US20230338913A1

US20230338913A1 - Devices and methods for oligonucleic acid library synthesis

Info

Publication number: US20230338913A1
Application number: US18/319,356
Authority: US
Inventors: William Banyai; Bill James Peck; Andres Fernandez; Siyuan Chen; Pierre Indermuhle; Eugene P. Marsh
Original assignee: Twist Bioscience Corp
Current assignee: Twist Bioscience Corp
Priority date: 2015-04-21
Filing date: 2023-05-17
Publication date: 2023-10-26
Also published as: US20160310927A1; US20180236425A1; US11691118B2; US20200330953A1; US9981239B2; WO2016172377A1; US10744477B2

Abstract

Devices and methods for de novo synthesis of large and highly accurate libraries of oligonucleic acids are provided herein. Devices include structures having a main channel and microchannels, where the microchannels have a high surface area to volume ratio. Devices disclosed herein provide for de novo synthesis of oligonucleic acids having a low error rate.

Description

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No. 16/921,712, filed Jul. 6, 2020, which is a continuation of U.S. patent application Ser. No. 15/960,319, filed on Apr. 23, 2018, now U.S. Pat. No. 10,744,477 issued Aug. 18, 2020, which is a continuation of U.S. patent application Ser. No. 15/135,434 filed Apr. 21, 2016, now U.S. Pat. No. 9,981,239 issued May 29, 2018, which claims the benefit of U.S. Provisional Application No. 62/150,795 filed Apr. 21, 2015, and U.S. Provisional Application No. 62/220,856 filed Sep. 18, 2015, each of which are herein incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on May 17, 2023, is named 44854_713_303_SL.xml and is 105,152 bytes in size.

BACKGROUND

Highly efficient chemical gene synthesis with high fidelity and low cost has a central role in biotechnology and medicine, and in basic biomedical research. De novo gene synthesis is a powerful tool for basic biological research and biotechnology applications. While various methods are known for the synthesis of relatively short fragments in a small scale, these techniques often suffer from scalability, automation, speed, accuracy, and cost.

BRIEF SUMMARY

Provided herein are devices for synthesizing oligonucleic acids, comprising: a plate; a main channel, wherein the main channel extends vertically into the plate from an opening on a top side of the plate, and wherein the main channel has a width of 0.5 to 2 mm; and a plurality of microchannels connected to the main channel, wherein each microchannel of the plurality of microchannels extends vertically from an opening on a bottom side of the plate into the main channel, and wherein each microchannel of the plurality of microchannels has a surface area to volume ratio of greater than 0.2 (1/um). Devices are further provided wherein the surface area to volume ratio provides for rapid exchange of chemical exposure during de novo synthesis of oligonucleic acids. Devices are further provided wherein the surface area to volume ratio is about 0.4 (1/um). Devices are further provided wherein the surface area to volume ratio is greater than 0.4 (1/um). Devices are further provided wherein the surface area to volume ratio is 0.41 (1/um). Devices are further provided wherein each microchannel of the plurality of microchannels has a surface area greater than 10,000 um². Devices are further provided wherein each microchannel of the plurality of microchannels has a surface area greater than 12,000 um². Devices are further provided wherein each microchannel of the plurality of microchannels has a surface area of about 13,000 um². Devices are further provided wherein the plurality of microchannels comprises 50 to 500 microchannels. Devices are further provided wherein the plurality of microchannels comprises 100 to 150 microchannels. Devices are further provided wherein a ratio of width to depth of a narrowest segment of each microchannel is from 0.5 to 0.01. Devices are further provided wherein a ratio of width to depth of a narrowest segment of each microchannel is about 0.05, 0.1, or 0.2. Devices are further provided wherein each microchannel of the plurality of microchannels has a total width of 30 um to 100 um. Devices are further provided wherein each microchannel of the plurality of microchannels has a total width of about 60 um. Devices are further provided wherein each microchannel of the plurality of microchannels has a depth of 10 to 500 um. Devices are further provided wherein each microchannel of the plurality of microchannels has a depth of about 30 um. Devices are further provided wherein the main channel has a width from 0.5 to 1.5 mm. Devices are further provided wherein the main channel has a width of about 1.2 mm. Devices are further provided wherein the main channel has a width of 1.15 mm. Devices are further provided wherein the device comprises more than 250 main channels. Devices are further provided wherein the device comprises more than 10,000 main channels. Devices are further provided that further comprising a first molecule, wherein the first molecule is bound to an interior surface of the plurality of microchannels and comprises a reactive group that binds to a nucleoside phosphoramidite. Devices are further provided wherein the first molecule is a silane. Devices are further provided wherein the first molecule is an aminosilane. Devices are further provided wherein the first molecule is 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane or N-(3-triethoxysilylpropyl)-4-hydroxybutyramide. Devices are further provided further comprising a second molecule, wherein the second molecule is bound to an interior surface of the main channel and lacks a reactive group that binds to a nucleoside phosphoramidite. Devices are further provided wherein the second molecule is a fluorosilane. Devices are further provided wherein the fluorosilane is (tridecafluorotetrahydrooctyl)-triethoxysilane. Devices are further provided wherein the first molecule has a higher surface energy than the second molecule, and wherein the first molecule has a greater hydrophobicity than the second molecule. Devices are further provided wherein the plate is a silicon plate. Devices are further provided wherein the device comprises at least 30,000 microchannels. Devices are further provided wherein the device comprises at least 700,000 microchannels.
Provided herein are devices for synthesizing oligonucleic acids, comprising: a plate; a main channel, wherein the main channel extends vertically into the silicon plate from an opening on a top side of the plate, and wherein the main channel has a width of 0.5 to 2 mm; and a plurality of microchannels connected to the main channel, wherein each microchannel of the plurality of microchannels extends vertically from an opening on a bottom side of the plate into the main channel, and wherein each microchannel of the plurality of microchannels comprises a total width that is less than 100 um, a microchannel surface area greater than 10,000 um, and a maximum width for the narrowest segment of the microchannel of 10 um. Devices are further provided wherein each microchannel of the plurality of microchannels has a surface area greater than 12,000 um². Devices are further provided wherein each microchannel of the plurality of microchannels has a surface area of about 13,000 um². Devices are further provided wherein the plurality of microchannels comprises 50 to 500 microchannels. Devices are further provided wherein the plurality of microchannels comprises 100 to 150 microchannels. Devices are further provided wherein a ratio of width to depth of a narrowest segment of each microchannel is from 0.5 to 0.01. Devices are further provided wherein a ratio of width to depth of a narrowest segment of each microchannel is about 0.05, 0.1, or 0.2. Devices are further provided wherein each microchannel of the plurality of microchannels has a total width of 30 um to 100 um. Devices are further provided wherein each microchannel of the plurality of microchannels has a total width of about 60 um. Devices are further provided wherein each microchannel of the plurality of microchannels has a depth of 10 to 500 um. Devices are further provided wherein each microchannel of the plurality of microchannels has a depth of about 30 um. Devices are further provided wherein the main channel has a width from 0.5 to 1.5 mm. Devices are further provided wherein the main channel has a width of about 1.2 mm. Devices are further provided wherein the main channel has a width of 1.15 mm. Devices are further provided wherein the device comprises more than 250 main channels. Devices are further provided wherein the device comprises more than 10,000 main channels. Devices are further provided further comprising a first molecule, wherein the first molecule is bound to an interior surface of the plurality of microchannels and comprises a reactive group that binds to a nucleoside phosphoramidite. Devices are further provided wherein the first molecule is a silane. Devices are further provided wherein the first molecule is an aminosilane. Devices are further provided wherein the first molecule is 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane or N-(3-triethoxysilylpropyl)-4-hydroxybutyramide. Devices are further provided further comprising a second molecule, wherein the second molecule is bound to an interior surface of the main channel and lacks a reactive group that binds to a nucleoside phosphoramidite. Devices are further provided wherein the second molecule is a fluorosilane. Devices are further provided wherein the fluorosilane is (tridecafluorotetrahydrooctyl)-triethoxysilane. Devices are further provided wherein the first molecule has a higher surface energy than the second molecule, and wherein the first molecule has a greater hydrophobicity than the second molecule. Devices are further provided wherein the plate is a silicon plate. Devices are further provided wherein the device comprises at least 30,000 microchannels. Devices are further provided wherein the device comprises at least 700,000 microchannels.
Provided herein are methods for de novo oligonucleic acid synthesis, comprising: providing predetermined sequences for a plurality of non-identical oligonucleic acids; providing a device described herein; adding a droplet of fluid comprising an extension reaction reagent specific to a microchannel; allowing sufficient time for an extension reaction step to occur; and repeating steps (c) and (d) until the plurality of non-identical oligonucleic acids are synthesized, wherein each oligonucleic acid at least 10 bases in length and attached to an inside region of the microchannel, and wherein the synthesized non-identical oligonucleic acids encode sequences with an aggregate error rate of less than 1 in 2000 bases compared to the predetermined sequences. Methods are further provided wherein the synthesized non-identical oligonucleic acids encode sequences with an aggregate error rate of less than 1 in 3000 bases compared to the predetermined sequences. Methods are further provided wherein the synthesized non-identical oligonucleic acids encode sequences with an insertion error rate of less than 1 in 5000 bases compared to the predetermined sequences. Methods are further provided wherein the synthesized non-identical oligonucleic acids encode sequences with a deletion error rate of less than 1 in 2000 bases compared to the predetermined sequences. Methods are further provided that further comprising washing the surface with a washing reagent, and wherein washing removes greater than 95% of unincorporated extension reaction reagent. Methods are further provided wherein washing removes greater than 99% of unincorporated extension reaction reagent. Methods are further provided wherein the droplet of fluid is less than about 32 pL in volume. Methods are further provided wherein the method is completed in less than 24 hours. Methods are further provided wherein the synthesized plurality of non-identical oligonucleic acids are fluidically connected to a single main channel and collectively encode for a single gene. Methods are further provided wherein the oligonucleic acids collectively encode for at least 200 genes at least 1 kb in length. Methods are further provided further comprising: releasing the plurality of non-identical oligonucleic acids from the surface; and subjecting the plurality of non-identical oligonucleic acids to a polymerase chain assembly reaction to assemble at least 200 genes. Methods are further provided wherein the at least 200 genes have an aggregate error rate of less than 1 in 2000 bases compared to the predetermined sequences without correcting errors. Methods are further provided wherein the at least 200 genes have an aggregate error rate of less than 1 in 3000 bases compared to the predetermined sequences without correcting errors. Methods are further provided wherein each oligonucleic acid has a tether region 12 to 25 bases in length. Methods are further provided wherein the tether region is homopolymeric. Methods are further provided wherein each oligonucleic acid is at least 30 bases in length. Methods are further provided wherein each oligonucleic acid 50 to 500 bases in length.
Provided herein are devices for synthesizing oligonucleic acids, comprising a silicon plate; a main channel, wherein the main channel extends vertically into the silicon plate from an opening on a top side of the silicon plate, and wherein the main channel has a width of 0.5 to 2 mm; and 50 to 500 microchannels connected to the main channel, wherein each of the 50 to 500 microchannels extends vertically from an opening on a bottom side of the silicon plate into the main channel, and wherein each microchannel of the 50 to 500 microchannels has a surface area to volume ratio of greater than about 0.4 (1/um), and a ratio of width to depth of a narrowest segment of each microchannel is from 0.5 to 0.01.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications disclosed herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term disclosed herein and a term in an incorporated reference, the term herein controls.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a plate configured for oligonucleic acid synthesis comprising 24 regions, or sub-fields, each having an array of 256 clusters.

FIG. 2 illustrates a closer view of the sub-field in FIG. 1 having 16×16 of clusters, each cluster having 121 individual loci.

FIG. 3 illustrates a detailed view of the cluster in FIG. 2 , where the cluster has 121 loci.

FIG. 4A illustrates a front view of a plate with a plurality of microchannels.

FIG. 4B illustrates a sectional view of plate with a plurality of microchannels.

FIG. 5 illustrates three-dimensional arrangements for microchannels.

FIG. 6 illustrates a cluster having a plurality of loci with double comb shapes.

FIG. 7 illustrates a cluster having a plurality of loci with single comb shapes.

FIG. 8 illustrates a cluster having a plurality of loci with single serpentine shapes.

FIGS. 9A-9F illustrate a workflow for the passive and active functionalization of an etched substrate.

FIGS. 10A-10C illustrate reagent deposition directly into microchannels within a main channel, where: microchannels are actively functionalized (FIG. 10A), main channels are passively functionalized (FIG. 10B); and microchannels are actively functionalized and main channels are passively functionalized (FIG. 10C). The dotted lines indicate that the image depicts one main channel of many in a single substrate (e.g., a plate).

FIGS. 11A-11C illustrate reagent deposition directly into microchannels within a main channel, where a plate contains a silicon oxide later at a boundary between a main channel and a microchannel, where: microchannels are actively functionalized (FIG. 11A), main channels are passively functionalized (FIG. 11B); and microchannels are actively functionalized and main channels are passively functionalized (FIG. 11C). The dotted lines indicate that the image depicts one main channel of many in a single substrate (e.g., a plate).

FIG. 12 illustrates a workflow for de novo oligonucleotide synthesis.

FIG. 13 illustrates a computer system.

FIG. 14 is a block diagram illustrating architecture of a computer system.

FIG. 15 is a diagram demonstrating a network configured to incorporate a plurality of computer systems, a plurality of cell phones and personal data assistants, and Network Attached Storage (NAS).

FIG. 16 is a block diagram of a multiprocessor computer system using a shared virtual address memory space.

FIG. 17 depicts read counts from a sub-array having 256 clusters (left), and an image of a cluster having 121 loci (right).

FIG. 18 is a graphical representation of oligonucleic acid frequency versus abundance from an experiment where oligonucleic acids were synthesized on the substrate of FIG. 17 .

FIG. 19 is a graphical representation of oligonucleic acid frequency versus abundance for four representative clusters of the substrate of FIG. 17 .

FIG. 20 is a graphical representation of oligonucleic acid frequency versus error rate for oligonucleic acids synthesized on the substrate of FIG. 17 .

FIG. 21 is a graphical representation of oligonucleic acid frequency versus error rate for oligonucleic acids synthesized on four representative clusters of the substrate of FIG. 17 .

FIG. 22 is a representation of read counts from 240 assembled genes from a library of oligonucleic acids synthesized on a substrate.

FIGS. 23, 24, 25 and 26 provide digital images from gel electrophoresis of 240 assembled genes from a library of oligonucleic acids synthesized on the substrate of FIG. 17 .

FIG. 27 provides an output reading from next generation sequencing of 240 assembled genes from a library of oligonucleic acids synthesized on the substrate of FIG. 17 .

FIG. 28 provides a graphical representation of insertion/deletion (“indel”) error count as a function of read cycle for a synthesized library of oligonucleic acids.

FIG. 29 provides a digital image of an electrophoresis gel showing 25mer to 200mer oligonucleic acids synthesized using a substrate and methods provided herein.

DETAILED DESCRIPTION

The present disclosure provides compositions, devices, methods and systems for the de novo synthesis of a library of oligonucleic acids with low error rates. The oligonucleic acids are useful components, such as for the generation of larger nucleic acids, such as genes as part of gene libraries.
Described herein are devices having structural features that control the flow of fluid through small channels (“microchannels”) which also serve as locations for oligonucleic acid extension. Factors that can impact the flow of fluid throw the surface include, without limitation, the number of microchannels, microchannel size, the shape of the microchannels, the width of a main channel which a group of microchannels collectively connect, and the chemical properties of surfaces involved (e.g., hydrophobicity and surface energy). During oligonucleic acid synthesis, it is desirable to have channels to have enough width to support the extension of multiple oligonucleic acids, while at the same time be narrow enough to support rapid fluid transfer. Rapid fluid transfer is desirable to provide for efficient chemical exchange during various steps of the de novo nucleic acid synthesis process, and reduce unwanted side reactions that may lead to increased error rates. Devices described herein increase fluid transfer rate through a substrate and also increase the amount of surface available for nucleic acid extension is by having microchannels with a high surface area to volume ratio. Such devices also provide for synthesis of oligonucleic acids with low error rates.
In some cases, oligonucleic acids synthesized within a cluster of extension locations (“loci”) comprise specific predetermined sequences that are configured to be assembled to generate a larger nucleic acid. In this manner, the parallel generation of genes is done on a single substrate. The average error rates for oligonucleic acids synthesized within a library using the systems and methods provided are often less than 1 in 1000, and are preferably less than about 1 in 2000, 1 in 5000 or less often.

