediates, and/or products may be drawn into a device, moved through channels and operational stations, and/or removed from a device using any suitable method (e.g., positive pressure, negative pressure, capillary action, electrical force, osmotic pressure, electro-osmosis, acoustic liquid handling, etc., or any combination of two or more thereof). In some embodiments, assembly reagents, intermediates, and/or products may be drawn into a device, moved between operational stations, and/or removed from a device using any suitable acoustic liquid handler (e.g., an acoustic droplet ejector). In some embodiments, the sample is introduced into a station, well, or reservoir on a substrate through the action of an acoustic liquid handler (e.g., an acoustic droplet ejector) without using a sipper or a channel. Accordingly, in some embodiments a microfluidic device or substrate may be provided without a sipper and/or without one or more channels connecting sample wells, reagent reservoirs, and/or operational stations. FIG. IA illustrates a non-limiting example of a microfluidic device according to aspects of the invention. In FIG. IA, the device comprises the following components: 110: a sipper, or means for transferring reagents, buffers or other components into the microfluidic device; 115: a main channel that is in fluid connection with the sipper and other operational stations on the device; 120: a spitter, or a means for transferring reagents, buffers or other components out of the microfluidic device; 130 and 140: sample wells for the storage of reagent, buffer, or other components in the microfluidic device; 150: a separation station and/or a concentration station for the separation and/or concentration of assembly reagents, intermediates, and/or products. In some embodiments, the microfluidic device further comprises additional channels in which, liquids, solutions and other components can be transported between different operational stations. As discussed herein, a microfluidic device may further comprise reaction wells, thermocyclers, and other components for moving or manipulating liquids, fluids and chemical components. Aspects of the invention may be used for automating one or more steps of a multiplexed nucleic acid assembly procedure. While most assembly protocols start with pools of overlapping synthesized oligonucleotides and end with PCR amplification of the target nucleic acid (e.g., a synthetic gene), the pathway between those two points can be quite different. Single stranded (ss) PCR assembly makes use of unphosphorylated oligonucleotides, which undergo repetitive PCR cycling to extend and create a full length template. A variant of this method, termed double stranded (ds) PCR, involves combining all single strand PCR oligonucleotides and their reverse complement oligonucleotides for assembly. In the case of enzymatic ligation, the initial oligonucleotide population is required to have phosphorylated 5' ends that allows T4 or Pfu DNA or other ligase to covalently connect these "building blocks" together to form an initial product or template. In addition to these differences, the ligation process is more efficient with oligonucleotide concentrations in the μM (10~6) range whereas both ss and ds PCR options are efficient at much lower concentrations (e.g., in the nM, 10'9 range). FIG. IB illustrates non-limiting embodiments of different assembly strategies. In i) a plurality of at least partially overlapping assembly nucleic acids (e.g., oligonucleotides) are mixed together. For example, about 20 different oligonucleotides each about 50 nucleotides in length may be combined in a single reaction. However, fewer or greater numbers of assembly nucleic acids may be used. Also, each assembly nucleic acid may be smaller or larger (e.g., less than about 20, between about 20 and about 200, between about 200 and about 400, or more than about 400 nucleotides long). Each different assembly nucleic acid includes a portion of the sequence of a target nucleic acid of interest. Non-limiting alternative configurations for the overlapping assembly nucleic acids are illustrated in FIGS. 2B-2D. The assembly nucleic acids may be assembled by polymerase-mediated assembly in ii). During this procedure, a plurality of denaturing and extension cycles are performed (e.g., using a thermostable polymerase) to generate a mixture that contains progressively longer nucleic acid products in each cycle. After an appropriate number of cycles (e.g., 2-5, between about 5 and about 15, between about 15 and about 30, or more than about 30, depending in part on the number and length of the different assembly nucleic acids that are included in the reaction), a reaction mixture may contain a full length nucleic acid product corresponding to the target nucleic acid of interest. However, the reaction mixture also typically contains many different partially assembled nucleic acid products of different sizes, hi some embodiments, the full length nucleic acid may be rare in the reaction mixture relative to the partially assembled nucleic acid products. In certain embodiments, a polymerase- mediated assembly ii) may include an additional amplification step using a pair of flanking primers (e.g., with one amplification primer corresponding to each end of the target nucleic acid). This amplification step (e.g., by PCR) preferentially or selectively amplifies full length target nucleic acid that has both correct ends. However, this amplification step also may amplify (at least linearly) partially assembled nucleic acids that have one of the two target nucleic acid ends complementary to an amplification primer. The amplification step also may generate a mixture of primer dimers. The amplification step also may amplify (exponentially) incorrectly assembled nucleic acids that include both target nucleic acid ends complementary to both amplification primers, hi some embodiments, the assembly mixture is at least partially purified (e.g., using a column, a gel, beads, or other purification technique) prior to the amplification step. In some embodiments, the amplification mixture is at least partially purified (e.g., using a column, a gel, beads, or other purification technique) prior to any subsequent assembly step. In some embodiments, a product from ii) is used as an intermediate for further assembly. In some embodiments the concentration of one or more of the components may be dynamically adjusted (e.g., normalized) before, during or after any one of the steps of the assembly procedure. In some embodiments the adjustment may be automated. In iii) a plurality of products from ii) are combined. For example, 5 different approximately 400 bp products, each from an independent assembly ii) using a different pool of assembly nucleic acids i), are combined for further assembly. However, it should be appreciated that fewer or greater numbers of intermediate products may be combined in iii). For example, between about 5 and about 10, about 10 to about 25, about 25 to about 50, or more. Also, the size of each assembly intermediate may range from about 100 to about 1,000 nucleotides (e.g., single-stranded or double-stranded) or longer.

In some embodiments, assembly iv) may involve polymerase-based extension cycles as described in ii) if the intermediate nucleic acids in iii) have appropriate overlapping sequences. The intermediates in iii) may be used as double-stranded intermediates. However, in some embodiments, at least some of the intermediates may be denatured and appropriate single strands may be combined (e.g., with at least partially overlapping complementary sequences). In some embodiments, assembly iv) may involve ligation (e.g., chemical or enzymatic) if the intermediates have appropriate ends for ligation (e.g., generated via restriction enzyme digestion, for example, using a Type IIS restriction enzyme). Regardless of the assembly reaction, correct assembly product in iv) may be amplified using appropriate flanking primers as described for ii). Also, a correct assembly product may be partially purified as described for ii).

Yet additional assembly reactions may be performed using the nucleic acid products from iv) as intermediates in a further assembly reaction. For example, in v) nucleic acids from iv) are combined. These nucleic acids may then be assembled by ligation in vi) to generate a larger nucleic acid molecule. In vii) an approximately 4 kb nucleic acid product is illustrated. However, larger nucleic acids may be assembled using larger intermediates (e.g., greater than 1.5 kb, 1.5 kb to about 2 kb, 2 kb to about 4 kb, 4 kb to about 6 kb, 6 kb to about 10 kb, 10 kb to about 20 kb, 20 kb to about 40 kb, 40 kb to about 60 kb, 60 kb to about 100 kb or longer), a greater number of intermediates (e.g., more than 3, more than 4, more than 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, or more), or a combination thereof. In FIG. IB, i') and ii') show non-limiting examples of initial assembly steps where assembly nucleic acids (e.g., oligonucleotides) are combined in i') and assembled by ligation in ii'). In certain embodiments, complementary oligonucleotide in i') are hybridized to form duplexes (e.g., duplexes of oligonucleotides, wherein each oligonucleotide is between about 30 and about 200 nucleotides long, or longer). In some embodiments, pairs of 125 nucleotide long oligonucleotides are hybridized to form duplexes in i'). The duplexes in i') may be combined together in a single ligase reaction in ii'). However, the yield of correctly assembled product can be very low if too many duplexes are combined in a single reaction. Accordingly, in some embodiments, small numbers of duplexes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 duplexes) may be combined and ligated to form intermediate nucleic acids that are subsequently ligated to additional intermediates. Non-limiting examples of alternative ligation strategies are illustrated in FIGS. 4B-4D. As described above for ii), a full length product generated in ii') may be selectively amplified. For example, a complete full length nucleic acid expected to be generated in ii') may be selectively amplified (e.g., by PCR) using a pair of flanking primers that are complementary to sequences in the two end duplexes that were incorporated into the nucleic acid product. As described above for ii), final products from ii') and/or intermediate ligation products generated during ii') may be at least partially purified prior to further processing. Ligated nucleic acid products generated in ii') of FIG. IB may be used as intermediates for further assembly, for example as described herein for iii) — vii) of FIG. IB. hi some embodiments the concentration of one or more of the components may be dynamically adjusted (e.g., normalized) before, during or after any one of the steps of the assembly procedure. In some embodiments the adjustment may be automated. Aspects of the invention provide microfluidic devices for performing one or more assembly steps (e.g., one or more of i) — vii) of FIG. IB). According to some aspects of the invention, size selection, concentration, or a combination thereof (e.g., using a microfluidic device) during or after any step of a multiplex assembly (e.g., during or after any of the assembly procedures illustrated in FIG. IB) may be used to increase the efficiency, yield, and/or accuracy of an assembly reaction. Accordingly, in some embodiments one or more size selection and/or concentration steps may be performed on a microfluidic device and the remainder of the assembly steps may be performed outside the device. For example, a reaction mixture from any assembly step may be aspirated (e.g., from a reaction well on another device such as an array or a multi-well plate) onto a device, size separated and/or concentrated within the device, and removed (e.g., via a spitter) into a well (e.g., a sample well or reaction well) on another device outside the microfluidic device (e.g., on an array or a multi-well plate outside the microfluidic device). In some embodiments a reaction mixture from any assembly step may be transferred or deposited through the action of an acoustic liquid handler (e.g., an acoustic droplet ejector). However, it should be appreciated that additional or alternative assembly steps also may be performed on a microfluidic device. For example, sample mixing, reaction incubation, thermal cycling for extension and/or amplification, restriction enzyme digestion, phosphorylation, methylation, ligation, host cell transformation also may be performed in appropriate operational stations on a device. Certain aspects of the invention also may benefit from other advantages of using microfluidic devices to perform biological and chemical processes. For example, small sample volumes can easily be handled by microfluidic devices. Small sample volumes may have a chemical advantage in addition to a cost advantage. For example, higher concentrations can readily be achieved, increasing reaction rate and efficiency. In addition, small samples can be processed and transported rapidly, thereby decreasing assembly time relative to reactions performed in certain automated liquid handling devices. In some embodiments, the automated liquid handling device comprises an acoustic liquid handler (e.g., an acoustic droplet ejector).

In some embodiments, an acoustic liquid handler may be used to mix the starting nucleic acids (e.g., initial oligonucleotides) in step i) or i'). The starting nucleic acids may be transferred using acoustic liquid handling technology from one or more reagent wells (e.g., on a first multi-well plate) into one or more reaction wells (e.g., on a second multi-well plate). The resulting reaction mixtures may be exposed to reaction conditions (e.g., thermal cycling) directly in the reaction wells or after transfer from the reaction wells to a microfluidic substrate where subsequent reaction steps are performed. Transfer from a reaction well to a chamber or other station on a microfluidic substrate may be automated using acoustic technology (e.g., an acoustic droplet generator), robot handling technology (e.g., a robotic pipettor), or microfluidic technology (e.g., aspirated using a sipper on a microfluidic device). Acoustic devices are non-contact dispensing devices able to dispense small volume of fluid (e.g. picoliter to microliter), see for example Echo 550 from Labcyte (CA), HTS-01 from EDC Biosystems. Acoustic technologies and devices for acoustically ejecting a plurality of fluid droplets toward discrete sites on a substrate surface for deposition thereon have been described in a number of patents such as U.S. Patents 6, 416,164; 6,596,239; 6,802,593; 6, 932,097; 7,090,333 and US Patent Application 2002-0037579, the disclosure of which are incorporated herein by reference. The acoustic device includes an acoustic radiation generator or transducer that may be used to eject fluid droplets from a reservoir (e.g., microplate wells) through a coupling medium. The pressure of the focused acoustic waves at the fluid surface creates an upwelling, thereby causing the liquid to urge upwards so as to eject a droplet, for example from a well of a source plate, to a receiving plate positioned above the fluid reservoir. The volume of the droplet ejected can be determined by selecting the appropriate sound wave frequency.

Non-limiting examples of assembly devices and methods are described in the following paragraphs in connection with FIGS. 2-4.

FIG. 2 A illustrates a non-limiting example of a microfluidic device for polymerase assembly according to aspects of the invention. In FIG. 2 A, the device comprises the following components: 210: a sipper, or means for transferring reagents, buffers or other components into the microfluidic device; 215: a main channel that is in fluid connection with the sipper and other operational stations on the device; 220: a spitter, or a means for transferring reagents, buffers or other components out of the microfluidic device; 230 and 240: sample wells for the storage of reagent, buffer, or other components in the microfluidic device; 250: a separation station and/or a concentration station for the separation and/or concentration of assembly reagents, intermediates, and/or products; 260: reaction well for mixing and reaction reagents, buffers or components. In some embodiments the reaction well is a thermocycler. A thermocycler may be a well that is connected to a temperature controller that is used to cycle the contents of the well between different temperatures of interest. However, it should be appreciated that different configurations of microfluidic thermocyclers may be used in devices and systems of the invention. For example, some microfluidic thermocyclers include two or more wells at different temperatures. The wells are in fluid communication and thermocycling is achieved by physically moving reaction volumes back and forth between the different wells at different temperatures. In some embodiments, the microfluidic device further comprises additional channels in which, liquids, solutions and other components can be transported between different operational stations. As discussed herein, a microfluidic device may further comprise additional reaction wells, thermocyclers, and other components for moving or manipulating liquids, fluids and chemical components. Liquids, buffers and components may be transported from one component to the next by applying pressure, electric, capillary or other forces.

Non-limiting examples of polymerase assembly of nucleic acids are shown in Fig 2B-2D. In i) a plurality of at least partially overlapping nucleic acids are mixed together. Non-limiting embodiments of overlapping nucleic acid are illustrated by i) in Figures

2B-2D. Any number of nucleic acids may be combined and the nucleic acids may be of any length as described herein. Prior to mixing, each of the individual nucleic acids solutions may have been stored in the sample wells (240). In other embodiments one or more of the nucleic acids were premixed and stored as such prior to the assembly process. The nucleic acids may have been introduced into the microfluidic device through the action of a sipper or through any other transferring means (e.g., a pipettor such as a robotic pipettor). In some embodiments, the nucleic acids may have been introduced through the action of an acoustic liquid handler (e.g., an acoustic droplet ejector). The nucleic acids may have been introduced into the sample wells directly or may have originally been introduced into the central channel and subsequently distributed to the various sample wells. In some embodiments the nucleic acids are transported from the sample wells to the reaction well and the mixing of the nucleic acids is done in the reaction well. As used herein for any solutions, mixing may involve shaking, stirring or other techniques, hi some embodiments, mixing may be achieved by flowing solutions together. In some embodiments, mixing may be achieved using ultrasound or other similar techniques. After mixing of the nucleic acids a polymerase is added to the mixture. The polymerase and components necessary for the polymerase to function (nucleotides and the appropriate buffers) may have been stored in a sample well (230). The components may be stored separately from the polymerase or together with the polymerase. In some embodiments the polymerase and other components necessary for polymerase function were stored outside of the microfluidic device and introduced into the reaction well through the action of a sipper or by any other means of transfer. In some embodiments, the polymerase and other components necessary for polymerase function may have been introduced through the action of an acoustic liquid handler (e.g., an acoustic droplet ejector). Once the nucleic acids and polymerase are mixed together, polymerase extension cycles can be executed by varying the temperature in the thermocycler. In ii) a cycle may comprise changing the temperature to facilitate hybridization of the overlapping nucleic acids, changing the temperature to facilitate extension by the polymerase and changing the temperature to facilitate denaturing of the nucleic acids. A number of cycles may be performed and the temperatures may be changed between cycles to facilitate the assembly of increasingly larger nucleic acids. In some embodiments the nucleic acid assembly mixture is transported though a separation station (250) at any point during one of the thermocycles. The separation station separates nucleic acids by size or other sortable characteristic and allows for analysis of the reaction mixture, hi addition, the reaction mixture may be enriched for the desired nucleic acid in the separation station. Once the desired product has been detected, the reaction mixture can be enriched for this product by adding flanking primers and performing additional thermocycler rounds (iv and v). Flanking primers can be added directly to the original reaction mixture or can be added after the mixture has been passed to the separation station and enriched for the desired nucleic acid. In some embodiments a new aliquot of polymerase with buffer and components is added after the mixture has been passed through the separation station. After this second round of polymerase extension the reaction mixture may again be passed through the separation station to isolate the desired nucleic acid and remove the polymerase, salts, unreacted nucleotides and unwanted side products, hi some embodiments, the concentration of one or more of the components may be dynamically adjusted (e.g., normalized) before, during or after any one of the steps of the assembly procedure. In some embodiments, the adjustment may be automated. The isolated nucleic acid may subsequently be stored in any of the sample wells or may be transferred from the microfluidic device through the action of a sipper or any other means of transfer. In some embodiments, the nucleic acid may be transferred from the microfluidic device through the action of an acoustic liquid handler (e.g., an acoustic droplet ejector).

