WO2021142133A1

WO2021142133A1 - Dna assembly in microfluidics device having integrated solid-phase columns

Info

Publication number: WO2021142133A1
Application number: PCT/US2021/012517
Authority: WO
Inventors: Matthew Hill; Marc Unger
Original assignee: Elegen Corporation
Priority date: 2020-01-07
Filing date: 2021-01-07
Publication date: 2021-07-15

Abstract

Embodiments of the present invention implement DNA assembly using a microfluidics channel having an integrated solid phase column. The presence of a solid phase enables purification of nucleic acid products by selective capture and elution. Enzymatic reactions can be performed in channels or chambers, and the reaction mixture can be passed over the column to selectively capture or selectively elute the desired product. Additional embodiments provide a looped microfluidics channel. When closed, the loop in the channel allows DNA and reagent mixtures to be flowed multiple times over a bead column for efficient capture and enzyme/reagent usage. Integrated valves control the elution volume, meter the reactants, and facilitate mixing. Additional embodiments of the present invention provide a tethered DNA assembly process, where oligonucleotide fragments are tethered to beads during and after assembly.

Description

DNA ASSEMBLY IN MICROFLUIDICS DEVICE HAVING INTEGRATED

SOLID-PHASE COLUMNS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Appl. No.

62/958,153, titled “DNA Assembly in Microfluidics Device Having Integrated Solid- Phase Columns,” filed January 7, 2020, incorporated by reference herein in its entirety.

BACKGROUND

Field

[0002] Embodiments of the present invention relate to the field of synthetic biology, and more particularly to the field of DNA assembly.

Background

[0003] DNA assembly is a key step in synthetic biology, yet the timing and cost is a significant bottleneck to the industry. DNA assembly involves converting oligonucleotides (e.g., 50-200 bases) into long pieces of double-stranded DNA (e.g., 1-5 kilobases). Conventional DNA assembly schemas include, for example, Polymerase Cycling Assembly (PCA), Gibson Assembly, and Golden Gate Assembly. Each of these schemas make use of homogeneous reactions, where all molecules are in solution. This is typically followed by amplification, affinity purification, quantitation, concentration normalization, and then enzymatic reactions for error correction. DNA assembly is typically performed using microliter volumes and conventional fluid handling/robotics. Not only is this process time consuming, but it inherently results in significant waste of materials to support the assembly process.

SUMMARY

[0004] Embodiments of the present invention implement DNA assembly using a microfluidics channel having an integrated solid phase column. The presence of a solid phase enables purification of nucleic acid products by selective capture and elution. Enzymatic reactions (e.g. PCA, error correction, or amplification) can be performed in channels or chambers, and the reaction mixture can be passed over the column to selectively capture or selectively elute the desired product, thereby separating the product from reactants (input nucleic acids, nucleotides, buffers, enzymes) or side products which may interfere with subsequent steps.

[0005] Additional embodiments of the present invention provide a looped microfluidics channel. When closed, the loop in the channel allows DNA and reagent mixtures to be flowed multiple times over a bead column for efficient capture and enzyme/reagent usage. Integrated valves control the elution volume, meter the reactants, and facilitate mixing.

[0006] Additional embodiments of the present invention provide a tethered DNA assembly process. In a tethered DNA assembly process, oligonucleotide fragments are tethered to beads during and after assembly. In an embodiment, a tethered DNA assembly process includes capturing one or more oligonucleotide fragments on one or more beads, flowing additional fragments across the captured oligonucleotide fragments, providing assembly enzymes to generate assembled DNA strands, flowing an error correction enzyme across the assembled DNA strands to create cleaved DNA strands and whole DNA strands, and flowing amplification primers across the assembled DNA strands to create bead-free double stranded DNA, wherein the amplification primers only match the whole DNA strands. In an embodiment, the tethered DNA process further includes sequencing the bead-free double stranded DNA, selecting one of the sequenced bead-free double stranded DNA as the perfect DNA strand, and providing an amplification primer corresponding to the perfect DNA strand. In an embodiment, the tethered DNA process further includes flowing the amplification primer corresponding to the perfect DNA strand across the assembled DNA strands to create cloned strands of the perfect DNA strand. In an embodiment, the tethered DNA process further includes mixing the amplification primer corresponding to the perfect DNA strand with the bead- free double stranded DNA to create cloned strands of the perfect DNA strand.

[0007] In an embodiment, the tethered DNA assembly process is performed in a microfluidics device having a microfluidic channel, and each step of flowing in the tethered DNA assembly process is recursively repeated by flowing material through a loop in the microfluidic channel. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate embodiments of the present invention and, together with the description, further serve to explain the principles of the invention and to enable a person skilled in the pertinent art to make and use the invention. Embodiments of the present invention will be described with reference to the accompanying drawings.