Definitions

The present disclosure employs, unless otherwise indicated, conventional molecular biology techniques, which are within the skill of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which these inventions belong.
Throughout this disclosure, various instances are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any instances. Accordingly, the description of a range should be considered to have specifically described all the possible subranges as well as individual numerical values within that range to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically described subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention, unless the context clearly dictates otherwise.
Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers +/−10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.
The terminology used herein is for the purpose of describing particular instances only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.
As used herein, the term “comprising” is inclusive or open-ended and does not exclude additional unrecited elements, device features, compositional components, or method steps.
The term “locus” as used herein refers to a discrete region on a structure which provides support for extension of an oligonucleic acid.
As used herein, the terms “preselected sequence,” “predefined sequence” or “predetermined sequence” are used interchangeably. The terms refer to sequence of a polymer that is known and chosen before synthesis or assembly of the polymer. In particular, various aspects of the invention are described herein primarily with regard to the preparation of nucleic acid molecules, the sequence of the oligonucleotide or polynucleotide being known and chosen before the synthesis or assembly of the nucleic acid molecules.
Substrates, Sub-Fields, Clusters and Loci
Provided herein are devices having a substrate (e.g., a plate) for the generation of a library of oligonucleic acids. An exemplary substrate 100 is illustrated in FIG. 1 , wherein the substrate 100 has about the same size dimensions as a standard 96 well plate: 140 mm by 90 mm. The substrate 100 comprises clusters grouped in 24 regions or sub-fields 105, each sub-field 105 comprising an array of 256 clusters 110. An expanded view of an exemplary sub-field 105 is shown in FIG. 2 . In the expanded view of four clusters (FIG. 2 ), a single cluster 110, has a Y axis cluster pitch (distance from center to center of adjacent clusters) of 1079.210 um or 1142.694 um, and an X axis cluster pitch of 1125 um. An illustrative cluster 110 is depicted in FIG. 3 , where the Y axis loci pitch (distance from center to center of adjacent loci) is 63.483 um, and an X axis loci pitch is 75 um. The locus width at the longest part, e.g., diameter for a circular loci, is 50 um and the distance between loci is 24 um. The number of loci 305 in the exemplary cluster in FIG. 3 is 121.
Fluid Conditioning
Provided herein are substrates comprising features which are segregated to allow for efficient chemical exchange of reagents during de novo oligonucleic acid synthesis. An exemplary arrangement is illustrated in FIGS. 4A-4B where a plate 405 is illustrated comprising a main channel 410 and a plurality of microchannels 415 connected to the main channel 410. The connection between the main channel 410 and the plurality of microchannels 415 provides for a fluid communication for flow paths from the main channel 410 to the each of the plurality of microchannels 415. A plate 405 described herein can comprise multiple main channels 410. The plurality of microchannels 415 collectively forms a cluster within the main channel 410. In some cases, a library of oligonucleic acids is synthesized in a plurality of loci where the loci are collectively a plurality of microchannels 415 of a cluster where the cluster is within a main channel 410, followed by the assembly of the oligonucleic acids into a large nucleic acid such as gene, wherein the assembly of the large nucleic acid optionally occurs within a main channel of the cluster, e.g., by using PCA. In further cases, a different oligonucleic acid is grown in each of the microchannels 415 with a main channel 410, and the oligonucleic acids collectively encode for a single gene.
The structure is configured to allow for controlled flow for de novo oligonucleic acid synthesis by providing for rapid exchange of chemical exposure during de novo synthesis of oligonucleic acids. For example, in some cases, configuration described herein provide for the controlled and even distribution of mass transfer paths, chemical exposure times, and/or wash efficiency during oligonucleic acid synthesis. In some instances, the configuration of a substrate allows for increased sweep efficiency, for example by providing sufficient volume for a growing an oligonucleic acid such that the excluded volume by the growing oligonucleic acid does not take up more than 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or less of the initially available volume that is available or suitable for growing the oligonucleic acid.
In addition to the physical segregation between each of the microchannels 415 of the plurality of microchannels, chemical coatings also provide and additional means for segregating oligonucleic acid species situated within a microchannel. Segregation can be achieved by differential functionalization of the surface, for example by having active and passive regions for oligonucleic acid synthesis coated on the surface. Differential functionalization can also be achieved by alternating the hydrophobicity across the substrate surface, thereby creating water contact angle effects that may cause beading or wetting of the deposited reagents. Employing larger structures can decrease splashing and cross-contamination of distinct oligonucleic acid synthesis locations with reagents of the neighboring spots. A device, such as an oligonucleic acid synthesizer, may be used to deposit reagents to distinct oligonucleic acid synthesis locations.
Microchannels: Structural Features
Microchannels described herein provide chemical properties, dimensions (width, height/depth, or length) de novo synthesized oligonucleic acids having a low error rate. While the plurality of microchannels 415 in FIG. 4 are circular, various shapes can be used to enhance flow rates through the channel, e.g., microchannels with curves or combs. A microchannel having a shape providing an increased surface area to volume ratio may be desirable for several reasons. First, an increase in surface area provides an increase in area that is suitable for oligonucleic acid attachment and synthesis. Second, a locus in the shape of a microchannel with increased surface area to volume ratio requires less fluid for efficient flow through the channel, thereby allowing less reagent volume per reaction. Third, without wishing to be bound by theory, the efficient flow through a locus in the shape of a microchannel with increased surface area to volume ratio minimizes residual occupation of reagents during flow through the microchannel and enhances wash efficiency, thereby minimizing or essentially eliminating undesirable secondary reactions during the chemical steps involved in the oligonucleotide extension process. Minimizing undesirable secondary reactions is another factor for keeping error rate down during oligonucleic acid synthesis.
Exemplary microchannels are illustrated in FIG. 5 which shows a top view of a double comb 510, single comb 520 or serpentine microchannel shape 530. In each exemplary microchannel in FIG. 5 , the width of the microchannel at the narrowest segment is 5 um, and each of the microchannel comprises at least one turn greater than 90 degrees in total. In some cases, the microchannel comprises 1 to 10 or more turns greater than 90 degrees in total. In some cases, the turns are curved and the microchannel comprises 1 to 10 or more turns in total. In the case of the single comb 520 and double comb 510, the turns are 90 degrees, i.e. perpendicular fluid paths from a top view. In the case of the serpentine comb 530, the turns are curved and 180 degrees, i.e. a U turn is viewed from top view. In some cases, a turn in a microchannel fluid path is 45 to 180 degrees in total, when viewed from a top view.
In a first example, a main channel 600 comprises a cluster of double comb channels, where each double comb channel 510 has a total width of 57 um, the width of the microchannel at the narrowest segment is 5 um, and each stick of the “comb” is 14 um apart from the center of another stick (FIG. 6 ). In a second example, a main channel 700 comprises a cluster of single comb channels, where each single comb channel 520 has a total width of 49 um, the width of the microchannel at the narrowest segment is 5 um, and each stick of the “comb” is 14 um apart from the center of another stick (FIG. 7 ). In a third example, a main channel 800 comprises a cluster of serpentine shaped channels, where each serpentine shaped channel 530 has a total width of 54 um, the width of the microchannel at the narrowest segment is 5 um, and each turn of the shape results in a parallel region 14 um apart from another region (FIG. 7 ). In some instances, the microchannel extends vertically into the substrate, e.g., a plate.
In some instances, a total surface area for a locus (e.g., a microchannel) in a device described herein is greater than about 9000, 10000, 11000, 12000, 12500, 12600, 12700, 12800, 12900 or 13000 um². In some instances, the total surface area for a locus in a device described herein is about 10000 to about 15000 um². In some instances, the total surface area for a locus in a device described herein is about 12000 to about 13000 um². In some instances, the total surface area to volume ratio (1/um) for a locus in a device described herein is about 0.2 to 0.5. In some instances, the total surface area to volume ratio (1/um) for a locus in a device described herein is greater than 0.20. In some instances, the total surface area to volume ratio (1/um) for a locus in a device described herein is greater than about 0.40. In some instances, the total surface area to volume ratio (1/um) for a locus in a device described herein is about 0.40. In some instances, the total surface area to volume ratio (1/um) for a locus in a device described herein is 0.41.
In some instances, a microchannel described herein has a width to depth (or height) ratio of 1 to 0.01, wherein the width is a measurement of the width at the narrowest segment of the microchannel. In some instances, a microchannel described herein has a width to depth (or height) ratio of 0.5 to 0.01, wherein the width is a measurement of the width at the narrowest segment of the microchannel. In some instances, a microchannel described herein has a width to depth (or height) ratio of about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5, or 1.
In some instances, a substrate is provided comprises a plurality of microchannels corresponding to a plurality of loci within a cluster, wherein the height or depth of the microchannel is from about 5 um to about 500 um, from about 5 um to about 400 um, from about 5 um to about 300 um, from about 5 um to about 200 um, from about 5 um to about 100 um, from about 5 um to about 50 um, or from about 10 um to about 50 um. In some cases, the height of a microchannel is less than 100 um, less than 80 um, less than 60 um, less than 40 um or less than 20 um. In some cases, microchannel height is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 μm or more.
In some instances, the width of a locus (e.g., microchannel or microwell) is from about 0.5 μm to about 500 um, from about 1 um to about 200 um, from about 1 um to about 100 um, from about 5 um to about 100 um, or from about 0.1 um to about 100 um, for example, about 90 um, 80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 um, 10 um, 5 um, 1 um or 0.5 um. In some instances, the width of a locus (e.g., microchannel) is less than about 100 um, 90 um, 80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 um or 10 um. In some instances, the distance between the center of two adjacent loci is from about 1 um to about 500 um, from about 1 um to about 200 um, from about 1 um to about 100 um, from about 5 um to about 200 um, from about 5 um to about 100 um, from about 5 um to about 50 um, or from about 5 um to about 30 um, for example, about 20 um. In some instances, the total width of a microchannel is about 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um, or 100 um. In some instances, the total width of a microchannel is about 10 um to 100 um, 30 um to 100 um, or 50 um to 70 um.
In some cases, each locus supports the synthesis of a population of oligonucleic acids having a different sequence than a population of oligonucleic acids grown on another locus. Provided herein are surfaces which comprise at least 10, 100, 256, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters. Provided herein are surfaces which comprise more than 2,000; 5,000; 10,000; 20,000; 30,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 5,000,000; or 10,000,000 or more distinct loci (e.g., microchannels). In some cases, each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150, 200, 500 or more loci. In some cases, each cluster includes 50 to 500, 50 to 200, 50 to 150, or 100 to 150 loci. In some cases, each cluster includes 100 to 150 loci. In exemplary arrangements, each cluster includes 109, 121, 130 or 137 loci.
Provided herein are structures wherein the distance between the centers of two adjacent loci within a cluster is from about 1 um to about 500 um, from about 5 um to about 200 um, or from about 0.5 μm to about 100 um. Provided herein are structures wherein the distance between two centers of adjacent loci is about 0.5 μm, 20 um, 25 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um, or 100 um.
Provided herein are loci having a width at the longest segment of 5 to 100 um. In some cases, the loci have a width at the longest segment of about 30, 35, 40, 45, 50, 55 or 60 um. In some cases, the loci are microchannels having multiple segments, wherein each segment has a center to center distance apart of 5 to 50 um. In some cases, the center to center distance apart for each segment is about 5, 10, 15, 20 or 25 um.
Main Channels: Structural Features
Main channels described herein extend from a top surface of a substrate, e.g., a plate, into the plate until reaching an interface with a plurality of microchannels, each microchannel connecting to a bottom surface of the substrate. In some instances, the main channel extends vertically. Main channels, e.g., main channel 410, can be in circular, rectangular, tapered, or rounded shapes.
In some instances, a width (a diameter in the case of a circle) of a cluster or the width of a main channel comprising a cluster, or both, is between about 0.05 mm to about 50 mm, between about 0.05 mm to about 10 mm, between about 0.05 mm and about 5 mm, between about 0.05 mm and about 4 mm, between about 0.05 mm and about 3 mm, between about 0.05 mm and about 2 mm, between about 0.05 mm and about 1 mm, between about 0.05 mm and about 0.5 mm, between about 0.05 mm and about 0.1 mm, between about 0.1 mm and 10 mm, between about 0.2 mm and 10 mm, between about 0.3 mm and about 10 mm, between about 0.4 mm and about 10 mm, between about 0.5 mm and 10 mm, between about 0.5 mm and about 5 mm, between about 0.5 mm and about 1.5 mm, or between about 0.5 mm and about 2 mm. In some instances, the width of a cluster or main channel or both is less than or about 5 mm, 4 mm, 3 mm, 2 mm, 1.5 mm, 1.2 mm, 1.15 mm, 1 mm, 0.5 mm, 0.1 mm, 0.09 mm, 0.08 mm, 0.07 mm, 0.06 mm or 0.05 mm. In some instances, the width of a cluster or main channel is between about 1.0 and about 1.3 mm. In some instances, the width of a cluster or main channel, or both is about 1.150 mm. In some instances, the width of a cluster or main channel, or both is about 0.08 mm.
In some instances, the height (depth) of a main channel is from about 20 um to about 1000 um, from about 50 um to about 1000 um, from about 100 μm to about 1000 um, from about 200 μm to about 1000 um, from about 300 μm to about 1000 um, from about 400 um to about 1000 um, or from about 500 μm to about 1000 um. In some cases, the height of a main channel is less than about 1000 um, less than about 900 um, less than about 800 um, less than about 700 um, or less than about 600 um. In some cases, the height of a main channel is about 500 um. In some cases, the height of a main channel is about 450 um.
In some instances, the number of distinct nucleic acids or genes assembled from a plurality of oligonucleic acids synthesized on a substrate is dependent on the number of clusters available in the substrate. In some instances, the density of clusters within a substrate is at least or about 1 cluster per 100 mm², 1 cluster per 10 mm², 1 cluster per 5 mm², 1 cluster per 4 mm², 1 cluster per 3 mm², 1 cluster per 2 mm², 1 cluster per 1 mm², 2 clusters per 1 mm², 3 clusters per 1 mm², 4 clusters per 1 mm², 5 clusters per 1 mm², 10 clusters per 1 mm², 50 clusters per 1 mm²or more. In some instances, a substrate comprises from about 1 cluster per 10 mm²to about 10 clusters per 1 mm². In some instances, the distance between the centers of two adjacent clusters is less than about 50 um, 100 um, 200 um, 500 um, 1000 um, or 2000 um or 5000 um. In some cases, the distance between the centers of two adjacent clusters is between about 50 um and about 100 um, between about 50 um and about 200 um, between about 50 um and about 300 um, between about 50 um and about 500 um, and between about 100 μm to about 2000 um. In some cases, the distance between the centers of two adjacent clusters is between about 0.05 mm to about 50 mm, between about 0.05 mm to about 10 mm, between about 0.05 mm and about 5 mm, between about 0.05 mm and about 4 mm, between about 0.05 mm and about 3 mm, between about 0.05 mm and about 2 mm, between about 0.1 mm and 10 mm, between about 0.2 mm and 10 mm, between about 0.3 mm and about 10 mm, between about 0.4 mm and about 10 mm, between about 0.5 mm and 10 mm, between about 0.5 mm and about 5 mm, or between about 0.5 mm and about 2 mm.
In some instances, the number of distinct oligonucleic acids synthesized on a substrate is dependent on the number of distinct loci available in the substrate. In some instances, the density of loci within a cluster of a substrate is at least or about 1 locus per mm², 10 loci per mm², 25 loci per mm², 50 loci per mm², 65 loci per mm², 75 loci per mm², 100 loci per mm², 130 loci per mm², 150 loci per mm², 175 loci per mm², 200 loci per mm², 300 loci per mm², 400 loci per mm², 500 loci per mm², 1,000 loci per mm²or more. In some cases, a substrate comprises from about 10 loci per mm²to about 500 mm², from about 25 loci per mm²to about 400 mm², from about 50 loci per mm²to about 500 mm², from about 100 loci per mm²to about 500 mm², from about 150 loci per mm²to about 500 mm², from about 10 loci per mm²to about 250 mm², from about 50 loci per mm²to about 250 mm², from about 10 loci per mm²to about 200 mm², or from about 50 loci per mm²to about 200 mm². In some instances, the distance between the centers of two adjacent loci within a cluster is from about 10 um to about 500 um, from about 10 um to about 200 um, or from about 10 um to about 100 um. In some cases, the distance between two centers of adjacent loci is greater than about 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um or 100 um. In some cases, the distance between the centers of two adjacent loci is less than about 200 um, 150 um, 100 um, 80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 um or 10 um.
A device described herein may comprise multiple main channels. In some cases, a device described herein comprises 1 to 250, 2 to 250, 1 to 500 or more main channels. In some cases, a device described herein comprises about 2, 10, 50, 100, 150, 200, 250, 256, 500, 512, 1000, 2500, 3000, 4000, 5000, 6000, 6144, 10000 or more main channels. In some cases, a plate described herein comprises about 2, 10, 50, 100, 150, 200, 250, 256, 500, 512, 1000, 2500, 3000, 4000, 5000, 6000, 6144, 10000 or more main channels. In some cases, the plate is a silicon plate or a silicon on insulator (SOI) plate.
In some instances, a substrate comprises a surface that supports the synthesis of a plurality of oligonucleic acids having different predetermined sequences at addressable locations on a common support. In some instances, a substrate described herein provides support for the synthesis of more than 2,000; 5,000; 10,000; 20,000; 30,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more non-identical oligonucleic acids. In some cases, the substrate provides support for the synthesis of more than 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more oligonucleic acids encoding for distinct sequences. In some instances, at least a portion of the oligonucleic acids have an identical sequence or are configured to be synthesized with an identical sequence. In some instances, the substrate provides a surface environment for the growth of oligonucleic acids having at least about 50, 60, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 bases or more.
In some instances, oligonucleic acids are synthesized on distinct loci of a substrate, wherein each locus supports the synthesis of a population of oligonucleic acids. In some cases, each locus supports the synthesis of a population of oligonucleic acids having a different sequence than a population of oligonucleic acids grown on another locus. In some instances, the loci of a substrate are located within a plurality of clusters. In some instances, a substrate comprises at least 10, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters. In some instances, a substrate comprises more than 2,000; 5,000; 10,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000; 1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; or 10,000,000 or more distinct loci. In some cases, each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150 or more loci. In some instances, each cluster includes 50 to 500, 100 to 150, or 100 to 200 loci. In some instances, each cluster includes 109, 121, 130 or 137 loci. In some instances, each cluster includes 5, 6, 7, 8, 9, 10, 11 or 12 loci.
In some instances, oligonucleic acids from distinct loci within one cluster have sequences that, when assembled, encode for a contiguous longer oligonucleic acid of a predetermined sequence, for example, a gene. In some cases, a substrate comprising more than 20,000 loci (e.g., microchannels) is used for the synthesis of up to 200 distinct genes of predetermined sequence. In some cases, a substrate comprising more than 29,000 loci (e.g., microchannels) is used for the synthesis of about 240 distinct genes of predetermined sequence. In some cases, a substrate comprising more than 700,000 loci is used for the synthesis of about 6,000 distinct genes of predetermined sequence.
Substrate: Materials
Substrates provided may be fabricated from a variety of materials suitable for the methods and compositions described herein. In certain instances, substrate materials are fabricated to exhibit a low level of nucleotide binding. In some cases, substrate materials are modified to generate distinct surfaces that exhibit a high level of nucleotide binding. In some instances, substrate materials are transparent to visible and/or UV light. In some instances, substrate materials are sufficiently conductive, e.g., are able to form uniform electric fields across all or a portion of a substrate. In some instances, conductive materials may be connected to an electric ground. In some cases, the substrate is heat conductive or insulated. In some cases, the materials are chemical resistant and heat resistant to support chemical or biochemical reactions, for example oligonucleic acid synthesis reaction processes. In some instances, a substrate comprises flexible materials. Flexible materials include, without limitation, modified nylon, unmodified nylon, nitrocellulose, polypropylene, and the like. In some instances, a substrate comprises rigid materials. Rigid materials include, without limitation, glass, fuse silica, silicon, silicon dioxide, silicon nitride, plastics (for example, polytetraflouroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, and the like), and metals (for example, gold, platinum, and the like). In some instances, a substrate is fabricated from a material comprising silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), glass, or any combination thereof. The substrates may be manufactured with a combination of materials listed herein or any other suitable material known in the art. In some instances, substrates described herein are in the shape of a plate, film or tape.
Substrate: Structural Features
In some instances, a substrate is about the size of a standard 96 well plate, for example between about 100 and 200 mm by between about 50 and 150 mm. In some instances, a substrate has a diameter less than or equal to about 1000 mm, 500 mm, 450 mm, 400 mm, 300 mm, 250 nm, 200 mm, 150 mm, 100 mm or 50 mm. In some instances, the diameter of a substrate is between about 25 mm and 1000 mm, between about 25 mm and about 800 mm, between about 25 mm and about 600 mm, between about 25 mm and about 500 mm, between about 25 mm and about 400 mm, between about 25 mm and about 300 mm, or between about 25 mm and about 200. Non-limiting examples of substrate size include about 300 mm, 200 mm, 150 mm, 130 mm, 100 mm, 76 mm, 51 mm and 25 mm. In some instances, a substrate has a planar surface area of at least about 100 mm²; 200 mm²; 500 mm²; 1,000 mm²; 2,000 mm²; 5,000 mm²; 10,000 mm²; 12,000 mm²; 15,000 mm²; 20,000 mm²; 30,000 mm²; 40,000 mm²; 50,000 mm²or more. In some instances, the thickness of a substrate is between about 50 mm and about 2000 mm, between about 50 mm and about 1000 mm, between about 100 mm and about 1000 mm, between about 200 mm and about 1000 mm, or between about 250 mm and about 1000 mm. Non-limiting examples of substrate thickness include 275 mm, 375 mm, 525 mm, 625 mm, 675 mm, 725 mm, 775 mm and 925 mm. In some cases, the thickness of a substrate varies with diameter and depends on the composition of the substrate. For example, a substrate comprising materials other than silicon may have a different thickness than a silicon substrate of the same diameter. Substrate thickness may be determined by the mechanical strength of the material used and the substrate must be thick enough to support its own weight without cracking during handling.
In some instances, a substrate described herein comprises a plurality of smaller regions, for example, at least about 2, 4, 6, 8, 10, 16, 24, 39, 50, 100 or more regions, wherein each region may be used independently from another region. In some cases, regions of a substrate are sub-fields or chips of a substrate. In some instances, reference to a substrate includes a region of a substrate.
Active and Passive Functionalization
Selective deposition or selective functionalization refers to a process that produces two or more distinct areas on a structure, wherein at least one area has a different surface or chemical property that another area of the same structure. Such properties include, without limitation, surface energy, chemical termination, surface concentration of a chemical moiety, and the like. In some cases, active functionalization refers to a method comprising the functionalization of a surface that will be utilized for oligonucleic acid synthesis. In some cases, passive functionalization refers to a method comprising the functionalization of a surface that will render these areas ineffective for oligonucleic acid synthesis. Any suitable process that changes the chemical properties of the surface described herein or known in the art may be used to functionalize the surface, for example chemical vapor deposition of an organosilane. Typically, this results in the deposition of a self-assembled monolayer (SAM) of the functionalization species.
In some instances, a method for functionalizing a surface of a substrate for oligonucleic acid synthesis comprises a resist or photoresist coat. Photoresist, in many cases, refers to a light-sensitive material useful in photolithography to form patterned coatings. It can be applied as a liquid to solidify on a substrate as volatile solvents in the mixture evaporate. In some instances, the resist is applied in a spin coating process as a thin film, e.g., 1 um to 100 um. In some cases, the coated resist is patterned by exposing it to light through a mask or reticle, changing its dissolution rate in a developer. In some cases, the resist cost is used as a sacrificial layer that serves as a blocking layer for subsequent steps that modify the underlying surface, e.g., etching, and then is removed by resist stripping. In some instances, the flow of resist throughout various features of the structure is controlled by the design of the structure. In some instances, a surface of a structure is functionalized while areas covered in resist are protected from active or passive functionalization.
In some instances, a substrate suitable for functionalization (e.g., an etched substrate comprising three-dimensional features) is deposited with resist, for example, by an oligonucleic acid synthesizer devices capable of delivering drops of fluid with micrometer accuracy. To resist coat only a small region of the substrate (e.g., lowered features such as a well and/or channel), a droplet of resist may be deposited into the lowered feature where it optionally spreads. In some instances, a portion of the resist is removed, for example, by etching (e.g., oxygen plasma etch) to leave a smooth surface covering only a select area. In some instances, the substrate is passively functionalized with a passive functionalization agent comprising a chemically inert moiety to create a surface having low surface energy. In some instances, the passively functionalized substrate is resist stripped, exposing areas of the substrate that were exposed during front-end processing (etched regions).