FIG. 3 A illustrates a non-limiting example of a microfluidic device for a MutS error removal procedure according to aspects of the invention, hi FIG. 3 A, the device comprises the following components: 310: a sipper, or means for transferring reagents, buffers or other components into the microfluidic device; 315: a main channel that is in fluid connection with the sipper and other operational stations on the device; 320: a spitter, or a means for transferring reagents, buffers or other components out of the microfluidic device; 330, 335 and 340: sample wells for the storage of reagent, buffer, or other components in the microfluidic device; 350 and 355: separation stations and/or a concentration stations for the separation and/or concentration of assembly reagents, intermediates, and/or products; 360 and 365: sample and reaction wells for mixing and storing of reaction reagents, buffers or components. In some embodiments the reaction well is a thermocycler. In some embodiments, the microfluidic device further comprises additional channels in which, liquids, solutions and other components can be transported between different operational stations. As discussed herein, a microfluidic device may further comprise additional reaction wells, thermocyclers, and other components for moving or manipulating liquids, fluids and chemical components. Liquids, buffers and components may be transported from one component to the next by applying pressure, electric, capillary or other forces.

A non-limiting example of a MutS error removal procedure is shown in Fig 3B. A preparation of de novo assembled or synthesized nucleic acids may contain one or more subsets of nucleic acids that have one or more sequence errors in addition to nucleic acids that have the correct desired sequence. In some embodiments, the sequence errors may be present on only one copy of a double-stranded heteroduplex nucleic acid molecule. In addition, or alternatively, the sequence errors may be present on both strands of a double-stranded homoduplex error-containing nucleic acid molecule (As shown in i)). According to the invention, denature ii) and re-annealing iii) procedures may be used to promote the formation of heteroduplex error-containing nucleic acids each incorporating one strand of an error-free nucleic acid and one strand of an error-containing nucleic acid (As shown in iv)). The amount or percentage of heteroduplex formation will depend on the relative amounts of homoduplex error- containing nucleic acids and homoduplex error-free nucleic acids that were denatured and re-annealed. In some embodiments the nucleic acids used in the MutS error removal procedure were generated in any of the above described polymerase assembly procedures. The nucleic acids can undergo the MutS error removal procedure at any stage of the polymerase assembly procedure. In preferred embodiments the MutS error removal procedure is executed after completion of a polymerase assembly. The nucleic acids to undergo the MutS error removal procedure can be stored in any of the sample wells. They can be transported in the sample wells directly after being isolated in the polymerase assembly procedure or they may be introduced by a sipper via the central channel or they may be introduced directly into the sample wells. In some embodiments, the nucleic acids may be introduced through the action of an acoustic liquid handler (e.g., an acoustic droplet ejector). In some embodiments the nucleic acids will be transported to a thermocycler reaction well. After introduction into the thermocycler the nucleic acids can be denatured ii) by changing the temperature or changing environmental conditions (e.g., addition of a chaotropic reagent). The nucleic acids can subsequently be re-annealed iii) by changing the temperature again or changing the buffer conditions. As long as the starting concentration of nucleic acids with correct sequences was higher then the starting concentration of nucleic acids with incorrect sequences, the denature and re- annealing process will result in the generation of heterogeneous complexes iv). MutS, a mismatch repair protein, binds nucleic acids with a heteroduplex formation v) and MutS will be added to the thermocycler well with the denatured and re-annealed nucleic acid sequences. The invention is not limited to MutS and embraces any protein that can bind to a heteroduplex nucleic acid. In aspects of the invention, one or more mismatch binding proteins may be used under conditions that promote a stable association between the mismatch binding protein(s) and one or more heteroduplex nucleic acids. The MutS protein and appropriate buffer may have been stored in one of the sample wells prior to addition to the nucleic acid mixture, or may be added by the sipper or any other transfer means. In some embodiments the temperature of the well containing the nucleic acid MutS mixture may be changed to optimize binding of MutS to the heteroduplex. Nucleic acids that are complexed with MutS will have a higher molecular weight and different electrostatic properties than nucleic acid without MutS. The MutS complexed nucleic acids can therefore be separated from unbound nucleic acids on one of the separation stations. In one embodiment the mixture of MutS complexed nucleic acids and "free" nucleic acids is transported through a separation station. Separation can be based on size, electrostatic interactions, affinity or other physic-chemical differences. In some embodiments the separation station is a sizing column, an ionic interaction column, or a column with anti-MutS antibodies. In some embodiments MutS is modified to increase retention on a column. A non-limiting example is a biotin modification which can be retained on an avidin column. In some embodiments the mixture is passed through one column multiple times. In other embodiments the mixture is passed through columns with different modes of separation (e.g., a sizing column in series with a MutS antibody column). Nucleic acids that were not complexed with MutS will not be retained in the column. The flow through fraction will therefore be enriched for nucleic acid without heteroduplexes. The MutS error removal procedure will therefore result in an increase in the amount of nucleic acid with the desired sequence vii). The enriched fraction can be stored in one of the sample wells or can be transferred form the microfluidic device by the spitter or any other means of transfer. In some embodiments, the enriched fraction may be transferred from the microfluidic device through the action of an acoustic liquid handler (e.g., an acoustic droplet ejector).

In some embodiments, an error correction procedure (e.g., using a CE-based separation on a microfluidic substrate) can be implemented on a microfluidic substrate by processing a reaction mixture that was assembled and/or combined with a mismatch recognition reagent as described herein prior to being introduced to the microfluidic substrate. Accordingly, such a microfluidic substrate may include only an input, a size separator, and an output that is configured and/or used in a preparative mode to recover a product of interest rather than discarding it as would be the case in an analytical configuration or use. In some embodiments, an error correction reaction (e.g., including one or more denaturing, reannealing, and/or binding to a mismatch recognition agent such as MutS) may be performed on a microfluidic substrate in a reaction station connected to a size-selection station (e.g., integrated on the same microfluidic substrate). FIG. 4A illustrates a non-limiting example of a microfluidic device for nucleic acid ligation according to aspects of the invention. In FIG. 4A, the device comprises the following components: 410: a sipper, or means for transferring reagents, buffers or other components into the microfluidic device; 415: a main channel that is in fluid connection with the sipper and other operational stations on the device; 420: a spitter, or a means for transferring reagents, buffers or other components out of the microfluidic device; 430, 435 and 440: sample wells for the storage of reagent, buffer, or other components in the microfluidic device; 450: a separation station and/or a concentration station for the separation and/or concentration of assembly reagents, intermediates, and/or products; 460 and 465: sample and reaction wells for mixing and storing of reaction reagents, buffers or components. In some embodiments the reaction well is a thermocycler. The microfluidic device further comprises additional channels in which, liquids, solutions and other components can be transported between different operational stations. As discussed herein, a microfluidic device may further comprise additional reaction wells, thermocyclers, and other components for moving or manipulating liquids, fluids and chemical components. Liquids, buffers and components may be transported from one component to the next by applying pressure, electric, capillary or other forces. Non-limiting examples of nucleic acid ligation assembly procedure are shown in

Figs 4B-D. The assembly of long fragments of nucleic acids consisting of multiple fragments i) (e.g., a ligation with more than two or three components), can be executed through iterative ligation ii). hi iterative ligation two or three nucleic acid sequences iii) are ligated first resulting in a larger nucleic acid v). This larger nucleic acid will subsequently be ligated with one or two additional nucleic acids vi), resulting in a new nucleic acid with the combined length of the both ligation steps vii). This process can be repeated multiple times to arrive at a nucleic acid with any desired length comprising any number of nucleic acid building blocks. The invention is not limited to the schemes depicted in Figs 4B-4D and embraces combinations of the depicted ligation schemes and any ligation, or coupling of two nucleic acids in general. The nucleic acids to be ligated may be transported from the sample wells into the reaction well. In some embodiments each nucleic acid to be ligated will be stored in a separate sample well. Separate storage will facilitate any kind of iterative combination scheme. Once two or more nucleic acids are combined in the reaction well and aliquot of ligase and appropriate buffer and components is added to the reaction well. The ligase and buffers may have been stored in sample wells or may be added by sipper via a channel (e.g., a central channel). In some embodiments, the ligase and buffers may be added through the action of an acoustic liquid handler (e.g., an acoustic droplet ejector). The invention embraces any ligase known to people of ordinary skill in the art. Once the nucleic acid and ligase and components are combined the nucleic acids will be ligated together. In some embodiments the temperature of the reaction well will be changed to facilitate the ligation reaction. New nucleic acids and/or new aliquots of ligase and components may be added to the reaction well at any time. In some embodiments aliquots of the reaction mixture or the complete reaction mixture can be transported through a separation station. In some embodiments the separation station is a sizing column. The lengths of the nucleic acids going through the column can be detected, allowing assessment of the completeness of the ligation reaction. In some embodiments only a small aliquot of the reaction mixture is sent through the separation station and used as an analytical tool to assess if the ligation reaction is complete. In other embodiments the complete ligation reaction mixture is sent through the separation station, separated by size, and the correct ligation product isolated. In some embodiments the isolated nucleic acids are returned to the reaction well to undergo the next step in the iterative ligation by addition of the next one or more nucleic acids. In some embodiments, the concentration of one or more of the components may be dynamically adjusted (e.g., normalized) before, during or after any one of the steps of the assembly procedure. In some embodiments, the adjustment may be automated. An iterative ligation assembly process may comprise multiple rounds of ligation, analytical detection and isolation. The final nucleic acid product can be stored in a sample well or transferred from the microfluidic device by a spitter or any other means. In some embodiments, the nucleic acid product is transferred from the microfluidic device through the action of an acoustic liquid handler (e.g., an acoustic droplet ejector). In some aspects of the invention, ligase-based assembly techniques do not work well with large pools of many different input nucleic acids. Ligation reactions can be very sensitive to the input oligonucleotide pool size. Accordingly, in some embodiments the complexity of a single ligation reaction is kept at less than 3 or 4 different oligonucleotides (e.g., 2, 3, or 4 different double-stranded input nucleic acids) to avoid requiring many subsequent sequencing reactions to identify a correct nucleic acid product after ligation. According to the invention, automated microfluidic techniques can be used to serially ligate a plurality of different starting nucleic acids, wherein each ligation reaction involves only a few different input nucleic acids (e.g., about 2-4). The product of each ligation reaction can be isolated by size separation on a microfluidic device and used for a subsequent ligation reaction. Accordingly, a ligation product can be "grown" through a series of low complexity ligation reactions. It should be appreciated that one or more of the input nucleic acids in a ligation reaction may be a synthesized oligonucleotide or other nucleic acid (e.g., a ligated nucleic acid from a prior ligation reaction). Accordingly, hierarchical ligation assemblies may involve branches of ligation reactions wherein the products of two ligation reactions are combined to form a ligation product that is combined with a ligation product from a parallel branch of ligation reactions as described herein. In each cycle of ligation, the product may be separated from the input nucleic acids using a size separation technique implemented on a microfluidic substrate. Such techniques avoid cloning and selection techniques (e.g., ligation, transfection, plating, colony picking, etc.) characteristic of a typical non- automated or semi-automated ligation assembly. The resulting assembly is faster and more efficient. FIG. 5 A illustrates a non-limiting embodiment of a micro fluidics-based assembly strategy in comparison to a process that involves cloning. The size selected products of a micro fluidics process may be fed into a further ligation reaction in the same microfluidic device (e.g., the same microfluidic chip) until a final product is generated an eluted off the chip without any intermediate cloning steps. The repeated ligation reactions may involve a recirculation geometry or a linear cascading geometry, or a combination thereof as described in more detail herein.

In some embodiments, a microfluidics-based solution for ligation-based assembly integrates a ligation assembly process in a step-wise manner. A microfluidics-based ligation-assembly system may include one or more of the following components i) an integrated microfluidics component for step-wise ligation assembly (optionally including a reusable fluidics substrate, an integrated chip-to-world interface, etc., or any combination thereof), ii) an oligonucleotide pooling and mixing component, iii) a ligation reaction component for performing a chemical ligation and/or an enzymatic ligation (e.g., a kinase and ligase reaction in a single step), iv) an error-correction component (e.g., a MutS filter), v) a size separation component (e.g., a capillary electrophoresis (CE) component), vi) a collection and/or concentration component (e.g., magnetic beads for fraction collection and concentration enhancement), and vii) a recirculating chip design, a cascaded chip design, or a chip design that incorporates recirculating and cascaded microfluidic components. It should be appreciated that a microfluidic device may include all or a subset of the components described above depending on the system configuration. Each component that is included in a microfluidic device may be a separate reaction station on a microfluidic substrate (e.g., a chip). However, some of the components may be included in the same reaction station (e.g., reaction mixing and temperature cycling, etc.). In some embodiments, the error correction component (e.g., the MutS filter) may be integrated with the ligation in the same reaction station and the product processed in a single size selection station to remove both un-ligated nucleic acids and error-containing ligated product. In some embodiments, capillary electrophoresis may include a combination of longitudinal and horizontal (e.g., transverse) electrophoresis as described herein.

In some embodiments, recirculating and/or cascaded microfluidic geometries may be used for step-wise assembly involving ligation of two nucleic acids in each step. In a recirculating configuration, the product of a first ligation reaction may be recirculated into the same microfluidic components on a chip and combined with a further input nucleic acid to form a second product that is again recirculated into the same microfluidic components on a chip and combined with yet a further input nucleic acid to form a third product. This recirculating technique may be repeated until a full length product is complete. In contrast, in a cascaded configuration the product of a first ligation reaction generated using a first set of microfluidic components may be delivered to a second set of microfluidic components where it is combined with a further input nucleic acid (e.g., from an earlier assembly using a different set of microfluidic components) to form a second product. This product in turn may be transferred to a further set of microfluidic components where it is ligated to yet a further nucleic acid. In some configurations, a series of parallel microfluidic components are connected in a hierarchical fashion (e.g., in a repeated geometry in which the output of two upstream sets of components are fed into the input of a single set of components immediately downstream) so that they "cascade" towards a single final set of components. In operation, a plurality of input nucleic acids are combined pairwise in a hierarchical series of ligation reactions to form a single final nucleic acid product after a series of steps (e.g., with the product of two reactions at a first step in the hierarchy being combined with the product of two separate reactions at the same first step in the hierarchy to form a larger product at a second step in the hierarchy that is combined with a further product at the second step to form a larger product for the third step, etc.).

It should be appreciated that this two-piece ligation approach is not economically efficient with traditional, non-microfluidic implementations due to time and labor costs associated with cloning. In contrast, by integrating a microfluidic system (e.g., with a unique capillary electrophoresis function) with other ligation reaction components, vector ligation and transfection can be eliminated from intermediate assembly cycles. Accordingly, integrated fluidics components are capable of repeatedly augmenting ligation product length until the final length is reached.