[0009] FIG. 1 illustrates an example microfluidics circuit having an integrated solid phase support chamber and a closeable loop.

[0010] FIG. 2 illustrates an example mixing flow path in the example microfluidics circuit of FIG. 1.

[0011] FIG. 3 illustrates an example assembly flow path in the example microfluidics circuit of FIG. 1.

[0012] FIG. 4 illustrates a process flow diagram for a tethered DNA assembly process, according to an embodiment of the invention.

[0013] FIG. 5 illustrates experimental results of a tethered hybridization performed according to an embodiment of the present invention.

[0014] FIG. 6 illustrates an example seven-oligo (three bridge) DNA strand.

[0015] FIG. 7 illustrates an example fifteen-oligo (seven bridge) DNA strand.

DETAILED DESCRIPTION

[0016] Although specific configurations and arrangements are discussed, it should be understood that this is done for illustrative purposes only. A person skilled in the pertinent art will recognize that other configurations and arrangements can be used without departing from the spirit and scope of the present invention. It will be apparent to a person skilled in the pertinent art that this invention can also be employed in a variety of other applications.

[0017] It is noted that references in the specification to "one embodiment," "an embodiment," "an example embodiment," etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases do not necessarily refer to the same embodiment. Further, when a particular feature, structure or characteristic is described in connection with an embodiment, it would be within the knowledge of one skilled in the art to effect such feature, structure or characteristic in connection with other embodiments whether or not explicitly described.

[0018] Current DNA synthesis methods can reliably produce oligonucleotide fragments having approximately 50-200 bases. For complete creation of synthetic DNA, however, those fragments must be assembled into larger DNA constructs that encode entire genes or metabolic pathways. Conventional DNA assembly schemas include, for example, Polymerase Cycling Assembly (PCA), Dual-Assymetric (DA) PCR, Overlap Extension (OE), Asymmetric PCR, Thermodynamically-Balanced Inside Out (TBIO), Two-Step (DA+OE), Polymerase Assembly Multiplexing (PAM), One-Step Simplified Gene Synthesis, Single-Molecule PCR, TopDown Real-Time Gene Synthesis, Shotgun ligation, Two-step Ligation and PCR, Ligase Chain Reaction, Brick-based assembly, Sequence- and Ligation-Independent Cloning (SLIC), Transformation-associated Recombination, Biobrick assembly, Gibson Assembly, and Golden Gate Assembly.

[0019] Using PCA as an example, one conventional DNA assembly process flow involves pooling oligonucleotide fragments in wells of a microtiter plate for a “one-pot” assembly reaction. If the oligonucleotide fragments have been constructed on a solid support, the fragments are released from the beads prior to assembly into longer chains.

A PCA process is performed on the fragments within each well, and the resultant, longer oligonucleotides undergo a first amplification process. SPRI (solid phase reversible immobilization) beads are used for DNA cleanup, and then the oligonucleotides are quantitated and normalized prior to error correction. After error correction, the DNA is clonally amplified. Typically bacteria are transfected with the DNA, allowed to grow, spread on plates, and allowed to form colonies. Several of these colonies are “picked”, allowed to grow further, and the DNA extracted. Each sample undergoes barcoding and library prep for sequencing, and then the samples are sequenced using Next Generation Sequencing to identify a sequence-perfect clone. The DNA extracted from a perfect clone is quantitated, normalized, and shipped.

[0020] Such a conventional process is not conducive to performance in a microfluidics environment. For quantitation, either quantitation means need to be integrated into the microfluidics and control instrumentation, which is difficult, or enough DNA needs to be made such that an aliquot can be removed for a macro-level quantitation, which is problematic for reactions at the microfluidic scale. Additionally, normalization may require a variable dilution for each reaction, which is difficult to implement in microfluidics. Clonal amplification using bacteria would also be very difficult to implement in microfluidics; the growth steps, colony production, and colony picking are all macroscopic operations which would require an impractical footprint to implement in a microfluidic chip. The need to pick multiple colonies from each reaction exacerbates the problem. Finally, the DNA would need to be extracted and sequenced, and the perfect clone quantitated and normalized (with difficulties as mentioned above).