Described herein are methods for the synthesis of oligonucleic acids having a low error rate compared to a predetermined sequence using a device configured to regulate the flow of reagents in a microfluidic environment. An exemplary method is illustrated in FIGS. 9A-9F, which depicts a workflow for active functionalization of a microchannels and passive functionalization of surrounding areas, e.g., main channels. An etched substrate 905 is prepared for active functionalization. In FIG. 9A, the substrate is wet cleaned, for example, using a piranha solution. In some instances, the substrate is plasma cleaned, for example, by dry oxygen plasma exposure. In FIG. 9B, the device layer is coated with photoresist 910, optionally after cleaning. In some instances, the photoresist is coated by a process governed by wicking into the device layer channels. In some instances, the photoresist is patterned using photolithography to expose areas that are desired to be passive (i.e., areas where oligonucleic acid synthesis is not designed to take place). Patterning by photolithography may occur by exposing the resist to light through a binary mask that has a pattern of interest. After exposure, the resist in the exposed regions may be removed in developer solution in FIG. 9C, and leaves photoresist at predetermined regions 915. A subsequent step, as shown in FIG. 9D, involves exposure to a passive functionalization agent 920 such as a low surface energy silane (e.g., fluorosilane gas vapor), for example, by chemical vapor deposition (CVD). Exposure to fluorosilane gas results in the deposition of a fluorocarbon on the surfaces without photoresist. In some cases, the substrate is exposed to a hydrocarbon silane. In some instances, passive functionalization comprises exposing a substrate to a passive functionalization agent such as one comprising silane. In some instances, the passively functionalized substrate are unresponsive to additional layers of functionalization agent (e.g., active functionalization agent) creating a monolayer on the surface. A substrate that has been coated with resist, and optionally patterned by photolithography and/or optionally passively functionalized, in many cases, is resist stripped. Resist stripping as shown in FIG. 9E leaves some surfaces passively functionalized while exposing regions of the substrate that was underneath the resist 925. For example, a resist is dissolved in an organic solvent. As another example, resist stripping of a substrate that was passively functionalized with a fluorosilane gas, leaves a surface having some regions of fluorination. In some cases, regions that were underneath the resist comprise silicon or silicon dioxide. In FIG. 9F, the surface is actively functionalized to prepare the surface for oligonucleic acid synthesis. An exemplary active functionalization agent is one that has a higher surface energy than the passive functionalization agent.
An exemplary workflow for the generation of differential functionalization patterns of a substrate is described herein, FIGS. 9A-F. The following workflow is an example process and any step or component may be omitted or changed in accordance with properties desired of the final functionalized substrate. In some cases, additional components and/or process steps are added to the process workflows embodied herein. In some instances, a substrate is first cleaned, for example, using a piranha solution. An example of a cleaning process includes soaking a substrate in a piranha solution (e.g., 90% H₂SO₄, 10% H₂O₂) at an elevated temperature (e.g., 120° C.) and washing (e.g., water) and drying the substrate (e.g., nitrogen gas). The process optionally includes a post piranha treatment comprising soaking the piranha treated substrate in a basic solution (e.g., NH₄OH) followed by an aqueous wash (e.g., water). In some instances, a substrate is plasma cleaned, optionally following the piranha soak and optional post piranha treatment. An example of a plasma cleaning process comprises an oxygen plasma etch. In some instances, the surface is deposited with an active functionalization agent following by vaporization. In some instances, the substrate is actively functionalized prior to cleaning, for example, by piranha treatment and/or plasma cleaning.
The process for substrate functionalization optionally comprises a resist coat and a resist strip. In some instances, following active surface functionalization, the substrate is spin coated with a resist, for example, SPR™ 3612 positive photoresist. The process for substrate functionalization, in various instances, comprises lithography with patterned functionalization. In some instances, photolithography is performed following resist coating. In some instances, after lithography, the substrate is visually inspected for lithography defects. The process for substrate functionalization, in some instances, comprises a cleaning step, whereby residues of the substrate are removed, for example, by plasma cleaning or etching. In some instances, the plasma cleaning step is performed at some step after the lithography step.
In some instances, a substrate coated with a resist is treated to remove the resist, for example, after functionalization and/or after lithography. In some cases, the resist is removed with a solvent, for example, with a stripping solution comprising N-methyl-2-pyrrolidone. In some cases, resist stripping comprises sonication or ultrasonication. In some instances, a resist is coated and stripped, followed by active functionalization of the exposed areas to create a desired differential functionalization pattern.
In various instances, the methods and compositions described herein relate to the application of photoresist for the generation of modified surface properties in selective areas, wherein the application of the photoresist relies on the fluidic properties of the substrates defining the spatial distribution of the photoresist. Without being bound by theory, surface tension effects related to the applied fluid may define the flow of the photoresist. For example, surface tension and/or capillary action effects may facilitate drawing of the photoresist into small structures in a controlled fashion before the resist solvents evaporate. In some instances, resist contact points are pinned by sharp edges, thereby controlling the advance of the fluid. The underlying structures may be designed based on the desired flow patterns that are used to apply photoresist during the manufacturing and functionalization processes. A solid organic layer left behind after solvents evaporate may be used to pursue the subsequent steps of the manufacturing process. Substrates may be designed to control the flow of fluids by facilitating or inhibiting wicking effects into neighboring fluidic paths. For example, a substrate is designed to avoid overlap between top and bottom edges, which facilitates the keeping of the fluid in top structures allowing for a particular disposition of the resist. In an alternative example, the top and bottom edges overlap, leading to the wicking of the applied fluid into bottom structures. Appropriate designs may be selected accordingly, depending on the desired application of the resist.
As illustrated in the detailed cross view of FIG. 10A, an exemplary described herein is coated with a layer of material comprising on or more active functionalization agent. FIG. 10A illustrates the deposition of reagents 1005 on a plate 405. The substrate 405 comprises a plurality of microchannels 415 in fluidic connection with a main channel 410. In this exemplary device, a single main channel 410 is depicted, and the dashed lines indicate that this is just one of many main channel 410/plurality of microchannels 415 connections in a single plate 405. Each of the plurality of microchannels 415 is coated with an active functionalization agent 1015. Each microchannel 415 of the plurality of microchannels in the example has a width of 5 um and a depth of 30 um. The main channel 410 has a diameter of 1150 um. The total height of the plate is 450 um. The main channel 410 encircles a cluster of microchannels. In some instances, as illustrated in FIG. 10B, a substrate described herein is coated with a layer of material comprising on or more passive functionalization agents. In this exemplary device, a passive functionalization agent 1020 coats surface of a plate 405. In some instances, as illustrated in FIG. 10C, a substrate described herein is coated with a layer of material comprising on or more passive functionalization agents and the microchannels of the substrate are coated with one or more active functionalization agents. In another exemplary device, the starting substrate is a silicon on insulator plate, where layers of silicon sandwich an insulator layer 1110, typically silicon dioxide. In some instances, the thickness of the insulator layer 1110 is from 1 to 50 um, e.g., 20 um. The remaining feature of this exemplary device, as shown in FIGS. 11A-11C, as the same as those depicted in FIGS. 10A-10C.
In some cases, only loci (i.e., microchannels) in a device described herein are coated with active functionalization agent. An active functionalization agent is a molecule that binds to the surface of the substrate and is also capable of binding to a nucleic acid monomer, thereby supporting a coupling reaction to the surface. Exemplary active functionalization agents are molecules having a hydroxyl group available for coupling with a nucleoside in a coupling reaction. In some cases, only main channels and/or surrounding areas (and not the microchannels) in a device described herein are coated with passive functionalization agent. A passive functionalization agent is a molecule that binds to the surface of the substrate and lacks a moiety available for coupling with a nucleoside in a coupling reaction.
Oligonucleic acids synthesized in the channels may be released for the purposes of generating longer nucleic acids. In some cases, following oligonucleic acid synthesis, oligonucleic acids within one cluster are released from their respective surfaces and pool into the main channel. In some cases, the pooled oligonucleic acids are assembled into a larger nucleic acid, such as a gene, within the main channel, so that the main channel functions as a reactor for nucleic acid assembly. In other cases, following oligonucleic acid synthesis, oligonucleic acids within one cluster are released from their respective surfaces and pool into a nanoreactor in fluidic communication with the microchannels.
In some embodiments, nucleic acid verification (e.g., sequencing of oligonucleic acids and/or assembled genes) is performed within a reactor or well. Nucleic acid assembly includes polymerase cycling assembly (PCA). In some cases, a capping element or other device is placed over open sides of the main channel to create an enclosed reactor. A substrate comprising a main channel that functions as a reactor for each cluster has the advantage that each cluster may have a different environment from another cluster in another reactor. As an example, sealed reactors (e.g., those with capping elements) may experience controlled humidity, pressure or gas content.
In some instances, a substrate is configured for both active and passive functionalization moieties bound to the surface at different areas of the substrate surface, generating distinct regions for oligonucleic acid synthesis to take place. In some instances, both active and passive functionalization agents are mixed within a particular region of the surface. Such a mixture provides a diluted region of active functionalization agent and therefore lowers the density of functionalization agent in a particular region.
Substrates described herein may comprise a high surface energy region at the site of active functionalization agent deposition. In some instances, the high surface energy region is coated with aminosilane. The silane group binds to the surface, while the rest of the molecule provides a distance from the surface and a free group at the end to which incoming bases attach. In some instances, the free group is a hydroxyl group. In some instances the high surface energy region includes an active functionalization reagent, e.g., a chemical that binds the substrate efficiently and also couples efficiently to monomeric nucleic acid molecules. In some cases, such molecules have a hydroxyl group which is available for interacting with a nucleoside in a coupling reaction. In some instances, the amino silane is selected from the group consisting of 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane and N-(3-triethoxysilylpropyl)-4-hydroxybutyramide. In some instances the high surface energy region includes a passive functionalization reagent, e.g., a chemical that binds the substrate efficiently but does not couple efficiently to monomeric nucleic acid molecules.
In some instances, described herein are substrates comprising a plurality of clusters, wherein each cluster comprises a plurality of loci that support the attachment and synthesis of oligonucleic acids. In some instances, a locus is on a three-dimensional surface, e.g., a well, microchannel, channel, or post. In some instances, a surface of a locus comprises a material that is actively functionalized to attach to at least one nucleotide for oligonucleic acid synthesis, or preferably, a population of identical nucleotides for synthesis of a population of oligonucleic acids. In some instances, oligonucleic acid refers to a population of oligonucleic acids encoding for the same nucleic acid sequence. In some cases, a surface of a substrate is inclusive of one or a plurality of surfaces of a substrate.
In some cases, the addition of a chemical layer on top of a surface (referred to as adhesion promoter) facilitates structured patterning of loci on a surface of a substrate. Exemplary surfaces which can benefit from adhesion promotion include, without limitation, glass, silicon, silicon dioxide and silicon nitride. In some cases, the adhesion promoter is a chemical with a high surface energy. In some instances, a second chemical layer is deposited on a surface of a substrate. In some cases, the second chemical layer has a low surface energy. The surface energy of a chemical layer coated on a surface can facilitate localization of droplets on the surface. Depending on the patterning arrangement selected, the proximity of loci and/or area of fluid contact at the loci can be altered.
In some instances, a substrate surface, or resolved loci, onto which nucleic acids or other moieties are deposited, e.g., for oligonucleic acid synthesis, are smooth or substantially planar or have raised or lowered features. In some instances, a substrate surface is modified with one or more different layers of compounds. Such modification layers of interest include, without limitation, inorganic and organic layers such as metals, metal oxides, polymers, small organic molecules and the like. Non-limiting polymeric layers include peptides, proteins, nucleic acids or mimetics thereof (e.g., peptide nucleic acids and the like), polysaccharides, phospholipids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyetheyleneamines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and any other suitable compounds described herein or otherwise known in the art. In some cases, polymers are heteropolymeric. In some cases, polymers are homopolymeric. In some cases, polymers comprise functional moieties or are conjugated.
In some instances, resolved loci of a substrate are functionalized with one or more moieties that increase and/or decrease surface energy. In some cases, a moiety is chemically inert. In some cases, a moiety is configured to support a desired chemical reaction, for example, one or more processes in an oligonucleic acid synthesis reaction. The surface energy, or hydrophobicity, of a surface is a factor for determining the affinity of a nucleotide to attach onto the surface. In some instances, a method for substrate functionalization comprises: (a) providing a substrate (e.g., a plate); and (b) silanizing the loci (e.g., microchannels) with a suitable silanizing agent described herein or otherwise known in the art, for example, an organofunctional alkoxysilane molecule. In some cases, the organofunctional alkoxysilane molecule comprises dimethylchloro-octodecyl-silane, methyldichloro-octodecyl-silane, trichloro-octodecyl-silane, trimethyl-octodecyl-silane, triethyl-octodecyl-silane, or any combination thereof. In some instances, a substrate surface comprises functionalized with polyethylene/polypropylene (functionalized by gamma irradiation or chromic acid oxidation, and reduction to hydroxyalkyl surface), highly crosslinked polystyrene-divinylbenzene (derivatized by chloromethylation, and aminated to benzylamine functional surface), nylon (the terminal aminohexyl groups are directly reactive), or etched with reduced polytetrafluoroethylene.
In some instances, a substrate surface is functionalized by contact with a derivatizing composition that contains a mixture of silanes, under reaction conditions effective to couple the silanes to the substrate surface, typically via reactive hydrophilic moieties present on the substrate surface. Silanization generally can be used to cover a surface through self-assembly with organofunctional alkoxysilane molecules. A variety of siloxane functionalizing reagents can further be used as currently known in the art, e.g., for lowering or increasing surface energy. The organofunctional alkoxysilanes are classified according to their organic functions. Non-limiting examples of siloxane functionalizing reagents include hydroxyalkyl siloxanes (silylate surface, functionalizing with diborane and oxidizing the alcohol by hydrogen peroxide), diol (dihydroxyalkyl) siloxanes (silylate surface, and hydrolyzing to diol), aminoalkyl siloxanes (amines require no intermediate functionalizing step), glycidoxysilanes (3-glycidoxypropyl-dimethyl-ethoxysilane, glycidoxy-trimethoxysilane), mercaptosilanes (3-mercaptopropyl-trimethoxysilane, 3-4 epoxycyclohexyl-ethyltrimethoxysilane or 3-mercaptopropyl-methyl-dimethoxysilane), bicyclohepthenyl-trichlorosilane, butyl-aldehydr-trimethoxysilane, or dimeric secondary aminoalkyl siloxanes. The hydroxyalkyl siloxanes can include allyl trichlorochlorosilane turning into 3-hydroxypropyl, or 7-oct-1-enyl trichlorochlorosilane turning into 8-hydroxyoctyl. The diol (dihydroxyalkyl) siloxanes include glycidyl trimethoxysilane-derived (2,3-dihydroxypropyloxy)propyl (GOPS). The aminoalkyl siloxanes include 3-aminopropyl trimethoxysilane turning into 3-aminopropyl (3-aminopropyl-triethoxysilane, 3-aminopropyl-diethoxy-methylsilane, 3-aminopropyl-dimethyl-ethoxysilane, or 3-aminopropyl-trimethoxysilane). The dimeric secondary aminoalkyl siloxanes can be bis (3-trimethoxysilylpropyl) amine turning into bis(silyloxylpropyl)amine. In some instances, the functionalizing agent comprises 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane and N-(3-triethoxysilylpropyl)-4-hydroxybutyramide.
In some instances, a substrate surface is contacting with a mixture of functionalization groups, e.g., aminosilanes, which can be in any different ratio. In some instances, a mixture comprises at least 2, 3, 4, 5 or more different types of functionalization agents. In some instances, the mixture comprises 1, 2, 3 or more silanes. In cases, the ratio of the at least two types of surface functionalization agents in a mixture is about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:3, 2:5, 2:7, 2:9, 2:11, 2:13, 2:15, 2:17, 2:19, 3:5, 3:7, 3:8, 3:10, 3:11, 3:13, 3:14, 3:16, 3:17, 3:19, 4:5, 4:7, 4:9, 4:11, 4:13, 4:15, 4:17, 4:19, 5:6, 5:8, 5:9, 5:11, 5:12, 5:13, 5:14, 5:16, 5:17, 5:18, 5:19, 6:7, 6:11, 6:13, 6:17, 6:19, 7:8, 7:9, 7:10, 7:11, 7:12, 7:13, 7:15, 7:16, 7:18, 7:19, 8:9, 8:11, 8:13, 8:15, 8:17, 8:19, 9:10, 9:11, 9:13, 9:14, 9:16, 9:17, 9:19, 10:11, 10:13, 10:17, 10:19, 11:12, 11:13, 11:14, 11:15, 11:16, 11:17, 11:18, 11:19, 11:20, 12:13, 12:17, 12:19, 13:14, 13:15, 13:16, 13:17, 13:18, 13:19, 13:20, 14:15, 14:17, 14:19, 15:16, 15:17, 15:19, 16:17, 16:19, 17:18, 17:19, 17:20, 18:19, 19:20, or any other ratio to achieve a desired surface representation of two groups. In some instances, a ratio of silanes is about 1:100, 1:1000, 1:2000 or 1:3000.
In some cases, an active functionalization agent comprises 11-acetoxyundecyltriethoxysilane. In some cases, an active functionalization agent comprises n-decyltriethoxysilane. In some cases, the active functionalization areas comprise one or more different species of silanes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more silanes. In some cases, one of the one or more silanes is present in the functionalization composition in an amount greater than another silane. For example, a mixed silane solution having two silanes comprises a 99:1, 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91:9, 90:10, 89:11, 88:12, 87:13, 86:14, 85:15, 84:16, 83:17, 82:18, 81:19, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45 ratio of one silane to another silane. In some instances, an active functionalization agent comprises 11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane (e.g., in a ratio from about 20:80 to about 1:99, or about 10:90 to about 2:98, or preferably about 5:95). In some cases, an active functionalization agent comprises glycidyloxypropyltriethoxysilane (GOPS). In some instances, the silane is a fluorosilane. In some instances, the silane is a hydrocarbon silane. In some cases, the silane is 3-iodo-propyltrimethoxysilane. In some cases, the silane is octylchlorosilane. In some instances, an active functionalization agent comprises N-(3-triethosysilylpropyl)-4-hydroxybutyramide. In some cases, the passive functionalization agent comprises a silane. In some cases, the passive functionalization agent comprises a mixture of silanes. In some cases, the passive functionalization agent comprises perfluorooctyltrichlorosilane.
In some instances, desired surface tensions, wettabilities, water contact angles, and/or contact angles for other suitable solvents are achieved by providing a substrate surface with a suitable ratio of functionalization agents. In some cases, the agents in a mixture are chosen from suitable reactive and inert moieties, thus diluting the surface density of reactive groups to a desired level for downstream reactions. In some instances, the density of the fraction of a surface functional group that reacts to form a growing oligonucleotide in an oligonucleotide synthesis reaction is about 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 7.0, 10.0, 15.0, 20.0, 50.0, 75.0, 100.0 μMol/m².
In some instances, a surface of a substrate is prepared to have a low surface energy. In some cases, a region of a surface of a substrate described herein is functionalized to enable covalent binding of molecular moieties that can lower the surface energy so that wettability can be reduced. In some instances, a region of surface of a substrate described herein is prepared to have a high surface energy and increased wettability. In some instances, a surface is modified to have a higher surface energy, or become more hydrophilic with a coating where the coating includes molecules having reactive hydrophilic moieties. By altering the surface energy of different parts of a substrate surface, spreading of a deposited reagent liquid (e.g., a reagent deposited during an oligonucleic acid synthesis method) can be adjusted, in some cases facilitated. In some instances, a droplet of reagent is deposited over a predetermined area of a surface with high surface energy. The liquid droplet can spread over and fill a small surface area having a higher surface energy as compared to a nearby surface. In some instances, a substrate surface is modified to comprise reactive hydrophilic moieties such as hydroxyl groups, carboxyl groups, thiol groups, and/or substituted or unsubstituted amino groups. Suitable materials include, but are not limited to, supports that can be used for solid phase chemical synthesis, e.g., cross-linked polymeric materials (e.g., divinylbenzene styrene-based polymers), agarose (e.g., Sepharose®), dextran (e.g., Sephadex®), cellulosic polymers, polyacrylamides, silica, glass (particularly controlled pore glass, or “CPG”), ceramics, and the like. The supports may be obtained commercially and used as is, or they may be treated or coated prior to functionalization.
In some instances, provided herein are methods for the manufacture of a substrate using a multilayer activation process. In some instances, one or more layers deposited during a multilayer activation process comprise one or more silanes. In some instances, a substrate, e.g., a silicon plate, is treated with a first layer of a material that modifies the surface to allow for adhesion of a photoresist. Non-limiting examples of materials for surface modification include 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane and N-(3-triethoxysilylpropyl)-4-hydroxybutyramide, and mixtures thereof, and/or other suitable materials described elsewhere herein or known in the art. In some instances, the modified substrate is treated with resist, exposed, developed, and residual resist is removed by plasma cleaning, exemplary details of which are described previously herein. In some instances, the substrate is passively functionalized, for example, with fluorosilane, to generate hydrophobic regions. In some instances, the substrate is stripped to remove remaining resist. In some instances, the first modified layer of the substrate is activated to change one or more functional groups of the surface modification material to a hydroxyl group. In a subsequent step, the hydroxyl surface of the substrate is treated with a second layer of surface modification material to dilute surface functional groups and increase coupling efficiency for nucleotide attachment. In some instances, the second layer is activated to change one or more functional groups to a hydroxyl group to support oligonucleic acid synthesis. In some instances, steps comprising the addition of a surface modification material followed by subsequent activation are repeated for one or more additional cycles (e.g., 1, 2, 3, 4, 5 or more additional cycles) to provide optimal spacing between oligonucleic acids during synthesis. In some instances, second and subsequent layers have purely organic chemistries of the general form A-R1-B and C—R2-D where A reacts with the terminal OH group. The system is then purged and C—R2-D reacts with the B group. R1 and R2 chemistries can be repeated to yield the desired film thickness in a molecular layer deposition process. The terminal groups B and D can be hydroxyl or could be converted to hydroxyl in the final step of the deposition.
In some cases, the substrate may be two-dimensional (e.g., substantially planar) or three-dimension (e.g., comprise wells and/or channels). In some instances, an actively functionalized surface comprises a specific concentration of hydroxyl groups to achieve a pre-determined surface density for oligonucleic acid synthesis. In some instances, active functionalization is achieved by a wet process using a solution comprising an active functionalization agent. In some cases, the active functionalization agent comprises a silane or mixed silanes. In an example, a surface to be actively functionalized is treated with a solution comprising an active functionalization agent (e.g., 1% solution of N-(3-triethoxysilylpropyl-4hydroxybutyramide in ethanol and acetic acid) and the substrate incubated at a high temperature (e.g., 150° C. for 14 hours). In another example, a chemical vapor deposition process is employed wherein an active functionalization agent is delivered to the surface in a gaseous state. In some cases, an active functionalization agent is delivered by CVD with a controlled deposition pressure (e.g., 200 mTor) and temperature (e.g., 150° C.). A CVD process allows for in-situ plasma cleaning and is well suited for producing highly ordered self-assembled monolayers (SAMs).
Hydrophilic and Hydrophobic Surfaces
The surface energy, or hydrophobicity of a surface, can be evaluated or measured by measuring a water contact angle. Water contact angle is the angle between the drop surface and a solid surface where a water droplet meets the solid surface. A surface with a water contact angle of smaller than 90°, the solid surface can be considered hydrophilic or polar. A surface with a water contact angle of greater than 90°, the solid surface can be considered hydrophobic or apolar.
Surface characteristics of coated surfaces can be adjusted in various ways suitable for oligonucleotide synthesis. In some cases, the surface is selected to be inert to the conditions of ordinary oligonucleotide synthesis; e.g. the solid surface may be devoid of free hydroxyl, amino, or carboxyl groups to the bulk solvent interface during monomer addition, depending on the selected chemistry. In some cases, the surface may comprise reactive moieties prior to the start of a first cycle, or first few cycles of an oligonucleotide synthesis process, wherein the reactive moieties can be quickly depleted to unmeasurable densities after one, two, three, four, five, or more cycles of the oligonucleotide synthesis reaction. The surface can further be optimized for well or pore wetting, e.g., by common organic solvents such as acetonitrile and the glycol ethers or aqueous solvents, relative to surrounding surfaces.
Without being bound by theory, the wetting phenomenon is understood to be a measure of the surface tension or attractive forces between molecules at a solid-liquid interface, and is expressed in dynes/cm². For example, fluorocarbons have very low surface tension, which is typically attributed to the unique polarity (electronegativity) of the carbon-fluorine bond. In tightly structured Langmuir-Blodgett type films, surface tension of a layer can be primarily determined by the percent of fluorine in the terminus of the alkyl chains. For tightly ordered films, a single terminal trifluoromethyl group can render a surface nearly as lipophobic as a perfluoroalkyl layer. When fluorocarbons are covalently attached to an underlying derivatized solid (e.g. a highly crosslinked polymeric) support, the density of reactive sites can be lower than Langmuir-Blodgett and group density. For example, surface tension of a methyltrimethoxysilane surface can be about 22.5 mN/m and aminopropyltriethoxysilane surface can be about 35 mN/m. Briefly, hydrophilic behavior of surfaces is generally considered to occur when critical surface tensions are greater than 45 mN/m. As the critical surface tension increases, the expected decrease in contact angle is accompanied with stronger adsorptive behavior. Hydrophobic behavior of surfaces is generally considered to occur when critical surface tensions are less than 35 mN/m. At first, the decrease in critical surface tension is associated with oleophilic behavior, i.e. the wetting of the surfaces by hydrocarbon oils. As the critical surface tensions decrease below 20 mN/m, the surfaces resist wetting by hydrocarbon oils and are considered both oleophobic as well as hydrophobic. For example, silane surface modification can be used to generate a broad range of critical surface tensions. Accordingly, the methods and compositions of the invention may use surface coatings, e.g. those involving silanes, to achieve surface tensions of less than 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 115, 120 mN/m, or higher. Further, the methods and compositions of the invention may use surface coatings, e.g. those involving silanes, to achieve surface tensions of more than 115, 110, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6 mN/m or less. The water contact angle and the surface tension of non-limiting examples of surface coatings, e.g., those involving silanes, are described in Table 1 and Table 2 of Arkles et al. (Silanes and Other Coupling Agents, Vol. 5v: The Role of Polarity in the Structure of Silanes Employed in Surface Modification. 2009), which is incorporated herein by reference in its entirety. The tables are replicated below.