FIGS. 5B-5E illustrate non-limiting examples of a recirculating architecture and illustrative components. The architectures are illustrated in a cross-sectional view seen from above showing the outlines of the microfluidic pathways and reaction stations on a microfluidic substrate. A feature of a recirculating architecture is the feedback path which takes the output of a first reaction and feeds it into the next reaction. This allows the device to operate cyclically and ligate nucleic acids in a stepwise fashion. In some embodiments, there are three main components to the architecture illustrated in FIG. 5B. A assembly station (e.g., a kinase/ligase reaction chamber) shown in (1) is connected to a separation station (e.g., a CE flow separator) shown in (2) which is connected to a selection station (e.g., a magnetic selection station) shown in (3). The output of the selection station is connected to the input of the assembly station (1) so that the product of a first reaction can be fed back into a second assembly reaction using the same set of microfluidic stations (e.g., on the same chip). Fluid flow may be driven by pressure, and valves may be used to control the flow between different components of each station and/or between stations. Accordingly, during operation, the following events may occur on-chip. During a first step, enzyme mix (kinase, ligase, and possibly MutS mix), first and second assembly oligonucleotides may be introduced into the reaction chamber in (1) of FIG. 5B, for example via the inlet or the feedback path. Mixing (e.g., ultrasonic mixing) may be performed. The kinase and ligase reactions may be performed under a controlled temperature and for a set time. FIG. 5C illustrates a non-limiting example of a reaction chamber showing a kinase/ligase mixture being introduced via an inlet in panel (a). Subsequently, in panel (b) one or more oligonucleotides may be introduced via the inlet (e.g., from an oligonucleotide tray). In panel (c), one or more oligonucleotides or other nucleic acids (e.g., a ligation product from a prior round of assembly) may be introduced via the feedback path (e.g., from the selection station). In panel (d) the volume is mixed (e.g., using ultrasound). In panel (e), after completion of the kinase/ligase reactions, the volume in the reactor is moved to the separation station (e.g., the transverse-CE in (2) of FIG. 5B). It should be appreciated that the order of addition of the reagents and substrates in panels (a) - (c) can be changed as the invention is not limited in this respect. Also, during addition of reagents or nucleic acids, the liquid flow out of the reaction chamber may be sent to a waste port (e.g., spitter) as illustrated. As described herein, the flow may directed using valves or other techniques known in the art for directing fluid flow through different pathways of a microfiuidic device.

FIG. 5D illustrates a non-limiting example of a separation station. In this embodiment, separation is performed using transverse-CE across a laminar flow fan-out region. In some embodiments, the dimensions of the straight and fan-out regions are between about 0.1 mm and about 10 mm. The lengths are typically about 10 mm long. However, other dimensions may be used as the invention is not limiting in this respect. The reaction solution from (1) of FIG. 5B is introduced under a controlled volumetric flow. In some embodiments, a simultaneous flow of CE buffer is introduced via the buffer inlet (e.g., from a buffer reservoir on a separate support such as a multi-well plate via the sipper, or from a buffer reservoir on the microfiuidic substrate). The ratio of the two flows will determine the laminar flow pattern in the fan-out region illustrated in FIG. 5D. An electrostatic (DC) or electrodynamic (AC) field may be applied in-plane perpendicular to the principle direction of the flow. In some embodiments, (DC) voltages from about 1 Volt to about 1,000 Volts or more (e.g., up to about 4,000 to about 8,000 V) may be used for electrophoretic separation (e.g., between about 10 and about 200 Volts). In some embodiments, (AC) voltages can be applied for pulsed electrophoresis, with an amplitude, measured from peak-to-peak of the voltage waveform, on the order of about 1 Volt to about 1,000 Volts (e.g., between about 10 and about 200 Volts) or more (e.g., up to about 4,000 to about 8,000 V). However, other suitable (DC) or (AC) voltages may be used as the invention is not limited in this respect. The field serves to cause electrophoretic migration of analyte (e.g., nucleic acids of different lengths), with a migration direction also perpendicular to the principle direction of the flow. According to the invention, analytes separated in the flow structure are subsequently divided into different regions for further processing. In operation, panel (a) of FIG. 5D shows a laminar flow pattern that is established when the analyte reaction volume (darker region) and the buffer (lighter region) are introduced into the separation component. By adjusting the flow ratio of the two input streams, the flow pattern can be controlled. By the principle of diffusion, the analyte will diffuse into the buffer where concentration is lower. However, instead of relying on only diffusive forces to act on the analyte molecules (nucleic acids), an external field (AC or DC) is applied to cause electrophoretic migration of charged analyte molecules (e.g., DNA). Accordingly, the flow pattern acts as a moving gel, and as the gel is moved further downstream, the separated CE bands are further spaced apart by the established laminar flow pattern, making downstream selection with channels possible. The separation can happen only in the neck (linear, non-triangular) region of the device (see panel (b) of FIG. 5D) or through the entire length of the separation station (see panel (c) of FIG. 5D). The electrodes can be designed as programmable (termed electrical field manipulation or EFM), further controlling the migration of the bands and leading to better elution channel selection. The elution channel selection can be done dynamically by feedback controlling the inlet stream flow ratios and electrical field manipulation. The bands formed by the transverse-CE can be "cut" and separated into a discrete number of elution channels. The elution channels can be tuned to the proper analyte length by controlling the flow ratio of inlet analyte to buffer and by EFM. FIG. 5D, panels (b) and (c) illustrate examples of different regions of the laminar flow being separated into different elution channels. Once in an elution channel, all of the analyte fragments can by isolated, for example, by passing the flow in the elution channel through one or more capture regions within the selection station (e.g., capture regions where pre-deposited magnetic beads bind to the analyte and immobilize it in the capture regions as described in more detail below). In some embodiments, after the volume of the reaction chamber (e.g., the entire volume or a fraction thereof) has passed through the separation station, different fractions may be collected and/or concentrated in the selection station. The fractions containing the product size of interest may be isolated and the product separated for further assembly (e.g., by recirculating).

It should be appreciated that in some embodiments, electrodes can be used for electrochemical sensing, with applied excitation voltage in the form of direct current with amplitudes from about 1 nVolt to about 1 Volt or more. In some embodiments, pulsed (or alternating current, AC) voltages can be applied for sensing purposes, with an amplitude, measured from peak-to-peak of the voltage waveform, on the order of about 1 nVolt to about 1 Volt or more. However, other sensing voltages may be used as the invention is not limited in this respect.

FIG. 5E illustrates a non-limiting embodiment of a selection station involving magnetic separation. Panel (a) shows how the station is prepared prior to use. A buffer with diluted magnetic beads is flown through the entire volume (through all the channels) of the magnetic fraction selection (MFS) pathway. Magnetic beads may be between about 1 nm and about 500 μm in diameter (e.g., between about 10 nm and about 10 μm in diameter). Magnetic beads may be obtained from any suitable source (e.g., Ferrotec (USA)). In some embodiments, the buffer with diluted magnetic beads is also flown through the separation station (e.g., the T-CE station described above). Permanent or electro-magnets are placed at collection sites to trap the magnetic beads in the buffer. Once a sufficient amount of beads are captured, clean buffer (without beads) is flown in the device to flush out any un-captured beads. Loading and flush volumes may be sent to a waste port as illustrated. Following the flush, the MFS is ready for analyte capture. Any suitable capture technique (e.g., affinity capture technique) may be used. For example, the capture mechanism may be via a reversible biotin-streptavidin-Poly(N- isopropylacrylamide) reaction. Unmodified streptavidin binds to biotin with high affinity, resulting in a nearly irreversible bond. However, an engineered streptavidin can be conjugated to a temperature sensitive polymer, such as poly(iV-isopropylacrylamide), to allow reversible binding. For example, biotin-streptavidin binds at room temperature and dissociates at 37 0C. In some embodiments, a biotin is placed only on the first oligonucleotide of a ligation assembly. Accordingly, desired full length biotin-labeled assembly products will be captured by the magnetic beads. However, any unassembled biotin-labeled oligonucleotides and/or undesired shorter (and/or longer) length assembly products (e.g., primer dimers, etc.) also can be captured by the magnetic beads. Panel (b) of FIG. 5E shows solutions of nucleic acids of different lengths in different elution channels from the end of the separation station flowing into the different channels of the MFS. Once the MFS capture phase is complete, the magnetic fields at all the undesirable capture sites are removed in panel (c), allowing the magnetic beads to be released from those sites and washed (e.g., to the waste outlet as shown). The magnetic fields at the desirable sites are maintained to retain the beads. After washing is complete, the desirable magnetic beads are released and in panel (d) and recaptured (e.g., at another collection point as shown). The analyte (e.g., nucleic acid of interest) can be dissociated from the magnetic beads at this point (e.g., using an elevated temperature). This elution step can be carried out under a controlled (small) volume, hence also achieving sample concentration. Finally, the eluted analyte can serve as a building nucleic acid for the next round of ligation assembly as shown in panel (e) where the eluted nucleic acid of interest is transferred back to the reaction chamber.

Reaction chambers (e.g., in FIG. 5C) and capture zones (e.g., in FIG. 5E) of the invention may have a surface area of between about 1 mm2 and about 100 mm2 with volumes of between 10 nl and about 10 μl. However, other dimensions and volumes may be used as the invention is not limited in this respect. Similarly, the cross-sectional shapes of the reaction chambers and capture zones may be any suitable shape (e.g., round, oval, rectangular, trapesoid, triangular, irregular, etc., or any combination thereof) and are not limited by the shapes shown in the figures.

It should be appreciated that different separation and isolation stations from those described above may be used as the invention is not limited in this respect. It should be appreciated that high resolution is not required since the typical techniques only needs to separate the unassembled starting nucleic acids from an assembled product of the starting nucleic acids (e.g., separating N-mers from 2N-mers). For example, the transverse-CE is not designed as a high resolution device as illustrated from the discrete number of elution or collection channels. The resolution of the device is not exceeded by the collection system.

In some embodiments, an error correction process may be incorporated into the ligation procedure. It should be appreciated that the relatively low resolution of transverse-CE also may be used for error correction using a mismatch binding agent (e.g., MutS). The mass and charge differences between error-containing bound nucleic acids (bound to a mismatch binding agent such as a MutS protein) and error-free unbound nucleic acids may be large, making a low resolution technique suitable for this application.

FIGS. 6A-6B illustrate a non-limiting example of a cascaded architecture. In the cascaded architecture, the reactions are performed in the microfluidic channels and a fixed architecture of the channels determines the length of the reaction (in combination with the flow rate, volume, etc.). Fluid may be driven (e.g., electroosmotically). However, other forces may be used to drive the fluid (e.g., pressure, etc.). A cascaded architecture may include the following three components: i) a mixer, ii) a reactor (e.g., kinase/ligase reactor), and iii) a separation station (e.g., a CE size selector). Non- limiting examples of these components are illustrated in FIGS. 6 A and 6B. In some embodiments, the mixer is a diffusive mixing device. Two input oligonucleotides and the enzyme mix may be introduced into the mixer simultaneously. After mixing, in a continuous fashion, the mixture enters the reactor, where a temperature controlled channel facilitates the assembly reactions (e.g., enzymatic reactions). The flow rate of the inlets will determine the allowed reaction time for a given size/geometry of the reactor. Once the reaction reaches the end of the reactor, a small plug of material is injected into the CE separation column. This may be accomplished by using a cross- flow of CE buffer. However, in some embodiments, one or more valves may be used. In some embodiments, waste volume from the mixer and reactor prior to the reaction volume may be sent to the waste outlet as shown. By monitoring the fluorescence signal of the desired analyte, such analyte is extracted at the end of the CE column by using the same method where sample is injected into the CE column (e.g., by applying a cross flow). Concentration of products can be improved by running multiple sample plugs down the CE column in a time-multiplexed fashion. It should be appreciated that any suitable CE size-selector may be used (including for example, a transverse CE component as described herein).

In some embodiments, in order to assemble more starting nucleic acids into a final construct, units of the device may be "cascaded" together to perform a larger assembly. For example, FIG. 6B illustrates a non-limiting example where three linked units of the functional block shown in FIG. 6 A are connected. Further rounds of assembly are possible by cascading more stages together in a similar fashion. In some embodiments, a concentration station is used in between rounds of assembly. For example, a concentration technique similar to the one described above for the recirculating technique may be used. In some embodiments, amplification also may be used in between rounds of assembly. It should be appreciated that the recirculating and cascaded architectures may be combined with components of both in a single microfluidic device (e.g., on the same chip or on different chips within the same system). Certain similarities and differences between the recirculating and cascaded architectures should be appreciated. In some embodiments, the number of assembly cycles (e.g., repeats) may be determined by the system operator (e.g., using an appropriate computer-implemented program) for a recirculating device whereas the cascaded architecture determines the number of repeats by the pattern that is etched onto the substrate (e.g., the number of assembly cycles may be hard-wired into the cascaded design). In some embodiments, the reactor component is a chamber for the recirculating architecture in contrast to a channel for the cascaded device.

In some embodiments, the recirculating architecture is most desirable for its ability to separate and capture a large number of molecules. Since the design is continuous flow (when separation takes place perpendicular to the flow), it is possible to capture almost all of the ligation products. In contrast, the cascaded design, where only a small amount of the reaction volume can be injected into the capillary, can lead to a small number of molecules separated and captured. It should be appreciated that the input concentrations and step-wise yields need to be sufficient to produce useful levels of final product at the final stage. A useful level may be an amount that can be eluted, amplified, cloned, or otherwise propagated for further use. In some embodiments, nucleic acid (e.g., oligonucleotide) concentrations input into a microfluidic reaction are on the order of 1 nM to 1 μM (e.g., more than about 1 nM, about 1 nM to about 10 nM, about 10 nM to about 100 nM, or more than about 100 nM) for extension assemblies such as PCR assemblies, 10 nM to 10 μM (e.g., more than about 10 nM, about 10 nM to about 100 nM, about 100 nM to about 1 μM, or more than about 1 μM) for ligation, and about 100 nM down to a single molecule for amplification reactions (e.g., PCR amplifications). However, other suitable concentrations may be used as the invention is not limited in this respect. Typical reaction volumes on a microfluidic device may be between 10 nl and 10 μl (e.g., more than about 10 nl, about 10 nl to about 100 nl, about 100 nl to about 1 μl, or more than about 1 μl). However, other suitable volumes may be used as the invention is not limited in this respect. Accordingly, extension assemblies may include between about 10 attomoles and about 10 pmoles of input nucleic acids (e.g., oligonucleotides). Ligation reactions may include between about 100 attomoles and 100 pmoles of input nucleic acids (e.g., oligonucleotides). Amplification reactions (e.g., PCR amplifications) may contain between about 1 molecules and 10 pmoles of input nucleic acids (e.g., oligonucleotides). Yields from microfluidic devices may be on the order of 1 pM to 1 μM concentration of final product. However, other concentrations (e.g., down to 1 fM or lower) may be produced and used. Typical volumes are on the order of 10 nl to 10 μl with the number of molecules of final product between a single molecule and 100 pmoles. However, other suitable volumes and numbers of final molecules may be obtained as the invention is not limited in this respect.

In some embodiments, one or more concentration and/or amplification procedures may be included in between assembly cycles (e.g., after every cycle or after a predetermined number of cycles) in order to maintain sufficient levels for assembly throughout the process. Accordingly, one or more concentration and/or amplification stations may be integrated into a microfluidic device. It should be appreciated that the need for concentration/amplification will depend on the amount of input nucleic acids, the length of the nucleic acids being assembled, the number of cycles, the reaction volumes, the material used for the microfluidic devices (e.g., the extent to which it binds nucleic acids), the lengths of the microfluidic channels being used, etc., or any combination thereof. One of ordinary skill can determine optimal concentrations of input nucleic acids and requirements for intermediate concentration or amplification following the teachings provided herein.

In some embodiments, initial reaction mixtures (e.g., mixtures of oligonucleotides) may be prepared using an acoustic liquid handler. Precise reaction mixtures may be prepared from reagent solutions and/or oligonucleotide solutions (e.g., from a multi-well plate). In some embodiments, the reaction solutions may be processed in an initial reaction (e.g., in a thermocycler using a multi-well plate) prior to transfer to a microfluidic substrate. In some embodiments, the reaction solutions may be transferred directly to a microfluidic device for processing (e.g., ligation etc., as described herein). In some embodiments, the transfer to the microfluidic device is performed using a sipper (e.g., an inlet port as described herein) that takes a defined volume from a well (e.g., in a multi-well plate). In some embodiments, a robotic liquid handler (e.g., a robotic pipettor) transfers a volume to an inlet port. In some embodiments, an acoustic liquid handling technique may be used to transfer a suitable volume to a receiving station (e.g., inlet region or port) on a microfluidic device.

It should be appreciated that error-removal according to the invention may be performed after any one or more cycles of assembly described herein (e.g., after any one or more cycles or extension and/or ligation-based assembly). Error-removal may be performed on a size-selected or otherwise isolated nucleic acid fragments of interest, on crude assembly reaction mixtures, and/or on cloned nucleic acid fragments. It should be appreciated that even though FIGS. 5-6 do not illustrate error correction (e.g., using MutS binding), it may be incorporated into either of the recirculating or cascaded architectures as described herein.