Use of Microfluidics for DNA Assembly

[0021] Microfluidics uses small volumes of reagents, typically nanoliters rather than microliters. This saves cost by reducing consumption of expensive reagents. The use of reduced volumes can also increase speed, because the oligonucleotide diffusion distance is constrained to be small. For both molecules and heat, reducing the distance by a factor of x reduces the time required for diffusion by a factor of x², so the use of microfluidics can often reduce the total time required for a reaction. According to some embodiments, a DNA assembly method as described below can be completed within 1 hour or less. In some embodiments, the DNA assembly method can be completed in 20 minutes or less, 5 minutes or less, or even 1 minute or less. According to some embodiments, an oligonucleotide diffusion distance in a DNA assembly device as described below is 500 microns or less. In some embodiments, the oligonucleotide distance is 200 microns or less, or even 100 microns or less.

[0022] Additionally, microfluidics offers automation and integration. Automation reduces both hands-on time and total protocol execution time. It also reduces or eliminates sample- and reagent-handling errors. Integration means more reaction steps can be integrated by a given device, which means that the sample throughput of microfluidic instrumentation is significantly higher than conventional robotics. As an example, a chip run on the Fluidigm BioMark™ instrument requires 192 pipetting steps to load, and can run 9216 RT-qPCR reactions in a single 90-minute run. If run in 384- well plates, both a pipetting robot and a RT-qPCR instrument are required, performing 18,432 pipetting steps and 2490-minute runs.

[0023] The use of small reaction volumes, resulting from the use of microfluidics, often increases reaction fidelity and reduces side products. This is due to the greater initial relative concentration of the molecule of interest compared to background molecules that can give side products. For instance, with PCR, a single molecule in 1 nL is 10,000 times the concentration of a single molecule in 10 pL. Since the chance of a side reaction starting and consuming the reactants required for a successful reaction is constant per a given volume of solution, this dramatically improves the odds of a successful PCR. Furthermore, since the reaction reaches its limiting concentration faster, there is less chance for amplification bias. Similar improvements have been demonstrated with whole genome amplification (WGA) reactions. Accordingly, running the same DNA assembly reactions in small volumes should result in similar advantages, particularly with regard to amplification and error correction. The probability of assembling the correct fragment vs. incorrect fragments should be constant per unit volume.

[0024] Running reactions in small volumes often means that premium reagents or higher concentrations of expensive reagents can be employed with minimal effect on cost. This can improve reaction fidelity with minimal cost impact. This may be particularly important for error correction steps, which often use expensive reagents (e.g., CorrectASE™). Improved fidelity of assembly reactions and amplification reactions may enable the routine steps of purification, quantitation, and/or concentration normalization to be removed from the DNA assembly process.

[0025] Additionally, the use of microfluidics may enable solid-phase DNA assembly.

Conventional DNA assembly techniques have an upper limit to the number and length of oligonucleotides that can be assembled at once, such as 10-20 oligonucleotides for a PC A reaction. Generally, this is due to the number of possible error interactions - each oligonucleotide can interact with every other oligonucleotide in solution, so there are n² interactions, but only n correct interactions. Solid-phase assembly allows stepwise assembly of DNA from oligonucleotides. This can be used to extend the maximum length of the assembled DNA. Solid-phase assembly may also be used to reduce assembly error rates. Smaller groups of oligonucleotides (or even single oligonucleotides) can be assembled per step, under more stringent conditions.

[0026] Nonetheless, it has been challenging up until now to perform such assembly processes using microfluidics. Most microfluidics options are flow-through type devices, without any ability to capture and hold DNA eluted from a bead column while the particular enzymes and other processing solutions are flowed through the device. Further information regarding DNA assembly using microfluidics devices without solid phase capture means can be found in Khilko, Yuliya, "Master’s Thesis: Development of DNA assembly and error correction protocols for a digital microfluidic device" (2017).

DNA Assembly in Microfluidics Having a Solid Phase Column

[0027] While the disclosure herein refers to DNA, a skilled artisan will recognize that embodiments apply also apply to genes, other nucleic acids, polynucleotides, and the like.

[0028] The presence of a solid phase enables purification of nucleic acid products by selective capture and elution. Enzymatic reactions (e.g. PCA, error correction, or amplification) can be performed in channels or chambers, and the reaction mixture can be passed over the column to selectively capture or selectively elute the desired product, thereby separating the product from reactants (input nucleic acids, nucleotides, buffers, enzymes) or side products which may interfere with subsequent steps. However, while flowing a reactant over a microfluidic bead column to elute the DNA, the reactant elution volume must be carefully controlled lest the released DNA flow out of the device. Additionally, as discussed above, the conventional assembly steps of quantitation and normalization are difficult to perform at the microfluidic scale, presenting a technological barrier to implementing microfluidic DNA assembly at a useful scale. In some embodiments of the present invention, the solid supports, such as beads, are arranged substantially in single file.