TABLE 1

Contact angles of water (degrees) on smooth surfaces

Heptadecafluorodecyltrimethoxysilane	113-115
Poly(tetrafluoroethylene)	108-112
Polypropylene	108
Octadecyldimethylchlorosilane	110
Octadecyltrichlorosilane	102-109
Tris(trimethylsiloxy)silylethyldimethylchlorosilane	103-104
Octyldimethylchlorosilane	104
Butyldimethylchlorosilane	100
Trimethylchlorosilane	90-100
Polyethylene	88-103
Polystyrene	94
Poly(chlorotrifluoroethylene)	90
Human skin	75-90
Diamond	87
Graphite	86
Silicon (etched)	86-88
Talc	82-90
Chitosan	80-81
Steel	70-75
Methoxyethoxyundecyltrichlorosilane	73-74
Methacryloxypropyltrimethoxysilane	70
Gold, typical (see gold, clean)	66
Intestinal mucosa	50-60
Kaolin	42-46
Platinum	40
Silicon nitride	28-30
Silver iodide	17
[Methoxy (polyethyleneoxy)propyl]trimethoxysilane	15-16
Sodalime glass	<15
Gold, clean	<10
Trimethoxysilylpropyl substituted	<10
poly(ethyleneimine), hydrochloride

TABLE 2

Critical surface tensions (mN/m)

	Heptadecafluorodecyltrichlorosilane	12
	Poly (tetrafluoroethylene)	18.5
	Octadecyltrichlorosilane	20-24
	Methyltrimethoxysilane	22.5
	Nonafluorohexyltrimethoxysilane	23
	Vinyltriethoxysilane	25
	Paraffin wax	25.5
	Ethyltrimethoxysilane	27.0
	Propyltrimethoxysilane	28.5
	Glass, sodalime (wet)	30.0
	Poly (chlorotrifluoroethylene)	31.0
	Polypropylene	31.0
	Poly (propylene oxide)	32
	Polyethylene	33.0
	Trifluoropropyltrimethoxysilane	33.5
	3-(2-Aminoethyl)aminopropyltrimethoxysilane	33.5
	Polystyrene	34
	p-Tolyltrimethoxysilane	34
	Cyanoethyltrimethoxysilane	34
	Aminopropyltriethoxysilane	35
	Acetoxy propyltrimethoxysilane	37.5
	Poly(methyl methacrylate)	39
	Poly(vinyl chloride)	39
	Phenyltrimethoxysilane	40.0
	Critical surface tensions (mN/m)
	Chloropropyltrimethoxysilane	40.5
	Mercaptopropyltrimethoxysilane	41
	Glycidoxypropyltrimethoxysilane	42.5
	Poly(ethylene terephthalate)	43
	Copper (dry)	44
	Poly(ethylene oxide)	43-45
	Aluminum (dry)	45
	Nylon 6/6	45-46
	Iron (dry)	46
	Glass, sodalime (dry)	47
	Titanium oxide (anatase)	91
	Ferric oxide	107
	Tin oxide	111