In another aspect, devices and methods of the invention may be used to monitor one or more assembly and/or error removal steps described herein. In some embodiments, an aliquot of an assembly reaction may be removed at a plurality (e.g., two, three, four, five, or more) of time points during a single assembly (e.g., after each denaturing/extension cycle during a polymerase-based assembly). In certain embodiments, an aliquot of an assembly reaction may be removed from the reaction product for each intermediate in an assembly reaction. An aliquot that is removed from any assembly reaction may be analyzed to determine the distribution of nucleic acids that are present in the reaction. An analysis may be an electrophoretic analysis, a mass spectrometry analysis, or any other suitable analysis (e.g., an analysis that can identify different incomplete assembly products in a reaction mixture).

In some embodiments, assembly monitoring may be used to analyze and improve assembly reactions. For example, a plurality of different reaction steps may be performed and/or analyzed (e.g., in parallel or in series) on a microfluidic device in order to systematically analyze and determine optimal assembly conditions (e.g., times, temperatures, concentrations, reagents, etc.). In some embodiments, different optimal assembly conditions may be analyzed and/or identified for different types of assembly reactions (e.g., extension-based, ligation-based, or a combination thereof), or for different types of nucleic acids (e.g., different lengths, different chemical modifications, different GC contents, different repeat frequencies, etc., or any combination thereof). It should be appreciated that the reactions being tested may be performed on a microfluidic substrate or on a larger scale substrate such as a multi-well plate or other reaction well. Accordingly, an aliquot of a larger scale assembly reaction may be processed using micro fluidic devices and methods of the invention (e.g., the size distribution of different reaction mixtures may be analyzed). It should be appreciated that error correction or removal reactions may be analyzed and/or optimized using similar methods to those described herein for assembly reactions. Error removal reaction parameters that may be modified may include one or more of the following non-limiting parameters: temperature, buffer, type of mismatch protein, screening or selection method, concentration of nucleic acids and/or mismatch protein, etc., or any combination thereof. In certain embodiments, assembly monitoring may be used to track the progress of an assembly and identify reaction steps or intermediates that are incorrectly or incompletely assembled. These reactions may be repeated or modified in order to complete a final target nucleic acid of interest. By identifying intermediate reaction steps or products that are incomplete or incorrect, production time and/or efficiency may be enhanced. For example, instead of detecting an error when the final product is analyzed, an error may be detected early during the assembly process allowing corrective measures to be taken before further steps are performed that would incorporate the error. This may avoid repeating unnecessary steps and only involve repeating or modifying intermediate steps that have produced error containing product. This aspect of the invention may be particularly time-saving if it avoids repeating cloning or cell growth steps that may be more time consuming that enzymatic reaction steps.

Accordingly, aspects of the invention provide methods, devices, and systems for dynamically controlling a nucleic acid assembly procedure. In some embodiments, the concentration of one or more of the components in an assembly procedure may be dynamically calibrated or adjusted (e.g., normalized) before, during or after any one of the steps of the assembly procedure in response to changes or differences in the level of one or more reaction components measured at one or more stages in the assembly procedure. In some embodiments, the adjustment may be automated. Dynamic adjustment may include monitoring reaction products at one or more steps during assembly (e.g., after one or more of the following steps: oligonucleotide synthesis, amplification, purification, assembly by extension, assembly by ligation, error removal - for example by MutS, cloning, or any combination thereof) and re-adjusting (e.g., re- normalizing) the concentrations of the intermediate products from one or more steps prior to combining them for a subsequent step. This is particularly useful in a hierarchical assembly procedure where multiple parallel reactions are being processed towards a final product and the products from one set of parallel reactions are combined in a subsequent step comprising a smaller number of parallel reactions etc., until a final product is reached.

In one aspect of the invention, aspect of dynamic adjustment can be automated. In some embodiments, dynamic adjustment is implemented on a microfluidic device. In some embodiments, manipulation of fluid is automated. In many instances, well plates are used to store a large number of fluids for processing. Well plates or microplates are typically single piece in construction and comprise a plurality of identical wells, wherein each well is adapted to contain a small volume of fluid. Such well plates are commercially available in standardized sizes and may contain, for example, 96, 384, 1536, or 3456 wells per well plate. In a preferred embodiment, acoustic technologies are advantageously employed in dynamic adjustment. According to aspects of the invention, a starting imbalance in relative oligonucleotide concentrations in an assembly reaction can significantly affect the assembly efficiency and/or fidelity. In some embodiments, a dynamic control of an assembly procedure may involve dynamic adjustments to oligonucleotide concentrations in response to detected imbalances in the concentrations of input nucleic acids. Accordingly, dynamic control may involve determining the concentration of each oligonucleotide and adjusting them if necessary (e.g., to generate normalized relative concentrations of oligonucleotides in an assembly reaction). In some embodiments, dynamic control of an assembly procedure also involves dynamic adjustments (e.g., dynamic normalizations) of other reagents such as nucleotides, buffers, enzymes, etc., or any combination thereof. Dynamic adjustment may involve adjusting the amount of certain reagents and/or the volume of certain reaction mixtures. In other embodiments, dynamic control of an assembly procedure may involve dynamic adjustments to the relative concentrations of intermediate nucleic acids that are combined together for further assembly (e.g., after an earlier assembly reaction and/or after one or more error correction and/or error removal steps (e.g., using a mismatch binging protein). In some embodiments, the concentrations of nucleic acids that are being combined for further assembly are measured and the relative amounts of each nucleic acid that is used is normalized so that the same amount of each nucleic acid is combined in the subsequent assembly reaction.

In some aspects, the concentration of each nucleic acid (e.g., starting nucleic acid or intermediate nucleic acid) that is combined in an assembly reaction is adjusted (e.g., normalized) to improve the assembly reaction. For example, certain oligonucleotides may be synthesized and/or amplified and/or isolated less efficiently than others. In addition, some oligonucleotides may include some sequence specific features or characteristics (e.g. nucleic acid length, nucleic acid strandedness: double-stranded or single-stranded, GC content, the presence and/or length and/or GC content and/or sequence of single-stranded overhangs in a double-stranded nucleic acid, the degree of predicted secondary structures, the number and length of inverted and/or direct repeats, etc., or any combination thereof) and therefore may amplified less efficiently than others. Similarly, certain intermediates may be assembled less efficiently than others in a first round of assembly. Accordingly, the concentration of each nucleic acid (or pool of nucleic acids if a pool of variant nucleic acids is synthesized to be assembled into a library) may be adjusted to approximately the same level when they are combined for an initial or subsequent round of assembly. However, in some embodiments, the concentration of different starting or intermediate nucleic acids may be set at different levels. For example, certain nucleic acids (such as hairpin loop nucleic acids) may be provided at higher concentrations than others if it is helpful for an assembly or other reaction. In some embodiments, the concentrations of one or more substrates or intermediates may be adjusted dynamically during an assembly process. For example, concentrations of different nucleic acids may be monitored continuously throughout the assembly procedure or after one or more predetermined assembly steps. The relative concentrations of different nucleic acids may be adjusted (e.g., normalized) at any stage during the assembly procedure resulting in a dynamic adjustment of different nucleic acid concentrations in response to measurements of nucleic acid levels during the assembly procedure. For example, dynamic adjustment (e.g., normalization) may include monitoring reaction products after one or more steps of the assembly process and re-adjusting (e.g., re-normalizing) the concentrations of one or more of the intermediate products from one or more steps prior to combining them for a subsequent step (e.g., by increasing or reducing the amount more of one or more nucleic acid samples that is added to a subsequent step and/or by increasing or reducing nucleic acid sample or reaction volumes). In some embodiments, in order to adjust the relative nucleic acid amounts in a reaction mixture (e.g., to normalize the relative amounts or concentrations of different nucleic acids that are combined in an assembly reaction), different volumes of the different nucleic acids may be added, because different nucleic acid preparations may have different nucleic acid concentrations. As a result, the volumes or concentrations of one or more additional reagents may be adjusted to account for the different nucleic acid volumes and produce a reaction mixture that has an appropriate predetermined final concentration of reaction components (including, e.g., nucleic acids, enzymes, NTPs, buffers, salts, etc., or any combination thereof). Dynamic adjustments may be automated and incorporated into an automated assembly procedure that includes an automated dynamic control of the different assembly steps. Automated assembly procedures including dynamic control features may be implemented on a robotic device, for example, an automated acoustic device or microfluidic device. In some embodiments, dynamic adjustments in reagent amounts and/or reaction volumes may be implemented using a microfluidic device, a robotic dispensing device, an acoustic liquid handling device (e.g., an Echo™ 550 device), any other suitable liquid handling device, or any combination thereof.

Assembly methods of the invention may be automated. Aspects of the invention provide for intermediate quality control steps, for example, in an automated assembly procedure. Methods of the invention may therefore reduce the cost and increase the speed and accuracy of nucleic acid assembly procedures, particularly automated assembly procedures.

Aspect of the invention comprises methods for dynamic quality control for gene assembly using acoustic technologies. An assembly procedure may include several parallel and/or sequential reaction steps in which a plurality of different nucleic acids or oligonucleotides are synthesized, amplified, purified and are combined in order to be assembled (e.g., by extension or ligation as described herein) to generate a longer nucleic acid product to be used for further assembly, error removal, cloning, or other applications. It should therefore be appreciated that gene assembly procedures require that a diverse set of source components or reagents from a source microfluidic substrate be correctly dispensed and in a designated location of the receiving microfluidic substrate. For example, if a component or reagent failed to be dispensed at the right location of a receiving microfluidic substrate, a gene may not assemble or assembly of a gene may be incorrect (e.g. large deletion, etc...). Incorrect assemblies may then be detected after one more step of the assembly process using costly and time-intensive high resolution biological methods such as high resolution capillary electrophoresis or DNA sequencing.

As used herein, "source microfluidic substrate" refers to any microfluidic substrate suitable for containing a pool of source fluid and which allows an acoustic wave to propagate from a first side or end of the substrate, through the substrate to the second side or end of the substrate, wherein the source fluid is contained on the second side or within the substrate. As used herein, "receiving microfluidic substrate" means a substrate towards which a droplet of source fluid is ejected, or with which the ejected droplet makes contact, the receiving microfluidic substrate being separate (i.e. not in contact) at a selected distance of the source microfluidic substrate. Suitable source and receiving microfluidic substrate include multi-well plates commonly used in molecular biology applications.

According to aspects of the invention, acoustic radiation may be used to monitor and/or assess the volume of one or more fluid reservoirs (e.g. wells of the source or the receiving microfluidic substrate). A number of patents describe the use of acoustic energy to assess the content and volume of fluid in a container. U.S. Patent No.

5,880,364, for example, describes a non-contact ultrasonic system for measuring the volume of liquid in a plurality of containers and U.S. Patent 6,932,097 discloses the use of acoustic device in fluid composition and volume monitoring. Depth of the well fluid (i.e. volume of a reservoir) may be calculated by measuring the time-of-flight of sound energy to the surface of liquid in the reservoir. In one embodiment, a small burst of acoustic wave is directed toward the surface of the liquid in a well, the sound wave travels through the liquid and reflects off the surface of the liquid, returning toward the source. Using the speed of sound in the liquid, one can measure the depth of the liquid (i.e. the volume) by measuring the time between the generation of the burst and the reception of the reflection wave.

In one embodiment, acoustic technologies are used to monitor the dispensing of a plurality of components or reagents during gene assembly. In a preferred embodiment, methods are provided to assess if components have been correctly dispensed at a specific location (e.g. specific well on a microfluidic substrate or microplate). The methods include monitoring the volume or liquid height of the source microfluidic substrate. These methods are particularly useful for quality control and gene assembly process control. In one embodiment, acoustic technologies can be used to determine the volume of each well in a source microfluidic substrate before dispensing reagents therefore establishing a reference database for each well of the source microfluidic substrate. Accordingly, acoustic technologies can be used to determine the volume of each well of a receiving microfluidic substrate before receiving the reagents therefore establishing a reference database for each well of the receiving microfluidic substrate. After addition of components or reagents to the receiving microfluidic substrate, the source microfluidic substrate can be scanned to determine if the correct volume has been ejected. In addition, the receiving microfluidic substrate can be scanned to ensure that the correct volume has been dispensed into each well. In one embodiment, manufacturing or assembly defects can be detected based on a difference between the reference databases and the volume of each well of the source microfluidic substrate after droplet ejection. Alternatively, assembly defects can be detected based on a difference between the reference databases and the volume of each well of the receiving microfluidic substrate after dispense of the reagents. The reference database may represent a source microfluidic substrate, a receiving microfluidic substrate and may include all previous manipulations of a specific well. The detection step may be performed at one or more steps, at selected steps or for every single gene assembly steps (oligonucleotide synthesis, dilution, amplification, purification, gene extension, error removal, cloning etc...). Minor changes, inconsistencies, errors and/or other variations may be detected by comparison of the actual volumes with the reference databases. In some embodiments, reaction volumes may be adjusted to stay within a narrow range of one or more predetermined volumes. For example, reaction volumes may be dynamically monitored and adjusted to stay within +/- 0.01%, +/- 0.1%, +/- 1%, +/- 2%, +/- 5%, +/- 10%, +/- 15%, +/- 20%, +/- 25% of one or more predetermined reaction volumes. Once an assembly defect is detected, correction measures can be taken.

Correction measures can include flagging the defective wells, stopping further assembly steps or re-dispense reagents or any combination thereof. In some embodiments, the concentration of one or more of the components may be dynamically adjusted before, during or after any one of the steps of the assembly procedure in response to measured volumes at microfluidic locations.

In some embodiments, liquids including solution comprising nucleic acids, enzymes, buffers, salts, NTPs etc...are added to each one of the defective location on a microfluidic substrate using an acoustic liquid handler (e.g. an acoustic droplet ejector). The liquids can be transferred to any position or selected position on the microfluidic substrate. In some embodiments, solutions are transferred to any position on the microfluidic substrate by "flipping" the microfluidic substrate (e.g. with its sample wells and reaction wells pointing downward) and positioning the microfluidic substrate above a reservoir or reservoir wells that are connected to an acoustic liquid handler. Solutions are subsequently transferred to selected locations of the microfluidic substrate by applying the appropriate sound waves to the reservoir wells.

In a preferred embodiment, the database analysis and the detection of assembly defects are performed automatically and incorporated into the assembly process. The data are automatically processed and correcting processing steps can be initiated automatically by a process control computer. Alternatively, some steps may be performed manually.

In one embodiment, emission of component or reagents in solution may be monitored using a high speed droplet imaging. The dispensing technology (e.g. acoustic, microfluidic) may be coupled with high speed droplet imaging to capture images of droplet emission or images of droplet placement. In this manner, droplet emission and/or droplet dispensing at the correct location may be monitored. The high speed imaging technology captures images of droplets using high resolution cameras (e.g. CCD camera) together with short pulse laser illumination to freeze the motion. The camera may be coupled with an image acquisition system on a computer. Software can then measure the number of droplets, the droplet size and shape and may calculate the volume distribution.

It should be apparent from the present description that a microfluidic device of the invention may comprise different combinations of operational stations connected via one or more microfluidic channels in an architecture that may be adapted for a particular assembly, analysis, monitoring, error-removal, or other processing of nucleic acids (e.g., during assembly). In some embodiments, the concentration of one or more of the assembly components may be dynamically calibrated or adjusted (e.g., normalized) before, during or after any one of the steps of the assembly procedure in response to changes or differences in the level of one or more reaction components measured at one or more stages in the assembly procedure. In some embodiments, the adjustment may be automated. Dynamic adjustment may include monitoring reaction products at one or more steps during assembly (e.g., after one or more of the following steps: oligonucleotide synthesis, amplification, purification, assembly by extension, assembly by ligation, error removal - for example by MutS, cloning, or any combination thereof) and re-adjusting (e.g., renormalizing) the concentrations of the intermediate products from one or more steps prior to combining them for a subsequent step. This is particularly useful in a hierarchical assembly procedure where multiple parallel reactions are being processed towards a final product and the products from one set of parallel reactions are combined in a subsequent step comprising a smaller number of parallel reactions etc., until a final product is reached. This aspect of dynamic adjustment can be automated. In some embodiments, dynamic adjustment is implemented on a microfluidic device. In some embodiments dynamic adjustment is automated on a microfluidic device.