DNA Assembly in Microfluidics Channel Having a Closed Loop

[0029] Use of a microfluidics chip having a closeable loop in the flow path may open the door to efficient, high-throughput microfluidics-based DNA assembly. By using a closed loop to repeatedly flow DNA, reagents, etc. over a bead column, as described below according to embodiments of the present invention, reaction efficiency may be significantly increased such that quantitation and normalization may be skipped during DNA assembly. This not only removes the technical difficulties of implementing DNA quantitation and variable dilution on a microfluidics chip, but it also decreases cost and increases throughput by removing two reagent- and time-consuming reaction steps.

[0030] The 48. Atlas™ integrated fluidics circuit (IFC), commercialized by Fluidigm

Corp. of South San Francisco, CA, is an example microfluidic chip having microfluidics channels with both a solid phase column and a closeable loop. The 48. Atlas™ chip is a sample processing chip used in conjunction with Fluidigm’ s Juno™ system. Specifically, the Juno™ system is currently used to perform RNA-Seq sample preparation. The 48. Atlas™ chip was created using a multilayer soft lithography technique to pattern sequential layers of silicone (such as polydimethylsiloxane (PDMS)) with integrated valves. In the Juno™ system, the 48. Atlas™ chip captures polyA RNA from 48 samples, washes and elutes the RNA, fragments the RNA, performs reverse transcription and template switching, amplifies the products, and exports the product. Chip reactions are controlled with the Juno™ chip controller, which is programmable.

[0031] In embodiments of the present invention, a microfluidics chip design having a channel with an integrated solid phase column and a closeable loop, such as that used in Fluidigm’s 48. Atlas™ circuit, may be repurposed for gene assembly. The closed loop allows for mixing to occur in a more thorough and reliable manner than in typical flow through devices, and also allows the mixture to be flowed repeatedly over a bead column.

[0032] FIG. 1 illustrates an example microfluidics circuit 100 that can be used in embodiments of the present invention. Microfluidics circuit 100 includes two interconnected fluidic loops, having a set of micro-valves 102 that control liquid handling within the circuit. Microfluidics circuit 100 includes a solid support chamber 104. Solid support chamber 104 may contain, for example, a bead column or other specifically prepared surface to selectively capture oligonucleotides. FIG. 2 illustrates a mixing loop 200. To create mixing loop 200, select valves 102 in microfluidics circuit 100 are closed so as to restrict the microfluidics circuit to the shaded fluidic pathway. Because mixing loop 200 is primarily used for mixing, mixing loop 200 does not flow through solid support chamber 104. Mixing loop 200 may be used, for example, for enzymatic reactions, such as PCA, error correction, or amplification. FIG. 3 illustrates an assembly loop 300, where certain valves 102 are closed so as to restrict the microfluidics circuit to the shaded fluidic pathway. Because assembly loop 300 is primarily used to advance the assembly process, assembly loop 300 allows fluid to flow across the solid supports in solid support chamber 104. Valve operation and design details of microfluidic channels with such loops are further described in Tan, Swee Jin, et al. “A microfluidic device for preparing next generation DNA sequencing libraries and for automating other laboratory protocols that require one or more column chromatography steps.” PLOS one 8.7 (2013): e64084. Assembly loop 300 may be used not only to selectively capture a desired product, but also to selectively elute the desired product, thereby separating the product from reactants which may interfere with subsequent steps. [0033] While the embodiment of FIGS. 1-3 illustrate two loops in the microfluidic circuit, a person of skill in the art will recognize that a microfluidic circuit having a single loop can also be used.

[0034] In order to use a microfluidics channel having a closed loop for DNA assembly, the oligonucleotides from which the DNA is to be assembled must be added to the individual wells (also referred to herein as “lanes”) of the chip. In an embodiment, the oligo pools are added as a solution. In another embodiment, beads with attached oligos are added to the individual input wells using manual or automated pipetting mechanisms, and freed from the beads before input into the microfluidic chip. In yet another embodiment, the beads are individually steered to the input wells using one of the techniques described in PCT Publication No. WO 2019/040599, titled “Positional Tracking an Encoding in Microfluidics Devices.” Such a steering technique allows tracking of which oligonucleotide beads are sent to which well in the multi-well chip.