The surface of the substrate or a portion of the surface of the substrate can be functionalized or modified to be more hydrophilic or hydrophobic as compared to the surface or the portion of the surface prior to the functionalization or modification. In some cases, one or more surfaces can be modified to have a difference in water contact angle of greater than 90°, 85°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15° or 10° as measured on one or more uncurved, smooth or planar equivalent surfaces. In some cases, the surface of the microstructures, channels, resolved loci, resolved reactor caps or other parts of the substrate may be modified to have a differential hydrophobicity corresponding to a difference in water contact angle that is greater than 90°, 85°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15° or 10° as measured on uncurved, smooth or planar equivalent surfaces of such structures. Unless otherwise stated, water contact angles mentioned herein correspond to measurements that would be taken on uncurved, smooth or planar equivalents of the surfaces in question.
In some cases, hydrophilic resolved loci can be generated by first applying a protectant, or resist, over each loci within the substrate. The unprotected area can be then coated with a hydrophobic agent to yield an unreactive surface. For example, a hydrophobic coating can be created by chemical vapor deposition of (tridecafluorotetrahydrooctyl)-triethoxysilane onto the exposed oxide surrounding the protected channels or wells. Finally, the protectant, or resist, can be removed exposing the loci regions of the substrate for further modification and oligonucleotide synthesis. In some instances, the initial modification of such unprotected regions may resist further modification and retain their surface functionalization, while newly unprotected areas can be subjected to subsequent modification steps.
Substrate Etching
A process for carving features out of a substrate (e.g., a silicon plate) may include providing a substrate having a device layer and a handle layer, wherein the device layer is optionally separated from the handle layer by an electrical insulator layer, e.g., a layer of silicon dioxide—an exemplary substrate is a SOI wafer. In some instances, the provided substrate is oxidized on both surfaces. Photolithography may be applied to the device side of the substrate to create a mask of photoresist. In a subsequent step, a deep reactive-ion etching (DRIE) step may be used to etch vertical side-walls (e.g., until an insulator layer in a substrate comprising an insulator layer) at locations devoid of the photoresist. In a following step, the photoresist may be stripped. In some instances, photolithography, DRIE and photoresist strip steps are repeated on the substrate handle side. In cases wherein the substrate comprises an insulator layer such as silicon dioxide, buried oxide (BOX) may be removed using an etching process. Thermal oxidation can then be applied to remove contaminating polymers that may have been deposited on the side walls during the method. In a subsequent step, the thermal oxidation may be stripped using a wet etching process. In some instances, this method is used to generate a substrate having the features of a substrate exemplified in FIGS. 4A-4B. As another example, the process for manufacturing a substrate comprises a front-end process method comprising providing a starting material substrate, oxidizing both the device and handle sides; performing photolithography, DRIE and stripping of photoresist on the handle side; performing photolithography, DRIE and stripping of photoresist on the device side; removal of the oxide layer (e.g., BOX); and oxide growth (e.g., oxide is coated on one or more surfaces of the substrate to create a silicon substrate having a plurality of features).
In some instances, the substrate starting material comprises silicon. In some instances, the substrate is oxidized on one or more surfaces. In some instances, photolithography is applied to the front-side, back-side or both the front and back sides of the substrate, for example, to the back-side, to create a mask of photoresist. As a next step, the substrate is etched at locations devoid of photoresist, in many cases, beyond the oxidized layer, to create wells. As an example, the back-side is etched. In a subsequent step, the photoresist is stripped. In some instances, wherein photolithography was first applied to one side of the substrate, following photoresist stripping, a second side of the substrate is subjected to photolithography. For example, the back-side was first subjected to photolithography followed by photolithography of the front-side of the substrate. In some examples, a deep reactive-ion etching (DRIE) is used to etch vertical side walls to a prescribed depth, for example, about 450 um. In some cases, DRIE is used on the front-side, back-side or both the front and back sides during photolithography. In some instances, only one side of a substrate is etched to create three-dimensional features. In some instances, two sides, e.g., device and handle sides, of a substrate is etched to create three-dimensional features. In some processes, as an alternative or supplement to etching by DRIE, a SOI substrate (silicon on insulator silicon wafer) is used and the handle layer is etched down to the buried oxide, wherein the buried oxide can serve as an etch stop. Following photolithography on a second side of a substrate, the photoresist is stripped to generate a desired pattern. For example, the front-end is resist stripped to generate three dimensional features. In some cases, contaminates of the process (e.g., fluoropolymers) are removed by thermal oxidation followed by stripping of the thermal oxidation by a wet etching process. The substrate processed may comprise a plurality of wells, where the distance from the center of one main channel to the center of another main channel is about 1.69 mm and the total height of the main channel/microchannel is about 450 um.
Fiducial Marks
A substrate described herein may comprise fiducial marks to facilitate alignment with other components of a system. In some cases, substrates have one or more fiducial marks, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fiducial marks. A fiducial mark may be located near the origin, where the fiducial mark is closer to the origin than any one cluster. In some instances, a fiducial mark is located near an edge of the substrate portion. The fiducial mark may be located from about 0.1 mm to about 10 mm from the edge of the substrate portion, e.g., about 0.5 mm from the edge. The fiducial mark may be located close to or distant to a cluster. For example, a fiducial is located from about 1 mm to about 10 mm form a cluster, e.g., 1.69 mm. In some instances, a distance from the center of a fiducial mark and a nearest corner of a substrate in one dimension is from about 0.5 mm to about 10 mm, e.g., about 1 mm. In some instances, a length of a fiducial mark in one dimension is from about 0.5 mm to about 5 mm, e.g., about 1 mm. In some instances, the width of a fiducial mark is from about 0.01 mm to about 2 mm, e.g., 0.05 mm. The substrates described herein, in some instances, comprise one or more regions for annotation. In some instances, a substrate may have a label or serial number which is located a distance (e.g., 4 mm) from the edge of the substrate with a length (e.g., 9 mm) and width (e.g., 1.5 mm).
Oligonucleic Acid Synthesis
Structures having modified surfaces described herein may be used for de novo synthesis processes. An exemplary workflow for one such process is divided generally into phases: (1) de novo synthesis of a single stranded oligonucleic acid library, (2) joining oligonucleic acids to form larger fragments, (3) error correction, (4) quality control, and (5) shipment, FIG. 12 . Prior to de novo synthesis, an intended nucleic acid sequence or group of nucleic acid sequences is preselected. For example, a group of genes is preselected for generation.
Once preselected nucleic acids for generation are selected, a predetermined library of oligonucleic acids is designed for de novo synthesis. Various suitable methods are known for generating high density oligonucleic acid arrays. In the workflow example, a surface layer 1201 is provided. In the example, chemistry of the surface is altered in order to improve the oligonucleic acid synthesis process. Areas of low surface energy are generated to repel liquid while areas of high surface energy are generated to attract liquids. The surface itself may be in the form of a planar surface or contain variations in shape, such as protrusions, microwells, or microchannels which increase surface area. In the workflow example, high surface energy molecules selected serve a dual function of supporting DNA chemistry, as described in International Patent Application Publication WO/2015/021280, which is herein incorporated by reference in its entirety.
In situ preparation of oligonucleic acid arrays is generated on a solid support and utilizes single nucleotide extension process to extend multiple oligomers in parallel. A device, such as an oligonucleic acid synthesizer, is designed to release reagents in a step wise fashion such that multiple oligonucleic acids extend, in parallel, one residue at a time to generate oligomers with a predetermined nucleic acid sequence 1202. In some cases, oligonucleic acids are cleaved from the surface at this stage. Cleavage may include gas cleavage, e.g., with ammonia or methylamine.
The generated oligonucleic acid libraries are placed in a reaction chamber. In this exemplary workflow, the reaction chamber (also referred to as “nanoreactor”) is a silicon coated main channel, containing PCR reagents and lowered onto the oligonucleic acid library 1203. Prior to or after the sealing 1204 of the oligonucleic acids, a reagent is added to release the oligonucleic acids from the surface. In the exemplary workflow, the oligonucleic acids are released subsequent to sealing of the nanoreactor 1205. Once released, fragments of single stranded oligonucleic acids hybridize in order to span an entire long range sequence of DNA. Partial hybridization 1205 is possible because each synthesized oligonucleic acid is designed to have a small portion overlapping with at least one other oligonucleic acid in the pool.
After hybridization, a PCA reaction is commenced. During the polymerase cycles, the oligonucleic acids anneal to complementary fragments and gaps are filled in by a polymerase. Each cycle increases the length of various fragments randomly depending on which oligonucleic acids find each other. Complementarity amongst the fragments allows for forming a complete large span of double stranded DNA 1206.
After PCA is complete, the nanoreactor is separated from the surface 1207 and positioned for interaction with a polymerase 1208. After sealing, the nanoreactor is subject to PCR 1209 and the larger nucleic acids are formed. After PCR 1210, the nanochamber is opened 1211, error correction reagents are added 1212, the chamber is sealed 1213 and an error correction reaction occurs to remove mismatched base pairs and/or strands with poor complementarity from the double stranded PCR amplification products 1214. The nanoreactor is opened and separated 1215. Error corrected product is next subject to additional processing steps, such as PCR and molecular bar coding, and then packaged 1222 for shipment 1223.
In some cases, quality control measures are taken. After error correction, quality control steps include for example interaction with a wafer having sequencing primers for amplification of the error corrected product 1216, sealing the wafer to a chamber containing error corrected amplification product 1217, and performing an additional round of amplification 1218. The nanoreactor is opened 1219 and the products are pooled 1220 and sequenced 1221. After an acceptable quality control determination is made, the packaged product 1222 is approved for shipment 1223.
Oligonucleic acids may be synthesized on a substrate described herein using a system comprising an oligonucleic acid synthesizer that deposits reagents necessary for synthesis. Reagents for oligonucleic acid synthesis include, for example, reagents for oligonucleic acid extension and wash buffers. As non-limiting examples, the oligonucleic acid synthesizer deposits coupling reagents, capping reagents, oxidizers, de-blocking agents, acetonitrile and gases such as nitrogen gas. In addition, the oligonucleic acid synthesizer optionally deposits reagents for the preparation and/or maintenance of substrate integrity. An oligonucleic acid synthesizer may comprise material deposition devices that can move in the X-Y direction to align with the location of the substrate. The oligonucleic acid synthesizer can also move in the Z direction to seal with the substrate, forming a resolved reactor. In some instances, a substrate having a plurality of clusters is configured to seal with a capping element having a plurality of caps, wherein when the substrate and capping element are sealed, each cluster is separate from another cluster to form separate resolved reactors for each cluster. In some instances, the capping element is not present in the system or is present and stationary. A resolved reactor is configured to allow for the transfer of fluid, including oligonucleic acids and/or reagents, from the substrate to the capping element and/or vice versa. Fluid may pass through either or both the substrate and the capping element and includes, without limitation, coupling reagents, capping reagents, oxidizers, de-blocking agents, acetonitrile and nitrogen gas. The oligonucleic acid synthesizer of an oligonucleic acid synthesis system may comprise a plurality of material deposition devices, for example from about 1 to about 50 material deposition devices. Each material deposition device, in various instances, deposits a reagent component that is different from another material deposition device. In some cases, each material deposition device has a plurality of nozzles, where each nozzle is optionally configured to correspond to a cluster on a substrate. For example, for a substrate having 256 clusters, a material deposition device has 256 nozzles and 100 μm fly height. In some cases, each nozzle deposits a reagent component that is different from another nozzle.
The substrates described herein comprise actively functionalized surfaces configured to support the attachment and synthesis of oligonucleic acids. Synthesized oligonucleic acids include oligonucleic acids comprising modified and/or non-canonical bases and/or modified backbones. In various methods, a library of oligonucleic acids having pre-selected sequences is synthesized on a substrate. In some cases, one or more of the oligonucleic acids has a different sequence and/or length than another oligonucleic acid in the library. In some instances, the stoichiometry of each oligonucleic acid synthesized on a substrate is controlled and tunable by varying one or more features of the substrate (e.g., functionalized surface) and/or oligonucleic acid sequence to be synthesized; one or more methods for substrate functionalization and/or oligonucleic acid synthesis; or a combination thereof. In many instances, controlling the density of a growing oligonucleic acid on a resolved locus of a substrate allows for oligonucleic acids to be synthesized with a low error rate.
An example of a synthesis method that is useful with the substrates provided herein is one based on phosphoramidite chemistry. In some instances, oligonucleic acid synthesis methods comprise coupling a linker to a surface of a substrate, for example, to an actively functionalized surface of a substrate. In some instances, a linker separates a synthesized oligonucleic acid from a surface of the substrate. A linker includes a cleavable linker, such as a photocleavable linker. In some instances, a synthesized oligonucleic acid comprises a cleavable moiety that is introduced during synthesis. In some cases, a synthesized oligonucleic acid does not comprise a linker. For example, the synthesized oligonucleic acid is separated from the linker by one or more cleavable moieties. In some instances, the synthesized oligonucleic acid comprises a primer and/or adapter sequence that connects to a linker.
Without wishing to be bound by theory, the distance between extending oligonucleic acids is a factor correlating to error rate occurrence in synthesis of an oligonucleic acid library. One way to reduce the frequency of error is to minimize chain interaction during extension. Polymer “wobble” is controlled by altering the length of the tethering group at the base of the extending polymeric structure. In some instances, regulating “wobble” reduces error rate of polymer over the course of the synthesis process. In some instances, a linker comprises one or more bases coupled to the surface of a substrate and a cleavable moiety, wherein the cleavable moiety is configured to connect to the synthesized oligonucleic acid. In some cases, a linker is referred to as a tether or a tether region. In some instances, a linker comprises about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more bases located between a surface of a substrate and a synthesized oligonucleic acid. In some instances, a linker is synthesized and extends 12 to 25 bases from a device surface.
In some instances, a linker comprises a cleavable moiety, wherein the cleavable moiety is a modified or non-canonical base. When a plurality of synthesized oligonucleic acids of a library are connected to a substrate surface by a plurality of linkers having the same cleavable moiety, the cleavable moiety is referred to as a universal moiety. Examples of cleavable moieties include, without limitation, thymidine-succinyl hexamide CED phosphoramidite and DMMA. In some instances, a cleavable moiety is gas cleavable. In some instances, the linker comprises thymidine-succinyl hexamide CED phosphoramidite or DMMA. In some instances, the linker comprises a photocleavable primer. In an example, a photocleavable linker allows for the synthesized oligonucleic acid to be removed from the substrate without cleaving the protecting groups on the nitrogenous functionalities of each base, for example, by irradiation with light at about 350 nm.
Oligonucleic acids synthesized using the methods and/or substrates described herein comprise, in various instances, at least about 50, 60, 70, 75, 80, 90, 100, 120, 150, 200, 300, 400, 500, 600, 700, 800 or more bases. In some instances, a library of oligonucleic acids is synthesized, wherein a population of distinct oligonucleic acids are assembled to generate a larger nucleic acid comprising at least about 500; 1,000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000; 10,000; 11,000; 12,000; 13,000; 14,000; 15,000; 16,000; 17,000; 18,000; 19,000; 20,000; 25,000; 30,000; 40,000; or 50,000 bases. In some instances, oligonucleic acid synthesis methods described herein are useful for the generation of an oligonucleic acid library comprising at least 500; 1,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000; 1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000; 2,200,000; 2,400,000; 2,600,000; 2,800,000; 3,000,000; 3,500,000; 4,00,000; or 5,000,000 distinct oligonucleic acids. In some instances, at least about 1 μmol, 10 pmol, 20 pmol, 30 pmol, 40 pmol, 50 pmol, 60 pmol, 70 pmol, 80 pmol, 90 pmol, 100 pmol, 150 pmol, 200 pmol, 300 pmol, 400 pmol, 500 pmol, 600 pmol, 700 pmol, 800 pmol, 900 pmol, 1 nmol, 5 nmol, 10 nmol, 100 nmol or more of an oligonucleic acid is synthesized within a locus.
Methods for oligonucleic acid synthesis on a surface provided herein allow for synthesis at a fast rate. As an example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, 200 nucleotides per hour, or more are synthesized. Nucleotides include adenine, guanine, thymine, cytosine, uridine building blocks, or analogs/modified versions thereof. In some instances, libraries of oligonucleic acids are synthesized in parallel on substrate. For example, a substrate comprising about or at least about 100; 1,000; 10,000; 100,000; 1,000,000; 2,000,000; 3,000,000; 4,000,000; or 5,000,000 resolved loci is able to support the synthesis of at least the same number of distinct oligonucleic acids, wherein oligonucleic acid encoding a distinct sequence is synthesized on a resolved locus. In some instances, a library of oligonucleic acids are synthesized on a substrate with low error rates described herein in less than about three months, two months, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours, 18 hours, 12 hours or less. In some instances, larger nucleic acids assembled from an oligonucleic acid library synthesized with low error rate using the substrates and methods described herein are prepared in less than about three months, two months, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours, 18 hours, 12 hours or less. In some instances, up to about 800,000 distinct oligonucleic acids having sizes up to about 130 base pairs in length each are synthesized with an error rate below 1:1000, 1:2000, 1:3000 or less on a substrate described herein and using a method described herein in a span of less than about 24 hours.
In some instances, oligonucleic acid error rate is dependent on the efficiency of one or more chemical steps of oligonucleic acid synthesis. In some cases, oligonucleic acid synthesis comprises a phosphoramidite method, wherein a base of a growing oligonucleic acid chain is coupled to phosphoramidite. In some instances, coupling efficiency of the base is related to error rate. For example, higher coupling efficiency correlates to lower error rates. In some cases, the substrates and/or synthesis methods described herein allow for a coupling efficiency greater than 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99%. In some cases, an oligonucleic acid synthesis method comprises a double coupling process, wherein a base of a growing oligonucleic acid chain is coupled with a phosphoramidite, the oligonucleic acid is washed and dried, and then treated a second time with a phosphoramidite. In some instances, efficiency of deblocking in a phosphoramidite oligonucleic acid synthesis method contributes to error rate. In some cases, the substrates and/or synthesis methods described herein allow for removal of 5′-hydroxyl protecting groups at efficiencies greater than 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99%. In some instances, error rate is reduced by minimization of depurination side reactions.
Oligonucleic acids synthesized using the methods and/or substrates described herein encode for, in various instances, at least about 50, 60, 70, 75, 80, 90, 100, 120, 150, 200, 240, 300, 400, 500, 600, 700, 800, 900, 1,000, 6000, 6144, 10,000, or more genes. In some instances, a library of oligonucleic acids encode for at least 200 genes. In some instances, a library of oligonucleic acids encode for genes at least 500 bases, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb or more in length.
Methods for oligonucleic acid synthesis, in various instances, include processes involving phosphoramidite chemistry. In some instances, oligonucleic acid synthesis comprises coupling a base with phosphoramidite. In some instances, oligonucleic acid synthesis comprises coupling a base by deposition of phosphoramidite under coupling conditions, wherein the same base is optionally deposited with phosphoramidite more than once, i.e., double coupling. In some instances, oligonucleic acid synthesis comprises capping of unreacted sites. In some cases, capping is optional. In some instances, oligonucleic acid synthesis comprises oxidation. In some instances, oligonucleic acid synthesis comprises deblocking or detritylation. In some instances, oligonucleic acid synthesis comprises sulfurization. In some cases, oligonucleic acid synthesis comprises either oxidation or sulfurization. In some instances, between one or each step during an oligonucleic acid synthesis reaction, the substrate is washed, for example, using tetrazole or acetonitrile. Time frames for any one step in a phosphoramidite synthesis method include less than about 2 min, 1 min, 50 sec, 40 sec, 30 sec, 20 sec and 10 sec.
Oligonucleic acid synthesis using a phosphoramidite method comprises the subsequent addition of a phosphoramidite building block (e.g., nucleoside phosphoramidite) to a growing oligonucleic acid chain for the formation of a phosphite triester linkage. Phosphoramidite oligonucleic acid synthesis proceeds in the 3′ to 5′ direction. Phosphoramidite oligonucleic acid synthesis allows for the controlled addition of one nucleotide to a growing nucleic acid chain per synthesis cycle. In some instances, each synthesis cycle comprises a coupling step. Phosphoramidite coupling involves the formation of a phosphite triester linkage between an activated nucleoside phosphoramidite and a nucleoside bound to the substrate, for example, via a linker. In some instances, the nucleoside phosphoramidite is provided to the substrate activated. In some instances, the nucleoside phosphoramidite is provided to the substrate with an activator. In some instances, nucleoside phosphoramidites are provided to the substrate in a 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100-fold excess or more over the substrate-bound nucleosides. In some instances, the addition of nucleoside phosphoramidite is performed in an anhydrous environment, for example, in anhydrous acetonitrile. Following addition of a nucleoside phosphoramidite, the substrate is optionally washed. In some instances, the coupling step is repeated one or more additional times, optionally with a wash step between nucleoside phosphoramidite additions to the substrate. In some instances, an oligonucleic acid synthesis method used herein comprises 1, 2, 3 or more sequential coupling steps. Prior to coupling, in many cases, the nucleoside bound to the substrate is de-protected by removal of a protecting group, where the protecting group functions to prevent polymerization. A common protecting group is 4,4′-dimethoxytrityl (DMT).
Following coupling, phosphoramidite oligonucleic acid synthesis methods optionally comprise a capping step. In a capping step, the growing oligonucleic acid is treated with a capping agent. A capping step is useful to block unreacted substrate-bound 5′—OH groups after coupling from further chain elongation, preventing the formation of oligonucleic acids with internal base deletions. Further, phosphoramidites activated with 1H-tetrazole may react, to a small extent, with the O6 position of guanosine. Without being bound by theory, upon oxidation with I₂/water, this side product, possibly via O6-N7 migration, may undergo depurination. The apurinic sites may end up being cleaved in the course of the final deprotection of the oligonucleotide thus reducing the yield of the full-length product. The O6 modifications may be removed by treatment with the capping reagent prior to oxidation with I₂/water. In some instances, inclusion of a capping step during oligonucleic acid synthesis decreases the error rate as compared to synthesis without capping. As an example, the capping step comprises treating the substrate-bound oligonucleic acid with a mixture of acetic anhydride and 1-methylimidazole. Following a capping step, the substrate is optionally washed.
In some instances, following addition of a nucleoside phosphoramidite, and optionally after capping and one or more wash steps, the substrate bound growing nucleic acid is oxidized. The oxidation step comprises the phosphite triester is oxidized into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester internucleoside linkage. In some cases, oxidation of the growing oligonucleic acid is achieved by treatment with iodine and water, optionally in the presence of a weak base (e.g., pyridine, lutidine, collidine). Oxidation may be carried out under anhydrous conditions. Oxidation may be carried out using, for example, tert-Butyl hydroperoxide or (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, a capping step is performed following oxidation. A second capping step allows for substrate drying, as residual water from oxidation that may persist can inhibit subsequent coupling. Following oxidation, the substrate and growing oligonucleic acid is optionally washed. In some instances, the step of oxidation is substituted with a sulfurization step to obtain oligonucleotide phosphorothioates, wherein any capping steps can be performed after the sulfurization. Many reagents are capable of the efficient sulfur transfer, including but not limited to, 3-(dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT, 3H-1,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent, and N,N,N′N′-tetraethylthiuram disulfide (TETD).
In order for a subsequent cycle of nucleoside incorporation to occur through coupling, the protected 5′ end of the substrate bound growing oligonucleic acid must be removed so that the primary hydroxyl group can react with a next nucleoside phosphoramidite. In some instances, the protecting group is DMT and deblocking occurs with trichloroacetic acid in dichloromethane. Conducting detritylation for an extended time or with stronger than recommended solutions of acids may lead to increased depurination of solid support-bound oligonucleotide and thus reduces the yield of the desired full-length product. Methods and compositions of the invention described herein provide for controlled deblocking conditions limiting undesired depurination reactions. In some cases, the substrate bound oligonucleic acid is washed after deblocking. In some cases, efficient washing after deblocking contributes to synthesized oligonucleic acids having a low error rate.
Methods for the synthesis of oligonucleic acids typically involve an iterating sequence of the following steps: application of a protected monomer to an actively functionalized surface (e.g., locus) to link with either the activated surface, a linker or with a previously deprotected monomer; deprotection of the applied monomer so that it can react with a subsequently applied protected monomer; and application of another protected monomer for linking. One or more intermediate steps include oxidation or sulfurization. In some cases, one or more wash steps precede or follow one or all of the steps.
Methods for phosphoramidite based oligonucleic acid synthesis comprise a series of chemical steps. In some instances, one or more steps of a synthesis method involve reagent cycling, where one or more steps of the method comprise application to the substrate of a reagent useful for the step. For example, reagents are cycled by a series of liquid deposition and vacuum drying steps. For substrates comprising three-dimensional features such as wells, microwells, channels, microchannels and the like, reagents are optionally passed through one or more regions of the substrate via the wells and/or channels. In some instances, reagents are passed through the substrate during synthesis. In some cases, reagents are passed horizontally through the substrate. In some cases, reagents are passed vertically through the substrate. In some instances, reagents are passed over a substrate having curved features to enhance flow. In some cases, reagents are delivered to the substrate through the use of photoresist. In some instances, reagents are delivered to the substrate without moving the substrate. For example, reagents are passed over resolved loci within the substrate by flowing them through the substrate from one surface to an opposite surface of the substrate. In some instances, the substrate is moved, for example, to a flow cell, for reagent application, where it is then optionally repositioned. In an example, the substrate is deposited with a nucleoside using an oligonucleic acid synthesizer, moved to a flow cell for treating the substrate to one or more select reagents, and then repositioned back to the oligonucleic acid synthesizer for deposition of a subsequent monomer. Reagent delivery approaches suitable for the synthesis methods of the disclosure include manual and automatic, including use of robotic devices and pulse jets. Reagents include any component of an oligonucleic acid synthesis method, including chemical moieties such as nucleosides, washing solutions, and gases such as nitrogen.
In some instances, one or more reagents applied to the surface of a substrate during oligonucleic acid synthesis comprise a solvent. In some cases, a solvent comprises propylene carbonate. In some cases, a solvent comprises 2-methylglutaronitrile and/or 3-methoxypropionitrile. In some cases, a solvent comprises glutaronitrile. In some cases, a solvent comprises adiponitrile. In some instances, the solvent allows for high surface tension for reagent deposition. In some instances, the solvent allows for low surface tension for reagent deposition.
In some instances, the volume of reagents applied to a surface of substrate during oligonucleic acid synthesis is selected on the size, location and/or density of the surface to which the reagent is applied (e.g., an actively functionalized locus). In some instances, the volume of a drop of reagent applied to a surface during oligonucleic acid synthesis (e.g., deposition of a nucleoside) is less than about 0.5 picoliters (pL), 1 pL, 5 pL, 10 pL, 50 pL, 100 pL, 500 pL, 1000 pL, 5000 pL, 10000 pL, 100000 pL, 1000000 pL or 10000000 pL. In some instances, the reagents are delivered in droplets that have a total volume of about 47 pL or less. In some instances, the reagents are delivered in droplets that have a total volume of about 30 to 50 pL. In some instances, the reagents are delivered in droplets that have a total volume of about 50, 49, 48, 47, 46, 44, 45, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, or 25 pL. In some instances, the rate at which a drop of reagent is applied is at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 50 or 100 m/sec.