In some embodiments, a single microfluidic substrate may comprise a plurality of PCR reactions wells and/or LCR reaction wells and/or ligase reaction wells (e.g., independently 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-50, 50-100, or more of each well). The microfluidic substrate also may comprise a plurality of nucleic acid isolation (e.g., separation) stations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-50, 50-100, or more). All or a subset of each of these operational stations (e.g., PCR wells, LCR wells, ligase wells, nucleic acid isolation stations, etc.) may be connected via one or more microfluidic channels in parallel or in series. In some embodiments, one or more microfluidic channels may loop back so that the reaction product from one operational station may be fed back into the same operational station or an earlier operational station. Any one of these configurations may be used to process large numbers of samples simultaneously, or essentially simultaneously. For example, large numbers of samples may be processed in parallel. However, large numbers of samples also may be processed in series. For example, a plurality of reaction volumes may be separated by buffer or other separation material within a single micro fluidic channel to form a chain of reaction mixtures. Accordingly, a plurality of reactions may be processed rapidly in series. One or more operational station may be washed after each reaction volume. In some embodiments, the buffer or separating material between each reaction mixture may contain suitable buffers, salts, detergents, etc., or any combination thereof, to wash an operational station between two samples. However, in some embodiments, a suitable wash buffer, salt, detergent, or any combination thereof, may be delivered through an operational station from a reservoir (e.g., on the microfluidic substrate) to wash the operational station in between samples. In some embodiments, a plurality of sequential assembly reactions may be performed using microfluidic devices. For example, a first plurality of assembly reactions, a first optional size selection step, an first optional error removal step, a second plurality of assembly reactions using the products of the first plurality of assembly reactions as substrates, a second optional size selection step, a second optional error removal step, a third round of assembly reactions, etc., may be performed on using microfluidic devices. In some embodiments, two or more (e.g., all) of the sequential assembly steps leading to a target nucleic acid may be performed on the same microfluidic substrate. In some embodiments, the substrate may contain a plurality of different operational stations dedicated to different steps in the assembly procedure (e.g., first assembly, first size selection , first error removal, second assembly, second size selection, second error removal, etc.). Products may be transferred from first stations to second stations and any subsequent stations as they are progressively assembled. In some embodiments, products may be transferred from first stations to second stations and any subsequent stations through the action of an acoustic liquid handler (e.g., an acoustic droplet ejector). However, it should be appreciated that a microfluidic substrate may contain an operational station that is used repeatedly during assembly. For example, a first plurality of assembly reactions may be processed (e.g., assembled, size-selected, and/or error corrected) using a first set of operational station(s) and the products of each of the reactions may be stored (e.g., individually or mixed in one or more storage wells on the microfluidic substrate). When the first intermediates or products are ready (e.g., they are all assembled, size-selected, and/or error-corrected, and distributed to appropriate storage stations), they may be removed from the storage station(s) and processed through the same first operational station(s) to produce a second product or plurality of products or intermediates. This process may be repeated until a final product is generated. It should be appreciated that a microfluidic substrate may contain different combinations of operational stations, some of which may be connected in parallel, some connected in series, including one or more microfluidic channels that feed back from an operational station outlet to an earlier position on the substrate (e.g., an inlet of an operational station). One or more assembly, error correction, isolation, or other reaction stations may be connected to one or more storage wells, reagent wells, buffer wells, etc., or any combination thereof. In some embodiments, one or more operational stations (including reactions stations, storage stations, buffer or reagent stations, etc.) may include one or more inlets and/or outlets (e.g., microfluidic channel connections). A substrate may include one or more valves, etc. to control operation. However, it should be appreciated that different aspects of the assembly procedure may be performed on different microfluidic substrates. However, the different substrates (e.g., chips) may be within the same microfluidic system or device and appropήate liquid channels or liquid handling devices are used to transfer reagents, intermediates, products, etc. from one assembly location to another. In some embodiments, the liquid handling device comprises an acoustic liquid handler (e.g., an acoustic droplet ejector).

In some embodiments, a nucleic acid (e.g., cloned into a vector) may be transformed into a cell (e.g., a host cell) within a microfluidic substrate. Accordingly, a microfluidic substrate may include one or more channels or wells that are sufficiently large to contain and/or transport one or more host cells. Transformation may involve electroporation. A microfluidic substrate may include one or more electroporation stations. Accordingly, in some embodiments a plurality of different assembly reactions in different stages of assembly (e.g., any one or more of i) - vii) of FIGS. IB, 2B-D, 3B, 4B-D, or any intermediate or additional stages, including transformed cells) may be present in different operational stations (e.g., in one or more assembly wells, error- correction wells, isolation wells such as size selection wells, storage wells, other microfluidic wells or channels, or any combination thereof) in a single microfluidic substrate. However, in some embodiments a plurality of different assembly reactions in different stages of assembly (e.g., any one or more of i) - vii) of FIGS. IB, 2B-D, 3B, 4B-D, or any intermediate or additional stages) may be present in different operational stations (e.g., in one or more assembly wells, error-correction wells, isolation wells such as size selection wells, storage wells, other microfluidic wells or channels, or any combination thereof) in different microfluidic substrates within the same microfluidic system. In some embodiments, multiple runs of a same reaction may be performed on the same microfluidic substrate, or on different substrates within the same microfluidic system. For example, duplicate, triplicate, etc. runs may be performed on the same microfluidic substrate or on different microfluidic substrates within the same microfluidic system. It should be appreciated that microfluidic substrates or systems may include a plurality (e.g., 2, 3, 4, 5, 5-10, 10-50, 50-100, or more) separate collection ports and/or distribution ports in order to process a plurality of samples and/or reagents. The different collection and/or distribution ports may be connected to the same or different parallel, serial, and/or other architectures adapted to methods described herein. In operation, samples (including reagents, buffers, oligonucleotides, intermediate nucleic acid products, other nucleic acids, etc.) may be collected via the collection port(s), transported through the appropriate operational stations via microfluidic channels and distributed via the distribution port(s). In some embodiments, the samples may be distributed using an acoustic liquid handler (e.g., an acoustic droplet ejector). The collection and distribution ports may be brought into contact with appropriate devices or systems (e.g., sample plates, oligonucleotide plates, reaction wells, multi-well plates, etc.) for collection or distribution using appropriate robotic or other devices (e.g., controlled by appropriate controllers, computers, software, etc.).

Microfluidic Devices, Substrates, and Systems

In some, but not all embodiments, all components of the systems and methods described herein are microfluidic. "Microfluidic," as used herein, refers to a device, apparatus or system including at least one fluid channel having a cross-sectional dimension of less than about 2 mm, less than 1 mm and a ratio of length to largest cross- sectional dimension of at least 3:1. A "microfluidic channel," as used herein, is a channel meeting these criteria. The "cross-sectional dimension" of the channel is measured perpendicular to the direction of fluid flow. Most fluid channels in components of the invention have maximum cross-sectional dimensions less than 2 mm, and in some cases, less than 1 mm. In one set of embodiments, all fluid channels containing embodiments of the invention are micro fluidic or have a largest cross sectional dimension of no more than 2 mm or 1 mm. In another embodiment, the fluid channels may be formed in part by a single component (e.g. an etched substrate or molded unit). Of course, larger channels, tubes, chambers, reservoirs, etc., can be used to store fluids in bulk and to deliver fluids to components of the invention. In one set of embodiments, the maximum cross-sectional dimension of the channel(s) containing embodiments of the invention are less than 500 microns, less than 200 microns, less than 100 microns, less than 50 microns, or less than 25 microns. Microfluidic channels may be closed. However, in some embodiments, microfluidic substrates may include one or more open channels.

As used herein, "integral" means that portions of components are joined in such a way that they cannot be separated from each other without cutting or breaking the components from each other.

A variety of materials and methods, according to certain aspects of the invention, can be used to form the fluidic or microfluidic system. For example, various components of the invention can be formed from solid materials, in which the channels can be formed via micromachining, film deposition processes such as spin coating and chemical vapor deposition, laser fabrication, photolithographic techniques, etching methods including wet chemical or plasma processes, and the like. See, for example, Scientific American, 248:44-55, 1983 (Angell, et al).

In one set of embodiments, at least a portion of the fluidic system is formed of silicon by etching features in a silicon chip. Technologies for precise and efficient fabrication of various fluidic systems and devices of the invention from silicon are known. In another embodiment, various components of the systems and devices of the invention can be formed of a polymer, for example, an elastomeric polymer such as polydimethylsiloxane ("PDMS"), polytetrafluoroethylene ("PTFE" or Teflon®), or the like. For instance, in some embodiments a system may be implemented by fabricating the fluidic system separately using PDMS or other soft lithography techniques (details of soft lithography techniques suitable for this embodiment are discussed in the references entitled "Soft Lithography," by Younan Xia and George M. Whitesides, published in the Annual Review of Material Science, 1998, Vol. 28, pages 153-184, and "Soft Lithography in Biology and Biochemistry," by George M. Whitesides, Emanuele Ostuni, Shuichi Takayama, Xingyu Jiang and Donald E. Ingber, published in the Annual Review of Biomedical Engineering, 2001, Vol. 3, pages 335-373; each of these references is incorporated herein by reference).

Different components can be fabricated of different materials. For example, a base portion including a bottom wall and side walls can be fabricated from an opaque material such as silicon or PDMS, and a top portion can be fabricated from a transparent or at least partially transparent material, such as glass or a transparent polymer, for observation and/or control of the fluidic process. Components can be coated so as to expose a desired chemical functionality to fluids that contact interior channel walls, where the base supporting material does not have a precise, desired functionality. For example, components can be fabricated as illustrated, with interior channel walls coated with another material. Material used to fabricate various components of the systems and devices of the invention, e.g., materials used to coat interior walls of fluid channels, may desirably be selected from among those materials that will not adversely affect or be affected by fluid flowing through the fluidic system, e.g., material(s) that is chemically inert in the presence of fluids to be used within the device (e.g., in the presence of nucleic acids such as ss or ds DNA, RNA, or other nucleic acids, and/or enzymes, and/or other reagents of the invention).

In some embodiments, various components of the invention are fabricated from polymeric and/or flexible and/or elastomeric materials, and can be conveniently formed of a hardenable fluid, facilitating fabrication via molding (e.g. replica molding, injection molding, cast molding, etc.). The hardenable fluid can be essentially any fluid that can be induced to solidify, or that spontaneously solidifies, into a solid capable of containing and/or transporting fluids contemplated for use in and with the fluidic network. In one embodiment, the hardenable fluid comprises a polymeric liquid or a liquid polymeric precursor (i.e., a "prepolymer"). Suitable polymeric liquids can include, for example, thermoplastic polymers, thermoset polymers, or mixture of such polymers heated above their melting point. As another example, a suitable polymeric liquid may include a solution of one or more polymers in a suitable solvent, which solution forms a solid polymeric material upon removal of the solvent, for example, by evaporation. Such polymeric materials, which can be solidified from, for example, a melt state or by solvent evaporation, are well known to those of ordinary skill in the art. A variety of polymeric materials, many of which are elastomeric, are suitable, and are also suitable for forming molds or mold masters, for embodiments where one or both of the mold masters is composed of an elastomeric material. A non-limiting list of examples of such polymers includes polymers of the general classes of silicone polymers, epoxy polymers, and acrylate polymers. Epoxy polymers are characterized by the presence of a three- membered cyclic ether group commonly referred to as an epoxy group, 1 ,2-epoxide, or oxirane. For example, diglycidyl ethers of bisphenol A can be used, in addition to compounds based on aromatic amine, triazine, and cycloaliphatic backbones. Another example includes the well-known Novolac polymers. Non-limiting examples of silicone elastomers suitable for use according to the invention include those formed from precursors including the chlorosilanes such as methylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc., or any combination thereof.

Silicone polymers are used in certain embodiments, for example, the silicone elastomer polydimethylsiloxane. Non-limiting examples of PDMS polymers include those sold under the trademark Sylgard by Dow Chemical Co., Midland, MI, and particularly Sylgard 182, Sylgard 184, and Sylgard 186. Silicone polymers including PDMS have several beneficial properties simplifying fabrication of the microfluidic structures of the invention. For instance, such materials are inexpensive, readily available, and can be solidified from a prepolymeric liquid via curing with heat. For example, PDMSs are typically curable by exposure of the prepolymeric liquid to temperatures of about, for example, about 65 0C to about 75 0C for exposure times of, for example, about an hour. Also, silicone polymers, such as PDMS, can be elastomeric and thus may be useful for forming very small features with relatively high aspect ratios, necessary in certain embodiments of the invention. Flexible (e.g., elastomeric) molds or masters can be advantageous in this regard.

One advantage of forming structures such as microfluidic structures of the invention from silicone polymers, such as PDMS, is the ability of such polymers to be oxidized, for example by exposure to an oxygen-containing plasma such as an air plasma, so that the oxidized structures contain, at their surface, chemical groups capable of cross-linking to other oxidized silicone polymer surfaces or to the oxidized surfaces of a variety of other polymeric and non-polymeric materials. Thus, components can be fabricated and then oxidized and essentially irreversibly sealed to other silicone polymer surfaces, or to the surfaces of other substrates reactive with the oxidized silicone polymer surfaces, without the need for separate adhesives or other sealing means. In most cases, sealing can be completed simply by contacting an oxidized silicone surface to another surface without the need to apply auxiliary pressure to form the seal. That is, the pre- oxidized silicone surface acts as a contact adhesive against suitable mating surfaces. Specifically, in addition to being irreversibly sealable to itself, oxidized silicone such as oxidized PDMS can also be sealed irreversibly to a range of oxidized materials other than itself including, for example, glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, and epoxy polymers, which have been oxidized in a similar fashion to the PDMS surface (for example, via exposure to an oxygen-containing plasma). Oxidation and sealing methods useful in the context of the present invention, as well as overall molding techniques, are described in the art, for example, in an article entitled "Rapid Prototyping of Microfluidic Systems and Polydimethylsiloxane," Anal. Chem., 70:474-480, 1998 (Duffy et al), incorporated herein by reference.

Another advantage to forming microfluidic structures of the invention (or interior, fluid-contacting surfaces) from oxidized silicone polymers is that these surfaces can be much more hydrophilic than the surfaces of typical elastomeric polymers (where a hydrophilic interior surface is desired). Such hydrophilic channel surfaces can thus be more easily filled and wetted with aqueous solutions than can structures comprised of typical, unoxidized elastomeric polymers or other hydrophobic materials. In one embodiment, a bottom wall is formed of a material different from one or more side walls or a top wall, or other components. For example, the interior surface of a bottom wall can comprise the surface of a silicon wafer or microchip, or other substrate. Other components can, as described above, be sealed to such alternative substrates. Where it is desired to seal a component comprising a silicone polymer (e.g., PDMS) to a substrate (bottom wall) of different material, the substrate may be selected from the group of materials to which oxidized silicone polymer is able to irreversibly seal (e.g., glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, epoxy polymers, and glassy carbon surfaces which have been oxidized). Alternatively, other sealing techniques can be used, as would be apparent to those of ordinary skill in the art, including, but not limited to, the use of separate adhesives, thermal bonding, solvent bonding, ultrasonic welding, etc. In some embodiments, the micro fluidic device may be made from or include other inert material. In some aspects the material is inert to the fluids and chemicals used in the micro fluidic device, and inert to physical changes like exposure to light and changes in pressure or temperature. Examples of inert materials are glass, quartz, silicon, plastics and polyacrylates. A microfluidic device can be made by combining two or more separate layers. These layers comprise a bottom portion and a top portion and optionally one or more interior portions. If the microfluidic device comprises a middle portion, this portion may contain the channels, reaction and sample wells, columns, and other functional elements. The components may also be etched in or laid out on the top or bottom part of the device. Methods for the manufacture of microfluidic devices are well known in the art (See e.g. US patent 6,322,753).

In some embodiments, due to the fabrication technology selected (PDMS and glass), extensive PDMS surface passivation and conditioning may be necessary to prevent loss of nucleic acids that would reduce the yield of assembled products during the assembly process. In some embodiments, the absorbing nature of the PDMS may render a device non-reusable since previously amplified DNA will be embedded into the PDMA walls after a reaction. Accordingly, although the PDMS-on-glass technology is less expensive (compared to glass-on-glass), and hence more disposable, the conditioning steps and the replacement of chips after each reaction makes this approach more expensive for a fully automated operation. In contrast, a glass-on-glass fabrication technology produces fluidic substrates that do not require passivation and are reusable. In some embodiments, a microfluidic device comprises a sipper. When aspects of the invention are described herein in connection with a sipper, it should be appreciated that any suitable collection port or other means for transferring components into a microfluidic device may be used as a substitute for the sipper. A sipper can be an integral part of the microfluidic device. The sipper can transfer components to the top of the fluid device, to the bottom or directly into channels or wells of the device. Alternatively, the sipper can be attached and detached from the microfluidic device when desired. A sipper can comprise a channel, a pipettor, a tube or any other means for moving a liquid or solid component into the microfluidic device. In some embodiments, the function of the sipper can be performed by an acoustic liquid handler (e.g., an acoustic droplet ejector). Transportation can be performed by fluid pressure, gas pressure, vacuum pressure, electronic modulators or physical means, like a piston directly pushing the component into the microfluidic device. The fluid, liquid, reagent, chemical or solid to be transferred can be transferred from vials, plates, slides, microfluidic devices or any device that can hold a liquid, fluid, chemical or solid.