[0035] Once the oligos for DNA assembly are pooled into the individual wells, steps to assemble the DNA may be carried out within each individual lane. For example, the enzymatic steps required for assembly (e.g., releasing any DNA fragments from the beads, PCA, amplification, error correction, and clonal amplification) may all be performed in each individual lane of the microfluidics chip, as long as the chip is capable of inline thermocycling. Each enzymatic step requires flowing in a different mixture of buffers, enzymes, primers, and/or washes. Each pool of the multi-well microfluidics chip needs to have 1-5 separate inlets for each pool (where one or more inlets are reused if the number of inlets is less than the number of enzymatic steps that occur), or a means allowing each of 1-5 inlets to dispense reagent to each lane without cross-contamination, such as that found on the 48. Atlas™ chip identified above. Multiple types of DNA assembly may be performed in a microfluidics chip having a closeable loop, such as the 48. Atlas™ chip.

[0036] In one embodiment, each lane of the chip can be used to perform “one pot synthesis,” where all reactions occur purely by adding the necessary enzymes/reagents to each lane, and recursively flowing the solution through the microfluidics loop to sufficiently equilibrate the reactions.

[0037] In another embodiment, each lane in the chip can be used to perform DNA assembly using successive solid phase capture/wash/release steps. During each step, DNA is mixed with input reagents, a reaction is allowed to occur, and the product DNA is captured on the solid support; reactants are then washed away, and the product DNA eluted from the solid support, ready for the next step. The DNA and reagents may be flowed around the loop to promote mixing. The mixture may be flowed around the loop through the solid phase column to promote capture of the product DNA. In this embodiment, the reactions take place in the small volumes of the microfluidics loop and any chamber that is present, such that lower volumes of reagents/enzymes can be used as compared to conventional flow-through systems.

[0038] In yet another embodiment, each lane in the chip can be used to perform a “tethered” DNA assembly process, where oligonucleotides remain tethered to a support, such as a bead, throughout the assembly process. The steps and advantages of a tethered DNA assembly process according to an embodiment of the present invention are further described below with respect to FIG. 4.

Tethered DNA Assembly

[0039] According to embodiments of the present invention, performing DNA assembly on microfluidics devices such as the 48. Atlas™ and the Mondrian™ digital microfluidics systems can be improved by using a tethered DNA assembly process.

[0040] In a tethered DNA assembly according to embodiments of the invention, oligonucleotides are captured on the surface of a solid support. In an embodiment, the solid support includes beads, such as, for example and without limitation, highly porous polymeric beads; glass or silica beads including, but not limited to fused silica (amorphous pure silica), quartz (crystalline pure silica), or other any other suitable beads known in the art, which can be packed into a chamber or column. In another embodiment, the solid support is a flat substrate. In yet another embodiment, the solid support includes walls of the microfluidic channel. When the tethered DNA assembly process is implemented in a closed loop microfluidic chip such as that described with respect to FIGS. 1-3, the solid support may be located in solid support chamber 104. The capture process on the solid support needs to be specific, such that one oligo is captured at one end, and the rest of the oligos assemble by hybridization. Specific attachment can be arranged via known methods, such as avidin/biotin link, dig/antidig link, nickel/his- tag, or similar means. In an embodiment, a bead is primed with a common capture sequence, and the first oligonucleotide to be captured has a sequence complementary to the common capture sequence. [0041] FIG. 4 illustrates an example process flow for tethered DNA assembly, according to an embodiment of the invention. Additional details regarding a tethered DNA assembly may be found in Application No. PCT/US2020/019761, filed February 25,

2020, incorporated herein by reference in its entirety.

[0042] In step 402, in a microfluidic cartridge containing beads in a bead column, a first mixture of oligonucleotide fragments (and/or primers) is flowed over the bead column to capture the fragments on the surfaces of the beads. Only one oligonucleotide fragment is captured on the bead surface; the rest of the oligonucleotide fragments assemble on the captured oligo. When a multi-lane chip such as the 48. Atlas™ is used, each lane can be used to process different samples. Each lane may be dedicated to a single capture sequence, or multiple capture sequences may be possible in a single lane. For example, a single lane may include more than one type of capture bead, with a different set of oligonucleotides assembled on each. In another example, multiple different assemblies may be present on a particular bead, so as to produce two or more assembled fragments in a given lane. In an embodiment, the capture means is unique for each lane in a multi-lane chip. In another embodiment, the same capture means (e.g., avidin/biotin link) is duplicated in multiple lanes of a multi-lane chip. In an embodiment, each molecule to be assembled on a surface of a bead has a unique molecular identifier (UMI) or barcode that allows that particular molecule to be identified.