Oligonucleic acid synthesis methods include methods for the application of reagents during one or more steps during synthesis. Controlled application of reagents, such as nucleoside monomers to distinct regions of a substrate is important to achieve low error rates. In some instances, a reagent is deposited directly into a microchannel, with little or no contamination to an adjacent microchannel. In some cases, the volume of a reagent to be deposited within a three-dimensional feature such as a well or channel is adjusted to a small enough size to minimize cross-contamination. In some instances, the reagents are delivered in droplets that have a diameter of less than about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 um. A non-limiting method to reduce cross-contamination includes bringing the device that deposits the reagent sufficiently close to the surface such the deposited droplet falls substantially within the selected feature.
In some instances, efficient washing to remove unincorporated nucleosides contributes to low error rate. In some instances, the composition of the wash contributes to low error rate. As described herein, washing during oligonucleic acid synthesis includes one or all wash steps performed during oligonucleic acid synthesis. In some cases, a wash step is performed wherein at least or about 60%, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of unincorporated nucleosides or extension reaction reagent are removed from the surface of the substrate. In some cases, a wash step is performed wherein at least or about 95.0, 95.1, 95.2, 95.3, 95.4, 95.5, 95.6, 95.7, 95.8, 95.9, 96.0, 96.1, 96.2, 96.3, 96.4, 96.5, 96.6, 96.7, 96.8, 96.9, 97.0, 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.9, 98.0, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100% of unincorporated nucleosides or extension reaction reagent are removed from the surface of the substrate. In some cases, the configuration of a substrate such as one with narrow microchannels, contributes to wash efficiency and error rate. In some cases, substrates having channels are washed by passage of a wash solution through the substrate, minimizing fluid contact of the growing oligonucleic acids. In some cases, the geometry of fluid flow during washing controls interfacial instability. For example, a substrate that is substantial planar, or two-dimensional, may have a curved surface to enhance wash efficiency and therefore error rate. In some instances, optimized wash conditions include those that minimize contact time between the wash reagent and the growing oligonucleic acid. For example, the passage of a wash solution through three-dimensional features allows for the effect washing of all surfaces in a short period of time. A water contact angle for the substrate, in particular, for regions of synthesis and/or surrounding areas, may be chosen in order to reduce depurination and/or speed of synthesis. In some instances, lower amount of depurination may be achieved on surfaces of higher surface energy, i.e. lower contact angle. For example, depurination occurs at a rate less than 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, 0.0005%, or 0.0001%.
In some instances, the surface properties of the substrate change during oligonucleic acid synthesis. Typically, the substrate at the beginning of synthesis can be relatively hydrophobic and while synthesis proceeds, may become increasingly hydrophilic. Oligonucleic acid features can gain substantial surface energy with increasing oligonucleotide length. Generally, these sites or features consisting of protected oligonucleotide acquire enough surface energy to become spontaneously wet to high surface tension organic solvents commonly used in phosphoramidite synthesis, such as acetonitrile or propylene carbonate, after about 10-20 synthesis cycles. The methods and compositions described allow for varying parameters, such as time, flow rate, temperature, volume, viscosity, and/or reagent concentration, during the synthesis of a growing oligonucleic acid as a function of length to account for the changing surface properties on loci of the surface. Such a variation may be applied by changing parameters in constant or varying increments at repeating cycles of the synthesis. Alternatively, parameters may be changed at only selected cycles of the synthesis and can optionally follow a pattern, such as every other cycle, every third, fourth, fifth, sixth, seventh, eighth, ninth, tenth cycle etc.
Oligonucleic acid synthesis methods described herein are suitable for the spatial control of oligonucleic acid synthesis within a small area of a substrate, e.g., a locus. In some instances, oligonucleic acid methods comprise phosphoramidite chemistry. In some instances, spatial control of oligonucleic acid synthesis is achieved using an oligonucleic acid synthesizer. In some instances, spatial control of oligonucleic acid synthesis is achieved using physical masks. In some instances, spatial control of oligonucleic acid synthesis is achieved by modulation of a 5′ hydroxyl deblocking during phosphoramidite synthesis. In some instances, spatial control of oligonucleic acid synthesis is achieved by photolithographic deprotection of photolabile monomers. In some instances, spatial control of oligonucleic acid synthesis is achieved by digital activation of photogenerated acids to carry out standard detritylation.
In some instances, the surface of the substrate that provides support for oligonucleic acid synthesis is chemically modified to allow for the synthesized oligonucleic acid chain to be cleaved from the surface. In some cases, the oligonucleic acid chain is cleaved at the same time as the oligonucleic acid is deprotected. In some cases, the oligonucleic acid chain is cleaved after the oligonucleic acid is deprotected. In an exemplary scheme, a trialkoxysilyl amine (e.g., (CH₃CH₂O)₃Si—(CH₂)₂—NH₂) is reacted with surface SiOH groups of a substrate, followed by reaction with succinic anhydride with the amine to create and amide linkage and a free OH on which the nucleic acid chain growth is supported.
In some instances, oligonucleic acids are synthesized with photolabile protecting groups, where the hydroxyl groups generated on the surface are blocked by photolabile-protecting groups. When the surface is exposed to UV light, e.g., through a photolithographic mask, a pattern of free hydroxyl groups on the surface may be generated. These hydroxyl groups can react with photoprotected nucleoside phosphoramidites, according to phosphoramidite chemistry. A second photolithographic mask can be applied and the surface can be exposed to UV light to generate second pattern of hydroxyl groups, followed by coupling with 5′-photoprotected nucleoside phosphoramidite. Likewise, patterns can be generated and oligomer chains can be extended. Without being bound by theory, the lability of a photocleavable group depends on the wavelength and polarity of a solvent employed and the rate of photocleavage may be affected by the duration of exposure and the intensity of light. This method can leverage a number of factors, e.g., accuracy in alignment of the masks, efficiency of removal of photo-protecting groups, and the yields of the phosphoramidite coupling step. Further, unintended leakage of light into neighboring sites can be minimized. The density of synthesized oligomer per spot can be monitored by adjusting loading of the leader nucleoside on the surface of synthesis.
Oligonucleotide Libraries with Low Error Rates
The term “error rate” may also be referred to herein as a comparison of the collective sequence encoded by oligonucleic acids generated compared to the aggregate sequence of a predetermined longer nucleic acid, e.g., a gene. Oligonucleic acids are synthesized on a substrate described herein in a process that minimizes the error rate. For example, error rate is less than 1 in 500 bases, 1 in 1000 bases, 1 in 1500 bases, 1 in 2000 bases, 1 in 2500 bases, 1 in 3000, 1 in 5000 bases or less. In some instances, low error rates are achieved for synthesized oligonucleic acid libraries having at least 20,000; 40,000; 60,000; 80,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 1,000,000; or 2,000,000 or more oligonucleic acids. In some cases, a subset of oligonucleic acids in the library has the same sequence. In some cases, one or more of the oligonucleic acids in the library comprises a different sequence. Error rates include mismatch error rate, deletion error rate, insertion error rate, indel error rate, and any combination thereof.
In some instances, low overall error rate or low error rates for individual types of errors are achieved. Individual types of error rates include deletions, insertions, or substitutions for an oligonucleic acid library synthesized on the substrate. In some instances, oligonucleic acids synthesized on the substrate have an average error rate of about 1:500, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, 1:20000, 1:30000, 1:40000, 1:50000 or less. In some instances, these error rates are for at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or more of the oligonucleic acids synthesized. In some instances, these error rates are for 100% of the oligonucleic acids synthesized.
In some instances, oligonucleic acids synthesized on the substrate have an average deletion error rate of about 1:500, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, 1:20000, 1:30000, 1:40000, 1:50000 or less. In some instances, oligonucleic acids synthesized on the substrate have an average insertion error rate of about 1:500, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, 1:20000, 1:30000, 1:40000, 1:50000 or less. In some instances, oligonucleic acids synthesized on the substrate have an average substitution error rate of about 1:500, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, 1:20000, 1:30000, 1:40000, 1:50000 or less. In some instances, overall error rate or error rates for individual types of errors such as deletions, insertions, or substitutions for each oligonucleotide synthesized on the substrate, for at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or more of the oligonucleotides synthesized on the substrate, or the substrate average may fall between about 1:500 and 1:50000, 1:500 and 1:40000; 1:500 and 1:30000; 1:500 and 1:20000; 1:500 and 1:10000; 1:500 and 1:9000; 1:500 and 1:8000; 1:500 and 1:7000; 1:500 and 1:6000; or 1:500 and 1:5000.
In some instances, the methods and systems described herein for oligonucleic acid synthesis results in minimal synthesis of truncation products that are less than the full length of the predetermined oligonucleic acid sequence. In some cases, a library of oligonucleic acids are synthesized with less than 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, or 0.0001% comprising truncation products. In some instances, the methods and systems described herein for oligonucleic acid synthesis result in minimal synthesis of products that are greater than predetermined oligonucleic acid sequence length. In some cases, a library of oligonucleic acids are synthesized with less than 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, or 0.0001% comprising greater than predetermined sequence length.
The oligonucleic acids synthesized using the systems and methods described herein are optionally evaluated for sequence accuracy prior to subsequent applications, for example, larger nucleic acid assembly. A common method for oligonucleic acid quality control comprises next generation sequencing.
Oligonucleic Acid Release and Assembly
Oligonucleic acids synthesized using the methods and substrates described herein, are optionally released from the surface from which they were synthesized. In some cases, oligonucleic acids are cleaved from the surface at this stage. Cleavage may include gas cleavage, e.g., with ammonia or methylamine. In some instances, all the loci in a single cluster collectively correspond to sequence encoding for a single gene, and optionally, when cleaved, remain on the surface of the loci. In some instances, the application of ammonia gas simultaneous deprotects phosphates groups protected during the synthesis steps, i.e., removal of electron-withdrawing cyano group. In some instances, once released from the surface, oligonucleic acids are assembled into larger nucleic acids. Synthesized oligonucleic acids are useful, for example, as components for gene assembly/synthesis, site-directed mutagenesis, nucleic acid amplification, microarrays, and sequencing libraries.
In some instances, oligonucleic acids of predetermined sequence are designed to collectively span a large region of a target sequence, such as a gene. In some instances, larger oligonucleic acids are generated through ligation reactions to join the synthesized oligonucleic acids. One example of a ligation reaction is polymerase chain assembly (PCA). In some cases, at least of a portion of the oligonucleic acids are designed to include an appended region that is a substrate for universal primer binding. For PCA reactions, the presynthesized oligonucleic acids include overlaps with each other (e.g., 4, 20, 40 or more bases with overlapping sequence). During the polymerase cycles, the oligonucleic acids anneal to complementary fragments and then are filled in by polymerase. Each cycle thus increases the length of various fragments randomly depending on which oligonucleic acids find each other. Complementarity amongst the fragments allows for forming a complete large span of double stranded DNA. In some cases, after the PCA reaction is complete, an error correction step is conducted using mismatch repair detecting enzymes to remove mismatches in the sequence. Once larger fragments of a target sequence are generated, they can be amplified. For example, in some cases, a target sequence comprising 5′ and 3′ terminal adaptor sequences is amplified in a polymerase chain reaction (PCR) which includes modified primers, e.g., uracil containing primers the hybridize to the adaptor sequences. The use of modified primers allows for removal of the primers through enzymatic reactions centered on targeting the modified base and/or gaps left by enzymes which cleave the modified base pair from the fragment. What remains is a double stranded amplification product that lacks remnants of adapter sequence. In this way, multiple amplification products can be generated in parallel with the same set of primers to generate different fragments of double stranded DNA.
In some instances, error correction is performed on synthesized oligonucleic acids and/or assembled products. An example strategy for error correction involves site-directed mutagenesis by overlap extension PCR to correct errors, which is optionally coupled with two or more rounds of cloning and sequencing. In certain instances, double-stranded nucleic acids with mismatches, bulges and small loops, chemically altered bases and/or other heteroduplexes are selectively removed from populations of correctly synthesized nucleic acids by affinity purification. In some instances, error correction is performed using proteins/enzymes that recognize and bind to or next to mismatched or unpaired bases within double stranded nucleic acids to create a single or double strand break or to initiate a strand transfer transposition event. Non-limiting examples of proteins/enzymes for error correction include endonucleases (T7 Endonuclease I, E. coli Endonuclease V, T4 Endonuclease VII, mung bean nuclease, Cell, E. coli Endonuclease IV, UVDE), restriction enzymes, glycosylases, ribonucleases, mismatch repair enzymes, resolvases, helicases, ligases, antibodies specific for mismatches, and their variants. Examples of specific error correction enzymes include T4 endonuclease 7, T7 endonuclease 1, S1, mung bean endonuclease, MutY, MutS, MutH, MutL, cleavase, CELI, and HINF1. In some cases, DNA mismatch-binding protein MutS (Therms aquaticus) is used to remove failure products from a population of synthesized products. In some instances, error correction is performed using the enzyme Correctase. In some cases, error correction is performed using SURVEYOR endonuclease (Transgenomic), a mismatch-specific DNA endonuclease that scans for known and unknown mutations and polymorphisms for heteroduplex DNA.
In various instances, a synthesized oligonucleic acid as described herein is amplified in an amplification reaction. In various instances, a nucleic acid assembled from an oligonucleic acid synthesized by the methods and systems described herein is amplified in an amplification reaction. As used herein, at least in some instances, an amplification reaction includes any method known in the art to amplify one or more nucleic acids. Provided herein, in various cases, are instances exemplifying polymerase chain reaction (PCR) as an amplification reaction.
In some instances, an amplification reaction, such as PCR, is based on repeated cycles of denaturation, oligonucleic acid primer annealing, and primer extension by thermophilic template dependent polynucleotide polymerase, resulting in the exponential increase in copies of a target nucleic acid sequence flanked by the primers. The two different PCR primers, which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete double stranded fragment whose length is defined by the distance between the 5′ ends of the oligonucleic acid primers.
Systems for Oligonucleic Acid Synthesis
Provided herein are systems for the synthesis of oligonucleic acid libraries on a substrate. In some instances, the system comprises the substrate for synthesis support, as described elsewhere herein. In some instances, the system comprises a device for application of one or more reagents of a synthesis method, for example, an oligonucleic acid synthesizer. In some instances, the system comprises a device for treating the substrate with a fluid, for example, a flow cell. In some instances, the system comprises a device for moving the substrate between the application device and the treatment device.
In one aspect, provided is an automated system for use with an oligonucleic acid synthesis method described herein that is capable of processing one or more substrates, comprising: a material deposition device for spraying a microdroplet comprising a reagent on a substrate; a scanning transport for scanning the substrate adjacent to the material deposition device to selectively deposit the microdroplet at specified sites; a flow cell for treating the substrate on which the microdroplet is deposited by exposing the substrate to one or more selected fluids; an alignment unit for aligning the substrate correctly relative to the material deposition device each time when the substrate is positioned adjacent to the material deposition device for deposition. In some instances, the system optionally comprises a treating transport for moving the substrate between the material deposition device and the flow cell for treatment in the flow cell, where the treating transport and said scanning transport are different elements. In other instances, the system does not comprise a treating transport.
In some instances, a device for application of one or more reagents during a synthesis reagent is an oligonucleic acid synthesizer comprising a plurality of material deposition devices. In some instances, each material deposition device is configured to deposit nucleotide monomers, for example, for phosphoramidite synthesis. In some instances, the oligonucleic acid synthesizer deposits reagents to the resolved loci, wells, and/or microchannels of a substrate. In some cases, the oligonucleic acid synthesizer deposits a drop having a diameter less than about 200 um, 100 um, or 50 um in a volume less than about 1000, 500, 100, 50, or 20 pl. In some cases, the oligonucleic acid synthesizer deposits between about 1 and 10000, 1 and 5000, 100 and 5000, or 1000 and 5000 droplets per second. In some instances, the oligonucleic acid synthesizer uses organic solvents.
In some instances, during oligonucleic acid synthesis, the substrate is positioned within or sealed within a flow cell. In some instances, the flow cell provides continuous or discontinuous flow of liquids such as those comprising reagents necessary for reactions within the substrate, for example, oxidizers and/or solvents. In some instances, the flow cell provides continuous or discontinuous flow of a gas, such as nitrogen, for drying the substrate typically through enhanced evaporation of a volatile substrate. A variety of auxiliary devices are useful to improve drying and reduce residual moisture on the surface of the substrate. Examples of such auxiliary drying devices include, without limitation, a vacuum source, depressurizing pump and a vacuum tank. In some cases, an oligonucleic acid synthesis system comprises one or more flow cells, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 and one or more substrates, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20. In some cases, a flow cell is configured to hold and provide reagents to the substrate during one or more steps in a synthesis reaction. In some instances, a flowcell comprises a lid that slides over the top of a substrate and can be clamped into place to form a pressure tight seal around the edge of the substrate. An adequate seal, includes, without limitation, a seal that allows for about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 atmospheres of pressure. In some cases, the lid of the flow cell is opened to allow for access to an application device such as an oligonucleic acid synthesizer. In some cases, one or more steps of an oligonucleic acid synthesis method are performed on a substrate within a flow cell, without the transport of the substrate.
In some instances, during oligonucleic acid synthesis, a capping element seals with the substrate, to form a resolved reactor. In some instances, a substrate having a plurality of clusters is configured to seal with a capping element having a plurality of caps, wherein when the substrate and capping element are sealed, each cluster is separate from another cluster to form separate resolved reactors for each cluster. In some instances, the capping element is not present in the system or is present and stationary. A resolved reactor is configured to allow for the transfer of fluid, including oligonucleic acids and/or reagents, from the substrate to the capping element and/or vice versa. In some instances, reactors are interconnected or in fluid communication. Fluid communication of reactors allows for washing and perfusion of new reagents for different steps of a synthesis reaction. In some cases, the resolved reactors comprise inlets and/or outlets. In some cases, the inlets and/or outlets are configured for use with a flow cell. As an example, a substrate is sealed within a flow cell where reagents can be introduced and flowed through the substrate, after which the reagents are collected. In some cases, the substrate is drained of fluid and purged with an inert gas such as nitrogen. The flow cell chamber can then be vacuum dried to reduce residual liquids or moisture to less than 1%, 0.1%, 0.01%, 0.001%, 0.0001%, or 0.00001% by volume of the chamber. In some instances, a vacuum chuck is in fluid communication with the substrate for removing gas.
In some instances, an oligonucleic acid synthesis system comprises one or more elements useful for downstream processing of the synthesized oligonucleic acids. As an example, the system comprises a temperature control element such as a thermal cycling device. In some instances, the temperature control element is used with a plurality of resolved reactors to perform nucleic acid assembly such as PCA and/or nucleic acid amplification such as PCR.
Computer Systems
Any of the systems described herein, may be operably linked to a computer and may be automated through a computer either locally or remotely. In various instances, the methods and systems of the invention may further comprise software programs on computer systems and use thereof. Accordingly, computerized control for the synchronization of the dispense/vacuum/refill functions such as orchestrating and synchronizing the material deposition device movement, dispense action and vacuum actuation are within the bounds of the invention. The computer systems may be programmed to interface between the user specified base sequence and the position of a material deposition device to deliver the correct reagents to specified regions of the substrate.
The computer system 1300 illustrated in FIG. 13 may be understood as a logical apparatus that can read instructions from media 1311 and/or a network port 1305, which can optionally be connected to server 1309 having fixed media 1312. The system, such as shown in FIG. 13 can include a CPU 1301, disk drives 1303, optional input devices such as keyboard 1315 and/or mouse 1316 and optional monitor 1307. Data communication can be achieved through the indicated communication medium to a server at a local or a remote location. The communication medium can include any means of transmitting and/or receiving data. For example, the communication medium can be a network connection, a wireless connection or an internet connection. Such a connection can provide for communication over the World Wide Web. It is envisioned that data relating to the present disclosure can be transmitted over such networks or connections for reception and/or review by a party 1322 as illustrated in FIG. 13 .
FIG. 14 is a block diagram illustrating a first example architecture of a computer system 1400 that can be used in connection with example instances of the present invention. As depicted in FIG. 14 , the example computer system can include a processor 1402 for processing instructions. Non-limiting examples of processors include: Intel Xeon™ processor, AMD Opteron™ processor, Samsung 14-bit RISC ARM 1176JZ(F)-S v1.0™ processor, ARM Cortex-A8 Samsung S5PC100™ processor, ARM Cortex-A8 Apple A4™ processor, Marvell PXA 930™ processor, or a functionally-equivalent processor. Multiple threads of execution can be used for parallel processing. In some instances, multiple processors or processors with multiple cores can also be used, whether in a single computer system, in a cluster, or distributed across systems over a network comprising a plurality of computers, cell phones, and/or personal data assistant devices.
As illustrated in FIG. 14 , a high speed cache 1404 can be connected to, or incorporated in, the processor 1402 to provide a high speed memory for instructions or data that have been recently, or are frequently, used by processor 1402. The processor 1402 is connected to a north bridge 1406 by a processor bus 1408. The north bridge 1406 is connected to random access memory (RAM) 1410 by a memory bus 1412 and manages access to the RAM 1410 by the processor 1402. The north bridge 1406 is also connected to a south bridge 1414 by a chipset bus 1416. The south bridge 1414 is, in turn, connected to a peripheral bus 1418. The peripheral bus can be, for example, PCI, PCI-X, PCI Express, or other peripheral bus. The north bridge and south bridge are often referred to as a processor chipset and manage data transfer between the processor, RAM, and peripheral components on the peripheral bus 1418. In some alternative architectures, the functionality of the north bridge can be incorporated into the processor instead of using a separate north bridge chip. In some instances, system 1400 can include an accelerator card 1422 attached to the peripheral bus 1418. The accelerator can include field programmable gate arrays (FPGAs) or other hardware for accelerating certain processing. For example, an accelerator can be used for adaptive data restructuring or to evaluate algebraic expressions used in extended set processing.
Software and data are stored in external storage 1424 and can be loaded into RAM 1410 and/or cache 1404 for use by the processor. The system 1400 includes an operating system for managing system resources; non-limiting examples of operating systems include: Linux, Windows™, MACOS™, BlackBerry OS™, iOS™, and other functionally-equivalent operating systems, as well as application software running on top of the operating system for managing data storage and optimization in accordance with example instances of the present invention. In this example, system 1400 also includes network interface cards (NICs) 1420 and 1421 connected to the peripheral bus for providing network interfaces to external storage, such as Network Attached Storage (NAS) and other computer systems that can be used for distributed parallel processing.
FIG. 15 is a diagram showing a network 1500 with a plurality of computer systems 1502 a, and 1502 b, a plurality of cell phones and personal data assistants 1502 c, and Network Attached Storage (NAS) 1504 a, and 1504 b. In example instances, systems 1502 a, 1502 b, and 1502 c can manage data storage and optimize data access for data stored in Network Attached Storage (NAS) 1504 a and 1504 b. A mathematical model can be used for the data and be evaluated using distributed parallel processing across computer systems 1502 a, and 1502 b, and cell phone and personal data assistant systems 1502 c. Computer systems 1502 a, and 1502 b, and cell phone and personal data assistant systems 1502 c can also provide parallel processing for adaptive data restructuring of the data stored in Network Attached Storage (NAS) 1504 a and 1504 b. FIG. 15 illustrates an example only, and a wide variety of other computer architectures and systems can be used in conjunction with the various instances of the present invention. For example, a blade server can be used to provide parallel processing. Processor blades can be connected through a back plane to provide parallel processing. Storage can also be connected to the back plane or as Network Attached Storage (NAS) through a separate network interface.
In some example instances, processors can maintain separate memory spaces and transmit data through network interfaces, back plane or other connectors for parallel processing by other processors. In other instances, some or all of the processors can use a shared virtual address memory space.
FIG. 16 is a block diagram of a multiprocessor computer system 1600 using a shared virtual address memory space in accordance with an example embodiment. The system includes a plurality of processors 1602 a-f that can access a shared memory subsystem 1604. The system incorporates a plurality of programmable hardware memory algorithm processors (MAPs) 1606 a-f in the memory subsystem 1604. Each MAP 1606 a-f can comprise a memory 1608 a-f and one or more field programmable gate arrays (FPGAs) 1610 a-f The MAP provides a configurable functional unit and particular algorithms or portions of algorithms can be provided to the FPGAs 1610 a-f for processing in close coordination with a respective processor. For example, the MAPs can be used to evaluate algebraic expressions regarding the data model and to perform adaptive data restructuring in example instances. In this example, each MAP is globally accessible by all of the processors for these purposes. In one configuration, each MAP can use Direct Memory Access (DMA) to access an associated memory 1608 a-f, allowing it to execute tasks independently of, and asynchronously from the respective microprocessor 1602 a-f. In this configuration, a MAP can feed results directly to another MAP for pipelining and parallel execution of algorithms.
The above computer architectures and systems are examples only, and a wide variety of other computer, cell phone, and personal data assistant architectures and systems can be used in connection with example instances, including systems using any combination of general processors, co-processors, FPGAs and other programmable logic devices, system on chips (SOCs), application specific integrated circuits (ASICs), and other processing and logic elements. In some instances, all or part of the computer system can be implemented in software or hardware. Any variety of data storage media can be used in connection with example instances, including random access memory, hard drives, flash memory, tape drives, disk arrays, Network Attached Storage (NAS) and other local or distributed data storage devices and systems.
In example instances, the computer system can be implemented using software modules executing on any of the above or other computer architectures and systems. In other instances, the functions of the system can be implemented partially or completely in firmware, programmable logic devices such as field programmable gate arrays (FPGAs) as referenced in FIG. 16 , system on chips (SOCs), application specific integrated circuits (ASICs), or other processing and logic elements. For example, the Set Processor and Optimizer can be implemented with hardware acceleration through the use of a hardware accelerator card, such as accelerator card 1322 illustrated in FIG. 13 .
The following examples are set forth to illustrate more clearly the principle and practice of instances disclosed herein to those skilled in the art and are not to be construed as limiting the scope of any claimed instances. Unless otherwise stated, all parts and percentages are on a weight basis.