In some embodiments the microfluidic device comprises a spitter. When aspects of the invention are described herein in connection with a spitter, it should be appreciated that any suitable distribution or dispensing port or means for transferring a fluid, liquid, reagent, chemical or solid from the microfluidic device into another device that can contain said fluid, liquid, reagent, chemical or solid may be used as a substitute for the spitter. A spitter can be an integral part of the microfluidic device. In some embodiments, the function of the spitter can be performed by an acoustic liquid handler (e.g., an acoustic droplet ejector). The spitter can transfer fluid, liquid, reagent, chemical or solid from the top of the fluid device, from the bottom or directly from the channels or wells, columns or other elements of the device. Alternatively, the spitter can be attached and detached from the microfluidic device when desired. A spitter can comprise a channel, a pipettor, a tube or any other means for moving a fluid, liquid, reagent, chemical or solid into the microfluidic device. Transportation can be performed by fluid pressure, gas pressure, vacuum pressure, electronic modulators or physical means, like a piston directly pushing the component into the microfluidic device. In some embodiments fluid, liquid, reagent, chemical or solid are transferred from the microfluidic device directly. Non-limiting examples of transferring methods are direct pipetting, pressure based displacement methods, and centrifugation or gravity based methods

In some embodiments, a microfluidic device is associated with an acoustic liquid handler (e.g., an acoustic droplet ejector). An acoustic liquid handler (e.g., an acoustic droplet ejector) is a device that can be used to transfer small droplets of liquids (e.g., on the order of a pi or a nl). The droplets of liquids are transferred by ultrasonic acoustic energy. Sound waves are transmitted through the bottom of a fluid reservoir resulting in the release of a droplet from the fluid reservoir. The volume of the droplet can be determined by selecting the appropriate sound wave frequency. The ejected droplet can subsequently be captured by a substrate or receiving device positioned above the fluid reservoir. An advantage of using acoustic droplet ejector is the elimination of the need for tips and other materials to transfer liquids from a reservoir to another plate. Acoustic liquid handlers (e.g., acoustic droplet ejectors) are well known in the art, see e.g., US Patent Nos. 6,416,164 and 6,802,593, the disclosures of which are incorporated herein by reference. In some embodiments, an Echo™ 550 acoustic liquid handler may be used. In some embodiments, acoustic liquid handling technology is integrated into a micro fluidic device so that it can be used to transfer small volumes of liquid between one or more positions on one or more microfluidic substrates (e.g., chips).

In some embodiments, liquids, for example solutions comprising enzymes and nucleic acids, are transferred onto the microfluidic device using acoustic liquid handling technology (e.g., a stand-alone acoustic droplet ejector or similar technology incorporated into a microfluidic system). The liquids can be transferred to any position on the microfluidic device including a reservoir, channel, sample well, reaction well, and/or any operational station. In one embodiment, liquids may be transferred to a microfluidic substrate by 'flipping' the microfluidic substrate (e.g., with its sample well and reaction wells pointing downward), and positioning the microfluidic substrate above reservoir wells (e.g., a multi-well plate) that are connected to an acoustic liquid handler (e.g., an acoustic droplet ejector). Liquids are subsequently transferred to selected components (e.g., sample wells) of the microfluidic device by applying the appropriate sound waves to the reservoir wells. In some embodiments, an acoustic liquid handler (e.g., an acoustic droplet ejector) is connected to the microfluidic device. In some embodiments, the acoustic liquid handler is a means for transferring liquids to the microfluidic device. In some embodiments, the function of the sipper is performed by an acoustic liquid handler (e.g., an acoustic droplet ejector). In some embodiments, acoustic liquid handling technology (e.g., an acoustic droplet ejector) is used to transfer liquids from a first microfluidic substrate to a second microfluidic substrate, a reservoir plate, or any receiving device. In some embodiments, acoustic liquid handling technology (e.g., an acoustic droplet ejector) is a means for transferring liquids from the microfluidic device. In some embodiments, the function of the spitter is performed by acoustic liquid handling technology (e.g., an acoustic droplet ejector). In some embodiments acoustic liquid handling technology is an integral part of the microfluidic device. In some embodiments, acoustic liquid handlers (e.g., acoustic droplet ejectors) are an integral part of a system comprising a plurality (e.g., a series) of microfluidic devices that are operably connected. In some embodiments, when acoustic liquid handling technology is used to transfer a reagent or product from a first location (e.g., a first well or reservoir or operational station) to a second location (e.g., a second well or reservoir or operational station), the first and second locations may be on different substrates (e.g., microfluidic substrates) so that the two locations can be aligned on opposing substrates for acoustic transfer between the two locations. Accordingly, a microfluidic device may include a plurality of different substrates that can be opposed to align different sample locations on opposing substrates for acoustic transfer. In some embodiments, traditional liquid handlers (e.g., robotic pipettors, etc.) may be used to transfer volumes of liquid from a first microfluidic substrate (e.g., a first chip) to a second microfluidic substrate (e.g., a second chip). Accordingly, a device or system may include a robotic handler that can move the two or more substrates as required for transfer between subsequent steps in an assembly reaction. In some embodiments, alternate assembly stations (including other processing stations, e.g., size selection stations) may be included on two substrates and sample may be passed back and forth between the two substrates for sequential assembly and/or processing steps.

Aspects of the invention provide a universal interconnect for connecting microfluidic devices and/or substrates (e.g., chips). Currently there exist few routes for directly connecting a first microfluidic device or substrate to a second microfluidic device or substrate. Integration of microfluidics with traditional liquid handling requires wasteful dead volumes, or dilutions of liquids processed on microfluidics to low or unusable concentrations in some embodiments. Therefore, it is very uncommon to see multiple separate microfluidic devices connected in a single pipeline. This slows integration of microfluidics in research practices because of the complications of getting in and out of a chip, and the extreme difficulty of transferring liquids from chip to chip. In order to optimize the use of microfluidics, complicated manufacturing is required to build all desired processes on a single chip, instead of across smaller elements that could be individually fabricated in a simpler manner. Furthermore, iterations in development of a process across chips could be greatly improved by allowing engineers to create and fabricate individual components and then string them together, much like a circuit board strings together multiple microchip components in the electronics industry

Certain embodiments of the invention are composed of polydimethylsiloxane elastomer and glass, and are formed with traditional lithographic and laser excimer techniques found in the microprocessor and microfluidics industries. However, other suitable materials may be used as the invention is not limited in this respect. In some embodiments, an interconnect includes a channel of PDMS on glass with holes etched through the PDMS. Microfluidic components may be designed in such a manner that they can be placed on a structure that confines them geometrically, especially confining their inputs and outputs, much like a breadboard in electronics. In this manner, discrete spacing between components exists, and PDMS/glass channels of defined sizes can be placed over the inputs/outputs of two or more microfluidics. When a compressive force is placed upon them, the PDMS seals to the fluidics like a gasket, and allows the PDMS microchannel to span between them. In this manner, the PDMS microchannels act like wires connecting the microfluidic devices. Similar channels can be made using other materials provided they provide an appropriate seal. Compressive force can be applied using any suitable compressive technique or device including weights, vacuum, clips, screws, etc., or other means of compressing interconnect channels to microfluidic inputs and/or outputs, or any combination thereof. FIG. 7 illustrates a non-limiting embodiment of a microfluidic interconnect shown in cross-sectional view. A channel formed between microfluidic substrate A and microfluidic substrate B is shown.

Liquids or materials can be transferred within or between microfluidic substrates by pressure gradients (vacuum, etc.), electrophoresis, dielectrophoresis, isotachophoresis, magnetism, electroosmosis, diffusion, etc., or any combination thereof.

In some embodiments, a channel can be made by exposing a silicon wafer covered in photoresist such as SU-8 in a pattern of the order of 1-10000 urn wide, any length and any size deep, with a general size around 10μmxl0μmx3cm. After coating and curing PDMS elastomer over the patterned Su-8 resist, the PDMS is stripped off leaving a channel embossed in the PDMS. The PDMS is then oxygen plasma treated, a glass wafer is oxygen plasma treated and the PDMS molecularly bonded to the glass. In this manner the channel is formed partly in glass, partly in PDMS. In certain embodiments, glass may be coated with a thin layer of PDMS to serve as the substrate, making channels composed entirely of PDMS. In some embodiments, holes to the channels could be drilled, laser etched, melted, formed by placing capillaries at the channel entrances before the PDMS is cast, etc., or any combination thereof. Aspects of the invention also relate to systems comprising multiple channels spanning many fluidic devices (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-50, 50-100 or more) arranged in series, in parallel, in other configurations, or any combination thereof. Embodiments include the use of a "bread board" to interface with any number of components, either to link them together or operate together. Aspects of the invention also comprises standardization of microfluidic components to be linked together with designated connector widths.

Accordingly, a microfluidic device may comprise one or more substrates and/or connectors that each comprise a number of channels. The channels can have various shapes and dimensions and offer routes of transportation for the liquids and reagents once introduced in the microfluidic device. A channel may have at least one cross- sectional dimension from between about 0.1 micrometer and about 500 micrometers (e.g., between about 1 and about 250 micrometers, between about 10 and about 100 micrometers). However, smaller or larger cross-sectional dimensions also may be used (e.g., up to 1 mm or 2 mm as described herein). The channels can be part of a network that includes T- and X- crossings. Liquids and reagents can be moved to any point within the microfluidic network, including channels, sample wells and functional elements, by applying the appropriate directional force, hi some embodiments the channels are heated to change the speed of the reagents and liquids moving through the channel and/or change the properties of the channel. In some embodiments a microfluidic device or system contains sample wells.

These wells are reservoirs for liquids, fluids and reagents. The reagents can be moved from the sample wells to various other parts of the channel network, including other sample wells, reaction wells, temperature chambers, separation stations or other functional elements. A microfluidic device may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and up to as many wells that can physically fit on the microfluidic device.

The microfluidic device may contain reaction wells. Reaction wells can facilitate chemical and/or biological reactions. Reagents may be transported from the sample wells to the reaction and well and the reaction may be executed. The reaction may occur spontaneously, upon mixing together of all the reagents or the reaction may be induced by a physical change, like light or a temperature change.

In some embodiments the microfluidic device contains separation stations. These may use electrophoretic and/or chromatographic separation to separate subject compounds based on size. Accordingly, compounds may be separated based on their physical or chemical characteristics, including size or charge. Size separation material may contain a resistance material, like a polymer, a sieve, a gel, or other material or combination of materials. When a mixture of molecules with different size is transported through a separation material, bigger molecules may travel faster or slower, depending on the coating used and the type of material used, allowing for separation. Aspects of the invention also include separation stations based on other molecular properties. For instance, molecules can be separated by charge (by using a charged column) or by affinity (by using an antibody column). In other embodiments, molecules and reagents can be separated or concentrated using isotachophoresis. This process relies on the mixing of two different buffer systems. This process can separate electrolytes based on charge.

The fluids and liquids within the microfluidic device can be transported and distributed by a variety of forces including electrokinetic forces, pressure based flow techniques, capillary forces, gravitational and centrifugal forces. The electrokinetic forces encompass both electro-osmotic and electrophoretic forces. These forces are best applied when the fluids and compounds to be transported have an electrostatic component. Electrostatic forces can be applied by standard electric circuitry, these techniques are known to people of ordinary skill in the art. Pressure based flow techniques encompass both vacuum and pressurized gas. Pressure and vacuum can be regulated by standard techniques including valves, vacuum pumps and pistons. Centrifugational forces can be applied by spinning the microfluidic device in a centrifuge. Fluid flow volumes and rates through microfluidic channels may be optimized for different applications. In some embodiments, flow rates between 0.1 nl and 1 μl per second maybe used (e.g., rates of about 1 nl, about 10 nl, about 100 nl, or about 1 μl per second). However, lower rates (less than 1 nl per second) or higher rates (e.g., about 5-10 μl per second or higher) may be used depending on the application. In some embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the functional stations described herein may be integrated into a single microfluidic substrate (e.g., a single chip). In some embodiment, a single chip may include a plurality of separate functional stations, or separate networks of functional stations, for performing a plurality of (e.g., 2-4, 4, 4-8, 8, 8-16, 16, 16-32, 32, 32-64, 64 or more) identical procedures (e.g., assembly, error correction, size selection, ligation, amplification, etc., or any combination thereof) in parallel. In some embodiments, different functional stations may be separated on to different substrates (e.g., different chips) and suitable techniques may be used to transfer the products from a first substrate to a second substrate as described herein.

In some embodiments the microfluidic device is coupled to processing elements. Processing elements comprise control elements like a computer or robotic device. Other processing elements include apparatus for analysis. Non-limiting examples are UV readers and other wavelength based detection methods, including fluorescence, phosphorescence or other signal readers. In addition, processing components may include sample manipulation components, for example, a liquid handling apparatus (e.g., with one or more robotic pipettors) and means for pre- and post procedure preparation and purification of samples. The microfluidic device also may be part of a network of microfluidic devices that can each have a separate function or have the same function. Examples of certain size selection stations, sippers, spitters, channels, and other operational stations may be found for example in US Patent Nos. 7,067,263; 6,849,411; 6,695,009; 6,670,153; 6,670,133; 6,649,358; 6,648,015; 6,630,353; 6,524,830; 6,174,675; the disclosures of which are incorporated herein by reference.

Accordingly, in some aspects of the invention a system is provided that takes input oligonucleotides and reagents, and outputs large fragments of properly assembled (e.g., ligated) nucleic acid (e.g., DNA). A system could be configured using one or more acoustic liquid handling devices, and/or one or more microfluidic devices (e.g., one or more microfluidic substrates and associated hardware), and/or one or more robotic liquid handling devices, and/or one or more additional robotic devices as described herein. Additional supporting hardware and/or software to house and control the functional components also may be provided. In some embodiments, a recirculating microfluidic system is provided with mechanical valving on a chip, hi some embodiments, a cascaded micro fluidic system is provided that allows for all control to be external to the chip (e.g., control of flow into and out of different ports on the chip). Certain devices also may include a platform that allows for high voltage application, controlled and measured electric currents, pressure applications and measurements, reagent aspiration, and reagent dispensing. Temperature controllers also may be provided to maintain the precise operating conditions required for the reactions and separations of DNA. hi addition to chip specific physical controls, systems may include robotic control for reagent addition into the chip, whether through one or more integrated capillary sippers, via one or more top mounted wells by a mechanical pipette head as is standard in liquid handling robots, or via other routes (e.g., acoustic delivery). In some embodiments, a gantry system (e.g., at least one three axis gantry system) and a mechanical pipettor (e.g., at least one mechanical pipette head) may be integrated into the system and device controller, hi some embodiments, a detector, e.g., a small photodiode based microscope and diode laser, may be provided to get signal feedback from the nucleic acid separations, to guide fragment separations.

On top of all of the hardware components for controlling the device and device controller, additional software packages may be provided (e.g., to record electric currents and diode measurements, controls valving, pressures, electric fields, robotic gantries, mechanical pipettors, etc., or any combination thereof). It should be appreciated that small amounts of DNA can be easily amplified or cloned, and this can be a problem when a piece of lab equipment, such as a DNA chip, is used several times. Accordingly, a protocol may be provided for properly cleaning a reusable device after each run to prevent well contamination, hi some embodiments, one or more DNAase and washing steps may be used and optimized for a robust protocol. It also should be appreciated that microfluidics may be scaled, for example, by processing multiple samples in parallel or in shorter time than traditional methods.

As described herein, a system may include one or more appropriate chip-to-world interfaces. The advantage of low volume processing on-chip can become a disadvantage when collecting post-processed material, as there is little material to capture. This is the main reason why commercial applications of lab-on-a-chip technologies usually end with digital readout, and subsequent sample disposal instead of fraction collection. Assembly procedures outlined herein involve recapture of post-processed material, and also may include several within-chip processes that could suffer from low step-wise yields. Therefore, the final output of a system may have very low concentrations of processed material. However, since nucleic acids can be readily amplified using standard molecular biology techniques, such as cloning, rolling circle amplification, or PCR, low yields are still acceptable provided there is sufficient product for subsequent processing.