[0043] Because the microfluidic cartridge has a looped fluid pathway bounded by valves connecting the pathway to inlet and outlet ports, the loop can be closed to recirculate the flowing fluid. Flowing the fluid around the loop induces mixing by folding the fluid, increasing reaction efficiency. The fluid can then be repeatedly flowed through the loop and across the beads in the bead column to improve capture efficiency. That is, each round of fluid can pass over the bead column multiple times to improve reaction efficiency and reduce waste. This allows the fluid volume necessary for sufficient and reliable reactions to be reduced as compared to flow-through techniques. In a flow through system, the molecule-to-bead ratio must be high and routinely leads to waste, as not all fragments will bind to beads on their first pass. By using a microfluidic system with a closed loop, however, a smaller volume of fluid may be used, as the loop ensures that each molecule will have multiple opportunities to bind to a bead in the bead column. This is particularly advantageous when the number of molecules is limited. [0044] Once the initial fragments (and/or primers) are captured by the beads, an outlet valve is opened to allow the remaining sample fluid to exit the bead channel. In an embodiment, the solid phase column is located in a chamber of the microfluidics device that forms part of the closed loop, where the channel has one or more valves that control access to one or more outlet ports. An example of a chamber for holding a solid phase column is illustrated in FIG. 3. As shown in FIG. 3, the outlet from solid support chamber 104 decreases in size so as to trap the beads within chamber 104, while still allowing fluid to circulate around assembly circuit 300.

[0045] It is to be understood that wash buffers/mixtures may be flowed across the bead column and/or through the closed loop as needed between processing steps. Additionally, any mixing of reagents, buffers, eluted oligos, etc. may be performed via mixing circuit 200 shown in FIG. 2, where mixing circuit 200 is created by closing select valves and preventing fluid flow through solid support chamber 104.

[0046] In steps 404, oligonucleotide fragments are assembled into longer strands. A mixture containing assembly enzymes/reagents is flowed through an inlet port and across the bead column, such as via assembly circuit 300 shown in FIG. 3. Any of a variety of assembly techniques and chemistries may be used, such as PCA or Gibson assembly. In the embodiment of FIG. 4, the assembly is split into first and second phases (steps 402 and 404, respectively), although one of skill in the art would understand that assembly could occur in a single phase through certain assembly techniques. The multi-pass nature of the looped microfluidics channel reduces waste, thereby increasing the assembly efficiency and reducing the fragment and enzyme volume needed for a high assembly percentage. This reduction in volume significantly reduces the cost of enzymes/reagents needed, allowing higher quality enzymes/reagents to be used, which in turn produces an improved and more accurate result. Once the mixture of fragments and enzymes/reagents has been flowed across the beads a sufficient number of times and for a sufficient length of time for assembly to take place, remaining fluid exits the channel through an outlet port.

[0047] Using a two-stage assembly technique for example, incubation and annealing occurs at step 402, while joining (e.g., gap-filling and sealing) of the fragments into longer strands occurs at step 404.

[0048] In step 406, an error correction reagent is flowed through the inlet port and across the bead column. The outlet valves are again closed so that the pathway forms a closed loop across the bead column. The assembled DNA is cleaved at mismatches. The error correction reagent may include, for example, CorrectASE™, produced by Thermo Fisher Scientific, Inc. of Waltham, MA. Once cleaving is complete, the cleaved fragments and remaining error correction reagent exits the channel through an outlet port.

[0049] In some embodiments, steps 402, 404, and 406 are repeated with a new pool of oligonucleotides to increase the length of the tethered product. In a closed loop chip such as that described with respect to FIGS. 1-3, valves may be closed and/or inlet ports may be opened in such a way that allows for multiple rounds of assembly.

[0050] In step 408, amplification primers capable of amplifying expected full-length strands are flowed through the inlet port and across the bead column, along with free nucleotides and necessary enzymes, and one or more thermocycling processes takes place. This creates double-stranded DNA (dsDNA) copies that are not tethered to the beads. Because the primers correspond only to the expected full-length strands, copies are not made of the shorter tethered fragments remaining after their end portions were cleaved off during the error correction step. In an embodiment, the primers may include UMI segments such that each tethered strand can be uniquely identified. In an embodiment, UMI-containing primers are only added if no UMI was added in the capturing step 402. In another embodiment, UMI-containing primers may be added even if the tethered strands already include a UMI from the capturing step 402. Once amplification is complete, the dsDNA is flowed out of the channel through an outlet port. If UMIs have been added to the tethered strands at any point during the assembly process, the amplified dsDNA will contain those respective UMIs.

[0051] In step 410, the dsDNA output in step 408 is sequenced, so that a perfect strand of

DNA can be selected. If UMIs were added during the assembly process, then the perfect strand of DNA will include the UMI of its matching tethered strand.