EXAMPLES

Example 1: Functionalization of a Substrate Surface

A substrate was functionalized to support the attachment and synthesis of a library of oligonucleic acids. The substrate surface was first wet cleaned using a piranha solution comprising 90% H₂SO₄and 10% H₂O₂for 20 minutes. The substrate was rinsed in several beakers with DI water, held under a DI water gooseneck faucet for 5 min, and dried with N₂. The substrate was subsequently soaked in NH₄OH (1:100; 3 mL:300 mL) for 5 min, rinsed with DI water using a handgun, soaked in three successive beakers with DI water for 1 min each, and then rinsed again with DI water using the handgun. The substrate was then plasma cleaned by exposing the substrate surface to O₂. A SAMCO PC-300 instrument was used to plasma etch O₂at 250 watts for 1 min in downstream mode.
The cleaned substrate surface was actively functionalized with a solution comprising N-(3-triethoxysilylpropyl)-4-hydroxybutyramide using a YES-1224P vapor deposition oven system with the following parameters: 0.5 to 1 Torr, 60 min, 70° C., 135° C. vaporizer.
The substrate surface was resist coated using a Brewer Science 200× spin coater. SPR™ 3612 photoresist was spin coated on the substrate at 2500 rpm for 40 sec. The substrate was pre-baked for 30 min at 90° C. on a Brewer hot plate. The substrate was subjected to photolithography using a Karl Suss MA6 mask aligner instrument. The substrate was exposed for 2.2 sec and developed for 1 min in MSF 26A. Remaining developer was rinsed with the handgun and the substrate soaked in water for 5 min. The substrate was baked for 30 min at 100° C. in the oven, followed by visual inspection for lithography defects using a Nikon L200. A plasma cleaning process was used to remove residual resist using the SAMCO PC-300 instrument to O₂plasma etch at 250 watts for 1 min.
The substrate surface was passively functionalized with a 100 μL solution of perfluorooctyltrichlorosilane mixed with 10 μL light mineral oil. The substrate was placed in a chamber, pumped for 10 min, and then the valve was closed to the pump and left to stand for 10 min. The chamber was vented to air. The substrate was resist stripped by performing two soaks for 5 min in 500 mL NMP at 70° C. with ultrasonication at maximum power (9 on Crest system). The substrate was then soaked for 5 min in 500 mL isopropanol at room temperature with ultrasonication at maximum power. The substrate was dipped in 300 mL of 200 proof ethanol and blown dry with N₂. The functionalized surface was activated to serve as a support for oligonucleic acid synthesis.