In some embodiments, the chip devices are provided with straightforward packaging that is capable of integration with automation hardware and lab microplates. For example, a small glass capillary fused to the fluidic circuit with epoxy may be used (as provided by Caliper Life Sciences). This has advantages in certain embodiments relative to the manifolds provided by other companies, such as Fluidigm. In some embodiments, a sipper can be operated in a reverse mode, so that material is deposited rather than collected. Devices and systems described herein can integrate a capillary sipper for the collection and deposition of material run on a microfluidic substrate. With appropriate dimensions, a single chip can integrate two or more of these capillaries to enable more complicated processing. Furthermore, wells can be positioned on the top of the devices such that they could be top loaded with standard multichannel pipettes, in this manner many difference types of reagents could be stored on the device for processing. Accordingly, a chip-to-world interface may include wells on top of a chip for reagent addition, and one or more capillaries capable of sipping/depositing reagents. In some embodiments, a fully automated system may include one or more robotic devices (e.g., robotic liquid handlers and other robotic devices) for transferring reaction liquid and/or reaction substrates (e.g., multi-well plates, reservoirs, microfluidic substrates, or any combination) between different components of the system. For example, initial oligonucleotide mixtures (optionally including reagents such as enzymes, buffers, etc.) may be prepared in a multi-well plate using an acoustic device, hi some embodiments, the oligonucleotide mixture is automatically transferred to an automated pipettor device where one or more reagents, salts, enzymes, etc. are added. In some embodiments, an oligonucleotide mixture along with suitable reagents etc. is automatically transferred to a temperature controlled device (e.g., a thermocycler) for an initial series of extension/denaturation reactions or for an initial ligation reaction. However, in some embodiments, an initial oligonucleotide mixture is automatically transferred directly to a microfluidic substrate as described herein for one or more cycles of assembly, error correction, or a combination thereof as described herein. Subsequently, an assembled product may be automatically ligated into a vector, and/or automatically transformed into a host cell (e.g., a prokaryotic or eukaryotic host cell such as a bacterial, yeast, insect, mammalian or other host cell). Accordingly, a system may include one or more components required for the processes described above along with suitable software, controllers, etc., or any combination thereof.

A system also may include device hardware (e.g., pipettors, spitters, channels, etc., or any combination thereof) and/or software and/or system controllers for monitoring and/or providing dynamic feedback during an assembly in order to optimize the assembly process. In some embodiments, if an assembly fails (e.g., observed at the end or at an intermediate step) one or more incorrect reaction products or volumes may be identified as described herein and the assembly may be reinitiated at the failed or incorrect stage or just prior to it (e.g., using corrected and/or adjusted reaction conditions or volumes or procedures) to obtain the final product. This increases the speed of the process, because the entire assembly process does not need to be reinitiated. In some embodiments, a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) different reaction conditions (e.g., different nucleic acid concentrations, temperatures, reaction times, reagent concentrations — for example different salt, buffer, or other reagent concentrations) are used in parallel reactions for one or more intermediate assembly steps and the resulting assembled products are evaluated. Appropriate volumes from successful assemblies are then used in subsequent assembly steps (e.g., in a plurality of parallel of different reactions or in single reactions). In some embodiments, the amount of input nucleic acid is normalized for each assembly step. In some embodiments, the amount of an assembly product obtained at an intermediate step is evaluated using an analytical microfluidic device. Accordingly, a system of the invention may include one or more analytical devices in addition to preparative devices, along with integrated robotics, software and controllers for automated the monitoring and dynamic control in addition to the underlying assembly procedures.

Oligonucleotide Synthesis and Assembly

As described above and throughout, the present invention provides microfluidic devices, systems and methods for synthesizing or assembling nucleic acids. The architecture of a device of the invention may include sites, chambers, wells, channels and other features for performing any one or more of the steps of described herein, including cell handling such as transformation and cloning, as well as polypeptide expression, isolation and purification. The present invention contemplates that one or more of the steps may be performed on one or more micro fluidic devices. According to the invention, in one embodiment of the construction of a single target nucleic acid, a sample may progress through a first microfluidic device, then be moved to a second microfluidic device for additional processing. In another embodiment, a sample may be removed from a microfluidic device for one or more steps then transferred back to the same device or moved to another device. Yet, in other embodiments, all of the steps including assembly, size separation and error filtration are performed in a single device.

Oligonucleotides used in assembly reactions of the invention may be synthesized using any suitable technique. For example, oligonucleotides may be synthesized on a column or other support (e.g., a chip). Examples of chip-based synthesis techniques include techniques used in synthesis devices or methods available from Combimatrix, Agilent, Affymetrix, or other sources. A synthetic oligonucleotide may be of any suitable size, for example between 10 and 1,000 nucleotides long (e.g., between 10 and 200, 200 and 500, 500 and 1,000 nucleotides long, or any combination thereof). An assembly reaction may include a plurality of oligonucleotides, each of which independently may be between 10 and 200 nucleotides in length (e.g., between 20 and 150, between 30 and 100, 30 to 90, 30-80, 30-70, 30-60, 35-55, 40-50, or any intermediate number of nucleotides). However, one or more shorter or longer oligonucleotides may be used in certain embodiments.

In one embodiment, the invention provides a microfluidic device comprising one or more reaction wells or compartments for on-chip synthesis of oligonucleotides. Such device may comprise a channel for transport of the synthesized oligonucleotides to a reaction well for purification or subsequent assembly steps.

Oligonucleotides may be provided as single stranded synthetic products. However, in some embodiments, oligonucleotides may be provided as double-stranded preparations including an annealed complementary strand. Oligonucleotides may be molecules of DNA, RNA, PNA, or any combination thereof. A double-stranded oligonucleotide may be produced by amplifying a single-stranded synthetic oligonucleotide or other suitable template (e.g., a sequence in a nucleic acid preparation such as a nucleic acid vector or genomic nucleic acid). Accordingly, a plurality of oligonucleotides designed to have the sequence features described herein may be provided as a plurality of single-stranded oligonucleotides having those feature, or also may be provided along with complementary oligonucleotides. In some embodiments, an oligonucleotide may be phosphorylated (e.g., with a 5' phosphate). In some embodiments, an oligonucleotide may be non-phosphorylated.

In some embodiments, an oligonucleotide may be amplified using an appropriate primer pair with one primer corresponding to each end of the oligonucleotide (e.g., one that is complementary to the 3' end of the oligonucleotide and one that is identical to the 5' end of the oligonucleotide). In some embodiments, an oligonucleotide may be designed to contain a central assembly sequence (designed to be incorporated into the target nucleic acid) flanked by a 5' amplification sequence (e.g., a 5' universal sequence) and a 3' amplification sequence (e.g., a 3' universal sequence). Amplification primers (e.g., between 10 and 50 nucleotides long, between 15 and 45 nucleotides long, about 25 nucleotides long, etc.) corresponding to the flanking amplification sequences may be used to amplify the oligonucleotide (e.g., one primer may be complementary to the 3' amplification sequence and one primer may have the same sequence as the 5' amplification sequence). The amplification sequences then may be removed from the amplified oligonucleotide using any suitable technique to produce an oligonucleotide that contains only the assembly sequence.

In some embodiments, a plurality of different oligonucleotides (e.g., about 5, 10, 50, 100, or more) with different central assembly sequences may have identical 5' amplification sequences and identical 3' amplification sequences. These oligonucleotides can all be amplified in the same reaction using the same amplification primers.

A preparation of an oligonucleotide designed to have a certain sequence may include oligonucleotide molecules having the designed sequence in addition to oligonucleotide molecules that contain errors (e.g., that differ from the designed sequence at least at one position). A sequence error may include one or more nucleotide deletions, additions, substitutions (e.g., transversion or transition), inversions, duplications, or any combination of two or more thereof. Oligonucleotide errors may be generated during oligonucleotide synthesis. Different synthetic techniques may be prone to different error profiles and frequencies. In some embodiments, error rates may vary from 1/10 to 1/200 errors per base depending on the synthesis protocol that is used. However, in some embodiments lower error rates may be achieved. Also, the types of errors may depend on the synthetic techniques that are used. For example, in some embodiments chip-based oligonucleotide synthesis may result in relatively more deletions than column-based synthetic techniques.

In some embodiments, one or more oligonucleotide preparations may be processed to remove (or reduce the frequency of) error-containing oligonucleotides. In some embodiments, a hybridization technique may be used wherein an oligonucleotide preparation is hybridized under stringent conditions one or more times to an immobilized oligonucleotide preparation designed to have a complementary sequence. Oligonucleotides that do not bind may be removed in order to selectively or specifically remove oligonucleotides that contain errors that would destabilize hybridization under the conditions used. It should be appreciated that this processing may not remove all error-containing oligonucleotides since many have only one or two sequence errors and may still bind to the immobilized oligonucleotides with sufficient affinity for a fraction of them to remain bound through this selection processing procedure.

In some embodiments, a nucleic acid binding protein or recombinase (e.g., RecA) may be included in one or more of the oligonucleotide processing steps to improve the selection of error free oligonucleotides. For example, by preferentially promoting the hybridization of oligonucleotides that are completely complementary with the immobilized oligonucleotides, the amount of error containing oligonucleotides that are bound may be reduced. As a result, this oligonucleotide processing procedure may remove more error-containing oligonucleotides and generate an oligonucleotide preparation that has a lower error frequency (e.g., with an error rate of less than 1/50, less than 1/100, less than 1/200, less than 1/300, less than 1/400, less than 1/500, less than 1/1,000, or less than 1/2,000 errors per base.

A plurality of oligonucleotides used in an assembly reaction may contain preparations of synthetic oligonucleotides, single-stranded oligonucleotides, double- stranded oligonucleotides, amplification products, oligonucleotides that are processed to remove (or reduce the frequency of) error-containing variants, etc., or any combination of two or more thereof.

In some aspects, synthetic oligonucleotides synthesized on an array (e.g., a chip) are not amplified prior to assembly. In some embodiments, a polymerase-based or ligase-based assembly using non-amplified oligonucleotides may be performed in a microfluidic device.

In some aspects, a synthetic oligonucleotide may be amplified prior to use. Either strand of a double-stranded amplification product may be used as an assembly oligonucleotide and added to an assembly reaction as described herein. A synthetic oligonucleotide may be amplified using a pair of amplification primers (e.g., a first primer that hybridizes to the 3' region of the oligonucleotide and a second primer that hybridizes to the 3' region of the complement of the oligonucleotide). The oligonucleotide may be synthesized on a support such as a chip (e.g., using an ink-jet- based synthesis technology). In some embodiments, the oligonucleotide may be amplified while it is still attached to the support. In some embodiments, the oligonucleotide may be removed or cleaved from the support prior to amplification. The two strands of a double-stranded amplification product may be separated and isolated using any suitable technique. In some embodiments, the two strands may be differentially labeled (e.g., using one or more different molecular weight, affinity, fluorescent, electrostatic, magnetic, and/or other suitable tags). The different labels may be used to purify and/or isolate one or both strands. In some embodiments, biotin may be used as a purification tag. In some embodiments, the strand that is to be used for assembly may be directly purified (e.g., using an affinity or other suitable tag), hi some embodiments, the complementary strand is removed (e.g., using an affinity or other suitable tag) and the remaining strand is used for assembly.

In some embodiments, a synthetic oligonucleotide may include a central assembly sequence flanked by 5' and 3' amplification sequences. The central assembly sequence is designed for incorporation into an assembled nucleic acid. The flanking sequences are designed for amplification and are not intended to be incorporated into the assembled nucleic acid. The flanking amplification sequences may be used as universal primer sequences to amplify a plurality of different assembly oligonucleotides that share the same amplification sequences but have different central assembly sequences, hi some embodiments, the flanking sequences are removed after amplification to produce an oligonucleotide that contains only the assembly sequence.

In some embodiments, one of the two amplification primers may be biotinylated. The nucleic acid strand that incorporates this biotinylated primer during amplification can be affinity purified using streptavidin (e.g., bound to a bead, column, or other surface). In some embodiments, the amplification primers also may be designed to include certain sequence features that can be used to remove the primer regions after amplification in order to produce a single-stranded assembly oligonucleotide that includes the assembly sequence without the flanking amplification sequences. In some embodiments, the non-biotinylated strand may be used for assembly.

The assembly oligonucleotide may be purified by removing the biotinylated complementary strand. In some embodiments, the amplification sequences may be removed if the non-biotinylated primer includes a dU at its 3' end, and if the amplification sequence recognized by (i.e., complementary to) the biotinylated primer includes at most three of the four nucleotides and the fourth nucleotide is present in the assembly sequence at (or adjacent to) the junction between the amplification sequence and the assembly sequence. After amplification, the double-stranded product is incubated with T4 DNA polymerase (or other polymerase having a suitable editing activity) in the presence of the fourth nucleotide (without any of the nucleotides that are present in the amplification sequence recognized by the biotinylated primer) under appropriate reaction conditions. Under these conditions, the 3' nucleotides are progressively removed through to the nucleotide that is not present in the amplification sequence (referred to as the fourth nucleotide above). As a result, the amplification sequence that is recognized by the biotinylated primer is removed. The biotinylated strand is then removed. The remaining non-biotinylated strand is then treated with uracil-DNA glycosylase (UDG) to remove the non-biotinylated primer sequence. This technique generates a single-stranded assembly oligonucleotide without the flanking amplification sequences. It should be appreciated that this technique may be used to process a single amplified oligonucleotide preparation or a plurality of different amplified oligonucleotides in a single reaction if they share the same amplification sequence features described above. In some embodiments, the biotinylated strand may be used for assembly. The assembly oligonucleotide may be obtained directly by isolating the biotinylated strand. In some embodiments, the amplification sequences may be removed if the biotinylated primer includes a dU at its 3* end, and if the amplification sequence recognized by (i.e., complementary to) the non-biotinylated primer includes at most three of the four nucleotides and the fourth nucleotide is present in the assembly sequence at (or adjacent to) the junction between the amplification sequence and the assembly sequence. After amplification, the double-stranded product is incubated with T4 DNA polymerase (or other polymerase having a suitable editing activity) in the presence of the fourth nucleotide (without any of the nucleotides that are present in the amplification sequence recognized by the non-biotinylated primer) under appropriate reaction conditions. Under these conditions, the 3' nucleotides are progressively removed through to the nucleotide that is not present in the amplification sequence (referred to as the fourth nucleotide above). As a result, the amplification sequence that is recognized by the non- biotinylated primer is removed. The biotinylated strand is then isolated (and the non- biotinylated strand is removed). The isolated biotinylated strand is then treated with UDG to remove the biotinylated primer sequence. This technique generates a single- stranded assembly oligonucleotide without the flanking amplification sequences. It should be appreciated that this technique may be used to process a single amplified oligonucleotide preparation or a plurality of different amplified oligonucleotides in a single reaction if they share the same amplification sequence features described above.

It should be appreciated that the biotinylated primer may be designed to anneal to either the synthetic oligonucleotide or to its complement for the amplification and purification reactions described above. Similarly, the non-biotinylated primer may be designed to anneal to either strand provided it anneals to the strand that is complementary to the strand recognized by the biotinylated primer.

In certain embodiments, it may be helpful to include one or more modified oligonucleotides in an assembly reaction. An oligonucleotide may be modified by incorporating a modified-base (e.g., a nucleotide analog) during synthesis, by modifying the oligonucleotide after synthesis, or any combination thereof. Examples of modifications include, but are not limited to, one or more of the following: universal bases such as nitroindoles, dP and dK, inosine, uracil; halogenated bases such as BrdU; fluorescent labeled bases; non-radioactive labels such as biotin (as a derivative of dT) and digoxigenin (DIG); 2,4-Dinitrophenyl (DNP); radioactive nucleotides; post-coupling modification such as dR-NHb (deoxyribose-NH2); Acridine (6-chloro-2- methoxiacridine); and spacer phosphoramides which are used during synthesis to add a spacer 'arm' into the sequence, such as C3, C8 (octanediol), C9, C12, HEG (hexaethlene glycol) and Cl 8.

Microfluidic devices may be used to amplify, purify, and/or process one or more oligonucleotides according to techniques described herein.