[0052] In step 412 and 414, the perfect strand of DNA is amplified using corresponding primers. In an embodiment, a mixture containing the primers, amplification enzymes, and free nucleotides is flowed through the inlet port and across the bead column. In another embodiment, amplification is performed on the pool of dsDNA output from step 408. If UMIs have been used, then the primers are chosen to correspond to only one of the tethered DNA strands. In this way, only perfect DNA is cloned. If amplification is performed using the tethered DNA in the bead column, then upon completion of the amplification process, the cloned DNA is flowed out of the microfluidics channel through an outlet port and collected. The original strands (as they existed post-error correction) remain tethered to the beads and are not mixed in with the cloned DNA.

[0053] While the above tethered assembly method has been described using a microfluidics channel having a closed loop, one of skill in the art would recognize that a similar tethered process can be performed using a flow-through microfluidics device, as long as the elution volume is carefully controlled, so that released DNA does not flow out of the device. In a microfluidics device having a closed loop, integrated valves control the elution volume and meter reactants mixing with the sample.

[0054] FIG. 5 illustrates experimental results from performance of a tethered hybridization method similar to that described with respect to FIG. 4. The experiment used a fluorescent (Texas Red) terminal oligonucleotide, and either included or omitted the penultimate oligonucleotide. Part (a) of FIG. 5 is a baseline image showing a capillary (i.e., a microfluidics channel) with beads. This baseline image shows no fluorescence. Part (b) of FIG. 5 shows, for comparison, a positive hybridization control with seven oligonucleotides. This seven-oligo strand is illustrated in FIG. 6. Parts (c) and (d) of FIG. 5 illustrate a fifteen-oligo hybridization that omits the penultimate oligonucleotide. This fifteen-oligo strand is illustrated in FIG. 7. As shown in part (c), at the start of hybridization some fluorescence can be seen in solution, though not on the beads. As shown in part (d), after hybridization, no fluorescence was observed. Parts (e) and (f) of FIG. 5 illustrate a fifteen-oligo hybridization that includes the penultimate oligonucleotide. As shown in part (e), fluorescence evolves quickly on the beads. As shown in part (f), fluorescence on the beads remains strong after wash, and is comparable to the seven-oligo hybridization shown in part (b). In the experimental example of FIG.

5, the per-oligo concentration in hybridization solution was 3mM. About 10 pL of solution was flowed over the beads. Fluorescence formed essentially immediately (in seconds.)

[0055] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary embodiments of the present invention as contemplated by the inventor(s), and thus, are not intended to limit the present invention and the appended claims in any way.

[0056] Embodiments of the present invention have been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0057] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0058] The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

WHAT IS CLAIMED IS:

1. A method of assembling a DNA strand from oligonucleotide fragments, comprising: providing one or more solid supports primed with a plurality of oligonucleotide capture elements in a microfluidic device; flowing an oligonucleotide fragment mixture across the one or more solid supports to induce annealing based on the plurality of oligonucleotide capture elements and oligonucleotide fragments in the oligonucleotide fragment mixture; and joining the annealed oligonucleotide fragments into a plurality of assembled DNA strands, each assembled DNA strand corresponding to a single capture element in the plurality of oligonucleotide capture elements.

2. The method of claim 1, further comprising: flowing an error correction enzyme across the plurality of assembled DNA strands to create cleaved DNA strands and whole DNA strands; and flowing amplification primers across the assembled DNA strands to create free double stranded DNA, wherein the amplification primers only match the whole DNA strands.

3. The method of claim 2, further comprising: sequencing the free double stranded DNA; selecting one of the sequenced bead-free double stranded DNA as a perfect DNA strand; and selecting an amplification primer corresponding to the perfect DNA strand.

4. The method of claim 3, further comprising flowing the amplification primer corresponding to the perfect DNA strand across the plurality of assembled DNA strands to create cloned strands of the perfect DNA strand.

5. The method of claim 3, further comprising mixing the amplification primer corresponding to the perfect DNA strand with the free double stranded DNA to create cloned strands of the perfect DNA strand.

6. The method of claim 1, wherein the tethered DNA assembly process is performed in a microfluidics device having a microfluidic channel, and one or more steps in the tethered DNA assembly process is performed by flowing material through a closed loop in the microfluidic channel.

7. The method of claim 1, wherein an oligonucleotide diffusion distance is 500 microns or less.

8. The method of claim 1, wherein an oligonucleotide diffusion distance is 200 microns or less.

9. The method of claim 1, wherein an oligonucleotide diffusion distance is 100 microns or less.

10. The method of claim 1, wherein the DNA assembly method is completed in 1 hour or less.

11. The method of claim 1, wherein the DNA assembly method is completed in 20 minutes or less.

12. The method of claim 1, wherein the DNA assembly method is completed in 5 minutes or less.

13. The method of claim 1, wherein the DNA assembly method is completed in 1 minute or less.

14. The method of claim 1, wherein the one or more solid supports are arranged substantially in single file.

15. A method of DNA assembly, comprising: disposing a solid phase support column in a chamber of a microfluidics circuit; performing an enzymatic reaction in a channel of the microfluidics circuit to produce a reaction mixture, the channel fluidly coupled to the chamber; and flowing the reaction mixture over the solid phase support column in the chamber to capture assembled oligonucleotides.

16. The method of claim 15, further comprising; flowing a wash mixture over the solid phase support column to remove remaining portions of the reaction mixture.

17. The method of claim 16, further comprising: flowing an elution fluid over the solid phase support column to elute the assembled plurality of oligonucleotides.

18. The method of claim 15, further comprising: flowing an elution fluid over the solid phase support column to elute the assembled plurality of oligonucleotides.

19. The method of claim 15, wherein the disposing, performing, and flowing steps are performed in a microfluidics device having a microfluidic channel, and one or more of the disposing, performing, or flowing steps are performed by flowing material through a closed loop in the microfluidic channel

20. The method of claim 15, wherein performing an enzymatic reaction comprises: selectively opening a first set of valves and closing a second set of valves in the microfluidics circuit to create a first closed loop pathway containing the channel of the microfluidics circuit; and mixing DNA with input reagents in the first closed loop pathway to produce a reaction mixture; and wherein flowing the reaction mixture over the solid phase support column comprises selectively opening a third set of valves and closing a fourth set of valves in the microfluidics circuit to create a second closed loop pathway containing both the channel and the chamber of the microfluidics circuit.

21. A method of DNA assembly, comprising: selectively opening a first set of valves and closing a second set of valves in a microfluidics circuit to create a first closed loop pathway in of the microfluidics circuit; performing a homogeneous enzymatic reaction between oligonucleotides carried in a solution and one or more reagents in the first closed loop pathway to create a reaction mixture; selectively opening a third set of valves and closing a fourth set of valves in the microfluidics circuit to create a second closed loop pathway of the microfluidics circuit; and performing a DNA assembly process using the reaction mixture in the second closed loop pathway of the microfluidics circuit.

22. A method of gene assembly, comprising: disposing a solid phase support column in a chamber of a microfluidics circuit; performing an enzymatic reaction in a channel of the microfluidics circuit to produce a reaction mixture, the channel fluidly coupled to the chamber; and flowing the reaction mixture over the solid phase support column in the chamber to capture assembled oligonucleotides.

23. The method of claim 22, further comprising; flowing a wash mixture over the solid phase support column to remove remaining portions of the reaction mixture.

24. The method of claim 23, further comprising: flowing an elution fluid over the solid phase support column to elute the assembled oligonucleotides.

25. The method of claim 22, further comprising: flowing an elution fluid over the solid phase support column to elute the assembled oligonucleotides.

26. The method of claim 22, wherein the disposing, performing, and flowing steps are performed in a microfluidics device having a microfluidic channel, and one or more of the disposing, performing, or flowing steps are performed by flowing material through a closed loop in the microfluidic channel

27. The method of claim 22, wherein performing an enzymatic reaction comprises: selectively opening a first set of valves and closing a second set of valves in the microfluidics circuit to create a first closed loop pathway containing the channel of the microfluidics circuit; and mixing a plurality of oligonucleotides with input reagents in the first closed loop pathway to produce a reaction mixture; and wherein flowing the reaction mixture over the solid phase support column comprises selectively opening a third set of valves and closing a fourth set of valves in the microfluidics circuit to create a second closed loop pathway containing both the channel and the chamber of the microfluidics circuit.

28. A method of gene assembly, comprising: selectively opening a first set of valves and closing a second set of valves in a microfluidics circuit to create a first closed loop pathway in of the microfluidics circuit; performing a homogeneous enzymatic reaction between oligonucleotides carried in a solution and one or more reagents in the first closed loop pathway to create a reaction mixture; selectively opening a third set of valves and closing a fourth set of valves in the microfluidics circuit to create a second closed loop pathway of the microfluidics circuit; and performing a gene assembly process using the reaction mixture in the second closed loop pathway of the microfluidics circuit.