In some embodiments, acoustic liquid handling devices are used to deposit precise volumes of individual oligonucleotides or mixtures of oligonucleotides (e.g., 2-4, 4-10, 10-40 different oligonucleotides) into a reaction mixture for a polymerase mediated assembly or for a ligation mediated assembly. In some embodiments, approximately 2.5 nl drops of each oligonucleotide or mixture of oligonucleotide subsets are deposited onto a target plate from a reservoir plate using an acoustic device. The resulting mixtures can be processed in as described herein for error-removal, amplification, polymerase- mediated assembly, ligase-mediated assembly, or any combination thereof as described herein.

Applications: Aspects of the invention may be useful for a range of applications involving the production and/or use of synthetic nucleic acids. As described herein, the invention provides methods for assembling synthetic nucleic acids with increased efficiency. The resulting assembled nucleic acids may be amplified in vitro (e.g., using polymerase chain reaction or PCR, ligase chain reaction or LCR, or any suitable amplification technique), amplified in vivo (e.g., via cloning into a suitable vector), isolated and/or purified. An assembled nucleic acid (alone or cloned into a vector) may be transformed into a host cell (e.g., a prokaryotic, eukaryotic, insect, mammalian, or other host cell). In some embodiments, the host cell may be used to propagate the nucleic acid. In certain embodiments, the nucleic acid may be integrated into the genome of the host cell. In some embodiments, the nucleic acid may replace a corresponding nucleic acid region on the genome of the cell (e.g., via homologous recombination). Accordingly, nucleic acids may be used to produce recombinant organisms. In some embodiments, a target nucleic acϊd may be an entire genome or large fragments of a genome that are used to replace all or part of the genome of a host organism. Recombinant organisms also may be used for a variety of research, industrial, agricultural, and/or medical applications.

Many of the techniques described herein can be used together, applying combinations of one or more extension-based and/or ligation-based assembly techniques at one or more points to produce long nucleic acid molecules. For example, concerted assembly may be used to assemble oligonucleotide duplexes and nucleic acid fragments of less than 100 to more than 10,000 base pairs in length (e.g., 100 mers to 500 mers, 500 mers to 1,000 mers, 1,000 mers to 5,000 mers, 5, 000 mers to 10,000 mers, 25,000 mers, 50,000 mers, 75,000 mers, 100,000 mers, etc.). In an exemplary embodiment, methods described herein may be used during the assembly of an entire genome (or a large fragment thereof, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) of an organism (e.g., of a viral, bacterial, yeast, or other prokaryotic or eukaryotic organism), optionally incorporating specific modifications into the sequence at one or more desired locations.

Any of the nucleic acid products (e.g., including nucleic acids that are amplified, cloned, purified, isolated, etc.) may be packaged in any suitable format (e.g., in a stable buffer, lyophilized, etc.) for storage and/or shipping (e.g., for shipping to a distribution center or to a customer). Similarly, any of the host cells (e.g., cells transformed with a vector or having a modified genome) may be prepared in a suitable buffer for storage and or transport (e.g., for distribution to a customer). In some embodiments, cells may be frozen. However, other stable cell preparations also may be used.

Host cells may be grown and expanded in culture. Host cells may be used for expressing one or more RNAs or polypeptides of interest (e.g., therapeutic, industrial, agricultural, and/or medical proteins). The expressed polypeptides may be natural polypeptides or non-natural polypeptides. The polypeptides may be isolated or purified for subsequent use.

Accordingly, nucleic acid molecules generated using methods of the invention can be incorporated into a vector. The vector may be a cloning vector or an expression vector. A vector may comprise an origin of replication and one or more selectable markers (e.g., antibiotic resistant markers, auxotrophic markers, etc.). In some embodiments, the vector may be a viral vector. A viral vector may comprise nucleic acid sequences capable of infecting target cells. Similarly, in some embodiments, a prokaryotic expression vector operably linked to an appropriate promoter system can be used to transform target cells. In other embodiments, a eukaryotic vector operably linked to an appropriate promoter system can be used to transfect target cells or tissues. Transcription and/or translation of the constructs described herein may be carried out in vitro (i.e., using cell-free systems) or in vivo (i.e., expressed in cells). In some embodiments, cell lysates may be prepared. Li certain embodiments, expressed RNAs or polypeptides may be isolated or purified. Nucleic acids of the invention also may be used to add detection and/or purification tags to expressed polypeptides or fragments thereof. Examples of polypeptide-based fusion/tag include, but are not limited to, hexa- histidine (His6) Myc and HA, and other polypeptides with utility, such as GFP, GST, MBP, chitin and the like. In some embodiments, polypeptides may comprise one or more unnatural amino acid residue(s).

In some embodiments, antibodies can be made against polypeptides or fragment(s) thereof encoded by one or more synthetic nucleic acids.

In certain embodiments, synthetic nucleic acids may be provided as libraries for screening in research and development (e.g., to identify potential therapeutic proteins or peptides, to identify potential protein targets for drug development, etc.)

In some embodiments, a synthetic nucleic acid may be used as a therapeutic (e.g., for gene therapy, or for gene regulation). For example, a synthetic nucleic acid may be administered to a patient in an amount sufficient to express a therapeutic amount of a protein. In other embodiments, a synthetic nucleic acid may be administered to a patient in an amount sufficient to regulate (e.g., down-regulate) the expression of a gene.

It should be appreciated that different acts or embodiments described herein may be performed independently and may be performed at different locations in the United States or outside the United States. For example, each of the acts of receiving an order for a target nucleic acid, analyzing a target nucleic acid sequence, identifying an assembly strategy, designing one or more starting nucleic acids (e.g., oligonucleotides), synthesizing starting nucleic acid(s), purifying starting nucleic acid(s), assembling starting nucleic acid(s), isolating assembled nucleic acid(s), confirming the sequence of assembled nucleic acid(s), manipulating assembled nucleic acid(s) (e.g., amplifying, cloning, inserting into a host genome, etc.), and any other acts or any parts of these acts may be performed independently either at one location or at different sites within the United States or outside the United States. In some embodiments, an assembly procedure may involve a combination of acts that are performed at one site (in the United States or outside the United States) and acts that are performed at one or more remote sites (within the United States or outside the United States).

Business applications:

Aspects of the invention may be useful to streamline nucleic acid assembly reactions. Accordingly, aspects of the invention relate to marketing methods, compositions, kits, devices, and systems for increasing nucleic acid assembly throughput involving microfluidic device-based assembly techniques described herein.

Aspects of the invention may be useful for reducing the time and/or cost of production, commercialization, and/or development of synthetic nucleic acids, and/or related compositions. Accordingly, aspects of the invention relate to business methods that involve collaboratively (e.g., with a partner) or independently marketing one or more methods, kits, compositions, devices, or systems for analyzing and/or assembling synthetic nucleic acids as described herein. For example, certain embodiments of the invention may involve marketing a procedure and/or associated devices or systems involving nucleic acid assembly techniques described herein. In some embodiments, synthetic nucleic acids, libraries of synthetic nucleic acids, host cells containing synthetic nucleic acids, expressed polypeptides or proteins, etc., also may be marketed.

Marketing may involve providing information and/or samples relating to methods, kits, compositions, devices, and/or systems described herein. Potential customers or partners may be, for example, companies in the pharmaceutical, biotechnology and agricultural industries, as well as academic centers and government research organizations or institutes. Business applications also may involve generating revenue through sales and/or licenses of methods, kits, compositions, devices, and/or systems of the invention.

According to aspects of the invention, a touchdown PCR assembly may be used to rescue an polymerase-mediated assembly reaction that does not work efficiently or correctly using a regular polymerase-based assembly reaction. Accordingly, stations and controllers of the invention may be adapted for performing a touchdown PCR reaction in somβ embodiments. It also should be appreciated that replicate reactions (e.g., 2, 3, 4, S, 6, 7 ,8, 9, 10 or more) may be performed using the same or different conditions as described above. In some embodiments, a touchdown PCR reaction is performed in one or more replicate conditions.

EXAMPLES

Microfluidic-based assembly, including size selection, sample concentration, and/or sample handling may be used for one or more of the reaction steps described in the following examples. However, smaller reaction volumes (or aliquots of these reaction volumes) may be collected for processing in a microfluidic substrate according to the invention. For example, reaction volumes of between about 5 nl and about 100 nl may be used. However, smaller or larger volumes may be used. In some embodiments, an acoustic liquid handler may be used for transferring volumes of less than 10 nl, for example less than 5 nl or about 1 nl or less. For example, small volumes of the reagents described in the following example may be collected and transported to a thermocycling well for assembly. In some embodiments, the thermocycling assembly may be performed (e.g., in a reaction tube or multi-well plate) using the volumes described below, and one or more aliquots of the reaction mixture (e.g., after thermocycling is complete, or after post assembly amplification is performed) may be collected by a microfluidic device through a collector port and transported along a microfluidic channel to a size-selection station where a nucleic acid of a predetermined size is isolated and then transported for further processing (e.g., error removal, or assembly) on the same microfluidic substrate or distributed (e.g., through a distribution port) for further processing on a different microfluidic substrate or in a different reaction format (e.g., a larger scale reaction tube or multi-well plate). Similarly, any of the other reactions or products described in the following example (or elsewhere herein) may be performed or processed on a microfluidic substrate after collection through one or more collection ports that are in fluid communication with appropriate operational station(s). The resulting product then may be further processed on the same substrate or distributed through one or more distribution ports that are in fluid connection with the operational station(s) where the products were generated (e.g., assembled, error-corrected, size- selected, or otherwise modified). Example 1. Nucleic acid fragment assembly.

Gene assembly via a 2-step PCR method: In step (1), a primerless assembly of oligonucleotides is performed and in step (2) an assembled nucleic acid fragment is amplified in a primer-based amplification.

A 993 base long promoter>EGFP construct was assembled from 50-mer abutting oligonucleotides using a 2-step PCR assembly.

Mixed oligonucleotide pools were prepared as follows: 36 overlapping 50-mer oligonucleotides and two 5' terminal 59-mers were separated into 4 pools, each corresponding to overlapping 200-300 nucleotide segments of the final construct. The total oligonucleotide concentration in each pool was 5 μM.

A primerless PCR extension reaction was used to stitch (assemble) overlapping oligonucleotides in each pool. The PCR extension reaction mixture was as follows: oligonucleotide pool (5 μM total) 1.0 μl (~ 25 nM final each) dNTP (10 mM each) 0.5 μl (250 μM final each)

Pfu buffer (1 Ox) 2.0 μl

PfU polymerase (2.5 U/μl) 0.5 μl dH2O to 20 μl

Assembly was achieved by cycling this mixture through several rounds of denaturing, annealing, and extension reactions as follows: start 2 min. 95°C

30 cycles of 95°C 30 sec, 65°C 30 sec, 72°C 1 min. final 720C 2 min. extension step

The resulting product was exposed to amplification conditions to amplify the desired nucleic acid fragments (sub-segments of 200-300 nucleotides). The following PCR mix was used: primerless PCR product 1.0 μl primer 5 ' (1.2 μM) 5 μl (300 nM final) primer 3 ' (1.2 μM) 5 μl (300 nM final) dNTP (10 mM each) 0.5 μl (250 μM final each)

Pfu buffer (1 Ox) 2.0 μl Pfu polymerase (2.5 U/μl) 0.5 μl dH2O to 20 μl The following PCR cycle conditions were used: start 2 min. 95°C 35 cycles of 95°C 30 sec, 65°C 30 sec., 72°C 1 min. final 72°C 2 min. extension step

The amplified sub-segments were assembled using another round of primerless PCR as follows. A diluted amplification product was prepared for each sub-segment by diluting each amplified sub-segment PCR product 1 :10 (4 μl mix + 36 μl dϊfcO). This diluted mix was used as follows: diluted sub-segment mix 1.0 μl dNTP (1OmM each) 0.5 μl (250 μM final each)

Pfu buffer (1Ox) 2.0 μl

Pfu polymerase (2.5 U/μl) 0.5 μl dH2O to 20 μl

The following PCR cycle conditions were used: start 2 min. 95°C

30 cycles of 95°C 30 sec, 65°C 30 sec, 72°C 1 min. final 72°C 2 min. extension step The full-length 993 nucleotide long promoter>EGFP was amplified in the following PCR mix : assembled sub-segments 1.0 μl primer 5' (1.2 μM) 5 μl (300 nM final) primer 3' (1.2 μM) 5 μl (300 nM final) dNTP (10 mM each) 0.5 μl (250 μM final each)

Pfu buffer (1 Ox) 2.0 μl

Pfu polymerase (2.5 U/μl) 0.5 μl dH2O to 20 μl

The following PCR cycle conditions were used: start 2 min. 95°C

35 cycles of 95°C 30 sec, 65°C 30 sec, 72°C 1 min. final 72°C 2 min. extension step

EQUIVALENTS

The present invention provides among other things methods for assembling large polynucleotide constructs and organisms having increased genomic stability. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

INCORPORATION BY REFERENCE

All publications, patents and sequence database entries mentioned herein, including those items listed below, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Claims

1. A microfluidic substrate comprising a first nucleic acid assembly station comprising a first input channel and a first output channel, a second nucleic acid assembly station comprising a second input channel and a second output channel, and a first size separation station comprising an input channel in fluid communication with the first output channel, and an output channel in fluid communication with the second input channel.
2. The microfluidic substrate of claim 1, wherein, the first and second nucleic acid assembly stations are the same, and a microfluidic loop is formed by: the nucleic acid assembly station, the channel connecting the output of the nucleic acid assembly station to the input of the size separation station, the size separation station, and the channel connecting the output of the size separation station back to the input of the nucleic acid assembly station.
3. The microfluidic substrate of claim 1, wherein the first and second assembly stations are separate stations in fluid communication with the size separation station.
4. The microfluidic substrate of claim 1, wherein one or both of the first assembly station and the second assembly station is a temperature controlled reaction chamber.
5. The microfluidic substrate of claim 1, wherein the size separation station is a capillary electrophoresis size separation component of the microfluidic substrate.
6. The microfluidic substrate of claim 1, further comprising a third nucleic acid assembly station comprising a third input channel and a third output channel, a fourth nucleic acid assembly station comprising a fourth input channel and a fourth output channel, and a second size separation station comprising an input channel in fluid communication with the third output channel, and an output channel in fluid communication with the fourth input channel, wherein the first and second assembly stations are not in fluid communication with the third and fourth assembly stations on the microfluidic substrate.
7. The microfluidic substrate of claim 1, wherein the output channel of the first size selection station and/or the second output channel of the second assembly station is connected to an input channel of a second microfluidic substrate via a microfluidic connector.
8. A method for assembling a nucleic acid, the method comprising introducing a first nucleic acid assembly mixture to a first assembly station on a first microfluidic substrate, incubating the first nucleic acid assembly mixture in the first assembly station under conditions to generate a first assembled nucleic acid product, transferring the mixture comprising the first assembled nucleic acid product to a size separation station in fluid connection with the first assembly station on the first microfluidic substrate, isolating the first assembled nucleic acid product from non-assembled nucleic in the size separation station, recovering the isolated first assembled nucleic acid product from the size separation station for further assembly in a second assembly reaction.
9. The method of claim 8, wherein the second assembly reaction is performed in the first assembly station.
10. The method of claim 8, wherein the second assembly reaction is performed in a second assembly station on the first microfludic substrate.
11. The method of claim 8, wherein the second assembly reaction is performed in a second assembly station on a second microfluidic substrate.
12. The method of claim 8, wherein the assembly reaction is a polymerase-based assembly reaction.
13. The method of claim 8, wherein the assembly reaction is a ligation.
14. The method of claim 8, further comprising repeating the assembly and size separations between 2 and SOO cycles using a series of additional input nucleic acids.
15. The method of claim 14, wherein the assembly and size separations are repeated for about 30 cycles using a series of thirty different input nucleic acids.
16. The method of claim 8, wherein the first nucleic acid assembly mixture comprises at least two different oligonucleotides of between 20 and 300 nucleotides in length.
17. The method of claim 8, wherein the first nucleic acid assembly mixture comprises at least two different oligonucleotides of between 30 and 200 nucleotides in length.
18. The method of claim 8, wherein a nucleic acid product of between 400 and 5,000 nucleotides in length is produced comprising the first intermediate nucleic acid product.
19. The method of claim 8, further comprising cloning a nucleic acid comprising the first assembled nucleic acid product.
20. A nucleic acid assembly system comprising an acoustic liquid handling device operably connected to a microfluidic device, wherein the microfluidic device comprises a microfluidic substrate comprising at least one size separation station.
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