Example 2: Preparation of Substrates Having Distinct Loci Configurations

Substrates were manufactured to comprise a plurality of clusters each comprising a plurality of distinct loci configured to provide structural support for oligonucleic acid synthesis. Substrate starting material was a 200 mm standard, double-sided polished silicon wafer having a 725 um thickness. Substrates were processed by a method comprising thermal oxidation at 1000 Å, photolithography using a Karl Suss MA6 mask aligner to generate fiducial structures; oxide etching down to the silicon; and resist stripping. Prepared substrates have 6,144 clusters, with each cluster having 121 reaction sites or loci for oligonucleic acid synthesis. The clusters are organized into 24 sub-fields, which each comprise a 16×16 array of clusters. A schematic of a substrate produced is shown in FIGS. 1-3 . As shown in FIG. 1 , the substrate has a dimension of 140.000 mm by 90.000 mm. As shown in FIG. 2 , the vertical distance between the centers of two adjacent clusters in one substrate is 1079.210 um and in another substrate 1142.694 um. The horizontal distance between the centers of two adjacent clusters in the substrate is 1125.0 um. An expanded view of a cluster of the substrate is shown in FIG. 3 . Each cluster has 121 loci, which are separated so that the horizontal distance between two adjacent loci is 75.000 um and the vertical distance between two loci is 63.483 um. The horizontal distance between the edges of two adjacent loci is 24.0 um.

Example 3: Maximization of Microchannel Surface Area

Substrates manufactured in this example were processed to generate three-dimensional loci having shapes configured to increase surface area to volume. Examples of locus shapes prepared using the methods described in this example are shown in FIG. 5 . A Silicon on Insulator (SOI) silicon wafer (sub-field size of 32.00×32.00 mm) was oxidized, and the device side processed by photolithography, deep RIE and photoresist stripping. The handle side of the substrate was processed by photolithography, deep RIE, photoresist stripping, and etching by removal of oxide layer (BOX etch). The processed substrate has a plurality of wells or holes within the handle layer, each having a width of 1.150 mm, wherein each channel has a plurality of microchannels having shapes that allow for an increase in surface area to volume. The smallest etch size for a feature of a shape of a microchannel within a substrate prepared in this example was 5 um. The distance between the centers of two adjacent clusters (wells) is 1.693 mm in all directions. The distance between the centers of two adjacent loci (microchannels) is 97.765 in a horizontal direction and 84.667 um in a vertical direction. The prepared substrate has a set of markings or fiducials of 0.5 mm diameter. The width of the main channel is 1.150 mm and the width of the microchannel is 5 um. Detailed features of substrates prepared using these methods are shown in FIGS. 5-8 .
A cluster of a processed substrate having double comb shaped loci is shown in a bird's eye view in FIG. 6 . The combined height of the two longest teeth is 57 um. The distance between two teeth of the comb is 14.0 um. The width of the comb handle is 5 um. The combined height of the two shortest teeth is 38.0 um. The width of the comb in the horizontal direction is 47.0 urn.
A cluster of a processed substrate having single comb shaped loci is shown in a bird's eye view in FIG. 7 . The height of the longest tooth is 49.0 um. The distance between two teeth of the comb is 14.0 um. The width of the comb handle is 5 um. The height of the shortest tooth is 39.0 um. The width of the comb in the horizontal direction is 47.0 um.
A cluster of a processed substrate having serpentine shaped loci is shown in a bird's eye view in FIG. 8 . The height of the loci shape is 54 um. The distance between two lines of the shape is 14 um. The width of a line of the shape is 5 um.
Detailed measurements for prepared substrates are shown in Table 3.

TABLE 3

Device depth (um)	30
Width of segments (um)	5

			Top surface		Total
		Internal	growth		projected	Total	Total
		area	area	Volume	area	Area	Volume
Segments	No.	(um²)	(um²)	(um³)	(um²)	(um²)	(um³)

Double comb
Lateral segments
	3	540	45	1350	135	1620	4050
End segments	2	1890	170	5100	340	3780	10200
Middle segments	2	2850	263	7875	525	5700	15750
Rounded ends	4	471	20	589	79	1885	2356
				Total	1079	12985	32356
Total growth area						15225
including top surface (um²)
Single comb
Lateral segments	1	2456	210	7050	210	2456	7050
End segments	2	2070	173	5175	345	4140	10350
Middle segments	2	2490	208	6225	415	4980	12450
Rounded ends	2	471	20	589	39	942	1178
				Total	1009	12518	31028
Total growth area						14827
including top surface (um²)
Serpentine
Vertical segments
	4	2100	175	5250	700	8400	21000
Annulus segments (whole)	1.5	2639	220	6597	330	3958	9896
Rounded ends	1	471	20	589	20	471	589
				Total	1050	12830	31485
Total growth area						15098
including top surface (um²)

The surface and volume parameters of loci having high surface area shapes (double comb, single comb, and serpentine) prepared using these methods were compared with the parameters of a locus having a revolver shape (barrel comprising 5 channels). The comparison is shown in Table 4. The loci having a comb or serpentine shape had a lower substrate volume than a substrate having revolver loci. The loci having a comb or serpentine shape had a greater surface area than a substrate having revolver loci. The loci having a comb or serpentine shape had a greater surface area to volume ratio than a substrate having revolver loci.

TABLE 4

	Revolver	Double comb	Single come	Serpentine

Total volume	47	32	31	31
(pL)
Total surface	9425	12985	12518	12830
area (um)
Surface area to	0.20	0.40	0.40	0.41
volume ratio
(1/um)
Total volume	1.00	0.69	0.66	0.67
relative to
revolver
Total surface	1.00	1.38	1.33	1.36
area relative to
revolver
Surface area to	1.00	2.01	2.02	2.04
volume ratio
relative revolver

Example 4: Synthesis of a 100-Mer Oligonucleic Acid on a Substantially Planar Substrate

A substantially planar substrate functionalized for oligonucleic acid synthesis was assembled into a flow cell and connected to an Applied Biosystems ABI394 DNA Synthesizer. In one experiment, the substrate was uniformly functionalized with N-(3-triethoxysilylpropyl)-4-hydroxybutyramide. In another experiment, the substrate was functionalized with a 5/95 mix of 11-acetoxyundecyltriethoxysilane and N-decyltriethoxysilane. Synthesis of 100-mer oligonucleic acids (“100-mer oligonucleotide”; 5′: CGGGATCCTTATCGTCATCGTCGTACAGATCCCGACCCATTTGCTGTCCACCAGT CATGCTAGCCATACCATGATGATGATGATGATGAGAACCCCGCAT##TTTTTTTTT T 3′ (SEQ ID NO: 1), where #denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 from ChemGenes)) were performed using the methods of Table 5.

TABLE 5

General DNA Synthesis		Time
Process Name	Process Step	(sec)

WASH (Acetonitrile Wash	Acetonitrile System Flush	4
Flow)	Acetonitrile to Flowcell	23
	N2 System Flush	4
	Acetonitrile System Flush	4
DNA BASE ADDITION	Activator Manifold Flush	2
(Phosphoramidite +	Activator to Flowcell	6
Activator Flow)	Activator +	6
	Phosphoramidite to
	Flowcell
	Activator to Flowcell	0.5
	Activator +	5
	Phosphoramidite to
	Flowcell
	Activator to Flowcell	0.5
	Activator +	5
	Phosphoramidite to
	Flowcell
	Activator to Flowcell	0.5
	Activator +	5
	Phosphoramidite to
	Flowcell
	Incubate for 25 sec	25
WASH (Acetonitrile Wash	Acetonitrile System Flush	4
Flow)	Acetonitrile to Flowcell	15
	N2 System Flush	4
	Acetonitrile System Flush	4
DNA BASE ADDITION	Activator Manifold Flush	2
(Phosphoramidite +	Activator to Flowcell	5
Activator Flow)	Activator +	18
	Phosphoramidite to
	Flowcell
	Incubate for 25 sec	25
WASH (Acetonitrile Wash	Acetonitrile System Flush	4
Flow)	Acetonitrile to Flowcell	15
	N2 System Flush	4
	Acetonitrile System Flush	4
CAPPING (CapA + B, 1:1,	CapA + B to Flowcell	15
Flow)
WASH (Acetonitrile Wash	Acetonitrile System Flush	4
Flow)	Acetonitrile to Flowcell	15
	Acetonitrile System Flush	4
OXIDATION (Oxidizer	Oxidizer to Flowcell	18
Flow)
WASH (Acetonitrile Wash	Acetonitrile System Flush	4
Flow)	N2 System Flush	4
	Acetonitrile System Flush	4
	Acetonitrile to Flowcell	15
	Acetonitrile System Flush	4
	Acetonitrile to Flowcell	15
	N2 System Flush	4
	Acetonitrile System Flush	4
	Acetonitrile to Flowcell	23
	N2 System Flush	4
	Acetonitrile System Flush	4
DEBLOCKING (Deblock	Deblock to Flowcell	36
Flow)
WASH (Acetonitrile Wash	Acetonitrile System Flush	4
Flow)	N2 System Flush	4
	Acetonitrile System Flush	4
	Acetonitrile to Flowcell	18
	N2 System Flush	4.13
	Acetonitrile System Flush	4.13
	Acetonitrile to Flowcell	15

Synthesized oligonucleic acids were extracted from the substrate surface and analyzed on a BioAnalyzer chip. Oligonucleic acid products were PCR amplified, cloned and Sanger sequenced. Table 6 summarizes the Sanger sequencing results for samples taken from spots 1-5 from one chip and spots 6-10 from a second chip.

TABLE 6

Spot	Error rate	Cycle efficiency

1	1/763 bp	99.87%
2	1/824 bp	99.88%
3	1/780 bp	99.87%
4	1/429 bp	99.77%
5	1/1525 bp	99.93%
6	1/1615 bp	99.94%
7	1/531 bp	99.81%
8	1/1769 bp	99.94%
9	1/854 bp	99.88%
10	1/1451 bp	99.93%

Overall, 89% (233/262) of the 100-mers that were sequenced had sequences without errors. Table 7 summarizes key error characteristics for the sequences obtained from the oligonucleic acid samples from spots 1-10.

TABLE 7

Sample ID/	OSA_	OSA_	OSA_	OSA_	OSA_	OSA_	OSA_	OSA_	OSA_	OSA_
Spot No.	0046/1	0047/2	0048/3	0049/4	0050/5	0051/6	0052/7	0053/8	0054/9	0055/10

Total	32	32	32	32	32	32	32	32	32	32
Sequences
Sequencing	25 of	27 of	26 of	21 of	25 of	29 of	27 of	29 of	28 of	25 of
Quality	28	27	30	23	26	30	31	31	29	28
Oligo	23 of	25 of	22 of	18 of	24 of	25 of	22 of	28 of	26 of	20 of
Quality	25	27	26	21	25	29	27	29	28	25
ROI Match	2500	2698	2561	2122	2499	2666	2625	2899	2798	2348
Count
ROI	2	2	1	3	1	0	2	1	2	1
Mutation
ROI Multi	0	0	0	0	0	0	0	0	0	0
Base
Deletion
ROI Small	1	0	0	0	0	0	0	0	0	0
Insertion
ROI Single	0	0	0	0	0	0	0	0	0	0
Base
Deletion
Large	0	0	1	0	0	1	1	0	0	0
Deletion
Count
Mutation:	2	2	1	2	1	0	2	1	2	1
G > A
Mutation:	0	0	0	1	0	0	0	0	0	0
T > C
ROI Error	3	2	2	3	1	1	3	1	2	1
Count
ROI Error	Err:~1	Err:~1	Err:~1	Err:~1	Err:~1	Err:~1	Err:~1	Err:~1	Err:~1	Err:~1
Rate	in 834	in 1350	in 1282	in 708	in 2500	in 2667	in 876	in 2900	in 1400	in 2349
ROI Minus	MP	MP	MP	MP	MP	MP	MP	MP	MP	MP
Primer	Err:~1	Err:~1	Err:~1	Err:~1	Err:~1	Err:~1	Err:~1	Err:~1	Err:~1	Err:~1
Error Rate	in 763	in 824	in 780	in 429	in 1525	in 1615	in 531	in 1769	in 854	in 1451

Example 5: Gene Assembly in Reactors Using PCA

Gene assembly within nanoreactors created using a three-dimensional substrate was performed. PCA reactions were performed using oligonucleic acids described in Table 8 (SEQ ID NOS: 2-61) to assemble the 3075 base LacZ gene (SEQ ID NO.: 62) using the reaction mixture of Table 9 within individual nanoreactors.

	TABLE 8

	Sequence Name	Sequence

	Oligo_1,	5′ATGACCATGATTACGGATTCACTGGCCG
	SEQ ID NO.: 2	TCGTTTTACAACGTCGTGACTGGGAAAACC
		CTGG3′

	Oligo_2,	5′GCCAGCTGGCGAAAGGGGGATGTGCTGC
	SEQ ID NO.: 3	AAGGCGATTAAGTTGGGTAACGCCAGGGTT
		TTCCCAGTCACGAC3′

	Oligo_3,	5′CCCCCTTTCGCCAGCTGGCGTAATAGCG
	SEQ ID NO.: 4	AAGAGGCCCGCACCGATCGCCCTTCCCAAC
		AGTTGCGCAGCC3′

	Oligo_4,	5′CGGCACCGCTTCTGGTGCCGGAAACCAG
	SEQ ID NO.: 5	GCAAAGCGCCATTCGCCATTCAGGCTGCGC
		AACTGTTGGGA3′

	Oligo_5,	5′CACCAGAAGCGGTGCCGGAAAGCTGGCT
	SEQ ID NO.: 6	GGAGTGCGATCTTCCTGAGGCCGATACTGT
		CGTCGTCCCCTC3′

	Oligo_6,	5′GATAGGTCACGTTGGTGTAGATGGGCGC
	SEQ ID NO.: 7	ATCGTAACCGTGCATCTGCCAGTTTGAGGG
		GACGACGACAGTATCGG3′

	Oligo_7,	5′CCCATCTACACCAACGTGACCTATCCCA
	SEQ ID NO.: 8	TTACGGTCAATCCGCCGTTTGTTCCCACGG
		AGAATCCGACGGGTTG3′

	Oligo_8,	5′GTCTGGCCTTCCTGTAGCCAGCTTTCAT
	SEQ ID NO.: 9	CAACATTAAATGTGAGCGAGTAACAACCCG
		TCGGATTCTCCGTG3′

	Oligo_9,	5′GCTGGCTACAGGAAGGCCAGACGCGAAT
	SEQ ID NO.: 10	TATTTTTGATGGCGTTAACTCGGCGTTTCA
		TCTGTGGTGCAACGG3′

	Oligo_10,	5′CAGGTCAAATTCAGACGGCAAACGACTG
	SEQ ID NO.: 11	TCCTGGCCGTAACCGACCCAGCGCCCGTTG
		CACCACAGATGAAACG3′

	Oligo_11,	5′CGTTTGCCGTCTGAATTTGACCTGAGCG
	SEQ ID NO.: 12	CATTTTTACGCGCCGGAGAAAACCGCCTCG
		CGGTGATGGTGCTG3′

	Oligo_12,	5′GCCGCTCATCCGCCACATATCCTGATCT
	SEQ ID NO.: 13	TCCAGATAACTGCCGTCACTCCAGCGCAGC
		ACCATCACCGCGAG3′

	Oligo_13,	5′AGGATATGTGGCGGATGAGCGGCATTTT
	SEQ ID NO.: 14	CCGTGACGTCTCGTTGCTGCATAAACCGAC
		TACACAAATCAGCGATTTC3′

	Oligo_14,	5′CTCCAGTACAGCGCGGCTGAAATCATCA
	SEQ ID NO.: 15	TTAAAGCGAGTGGCAACATGGAAATCGCTG
		ATTTGTGTAGTCGGTTTATG3′

	Oligo_15,	5′ATTTCAGCCGCGCTGTACTGGAGGCTGA
	SEQ ID NO.: 16	AGTTCAGATGTGCGGCGAGTTGCGTGACTA
		CCTACGGGTAACAGTTT3

	Oligo_16,	5′AAAGGCGCGGTGCCGCTGGCGACCTGCG
	SEQ ID NO.: 17	TTTCACCCTGCCATAAAGAAACTGTTACCC
		GTAGGTAGTCACG3′

	Oligo_17,	5′GCGGCACCGCGCCTTTCGGCGGTGAAAT
	SEQ ID NO.: 18	TATCGATGAGCGTGGTGGTTATGCCGATCG
		CGTCACACTACG3′

	Oligo_18,	5′GATAGAGATTCGGGATTTCGGCGCTCCA
	SEQ ID NO.: 19	CAGTTTCGGGTTTTCGACGTTCAGACGTAG
		TGTGACGCGATCGGCA3′

	Oligo_19,	5′GAGCGCCGAAATCCCGAATCTCTATCGT
	SEQ ID NO.: 20	GCGGTGGTTGAACTGCACACCGCCGACGGC
		ACGCTGATTGAAGCAG3′

	Oligo_20,	5′CAGCAGCAGACCATTTTCAATCCGCACC
	SEQ ID NO.: 21	TCGCGGAAACCGACATCGCAGGCTTCTGCT
		TCAATCAGCGTGCCG3′

	Oligo_21,	5′CGGATTGAAAATGGTCTGCTGCTGCTGA
	SEQ ID NO.: 22	ACGGCAAGCCGTTGCTGATTCGAGGCGTTA
		ACCGTCACGAGCATCA3′

	Oligo_22,	5′GCAGGATATCCTGCACCATCGTCTGCTC
	SEQ ID NO.: 23	ATCCATGACCTGACCATGCAGAGGATGATG
		CTCGTGACGGTTAACGC3′

	Oligo_23,	5′CAGACGATGGTGCAGGATATCCTGCTGA
	SEQ ID NO.: 24	TGAAGCAGAACAACTTTAACGCCGTGCGCT
		GTTCGCATTATCCGAAC3′

	Oligo_24,	5′TCCACCACATACAGGCCGTAGCGGTCGC
	SEQ ID NO.: 25	ACAGCGTGTACCACAGCGGATGGTTCGGAT
		AATGCGAACAGCGCAC3′

	Oligo25,	5′GCTACGGCCTGTATGTGGTGGATGAAGC
	SEQ ID NO.:	CAATATTGAAACCCACGGCATGGTGCCAAT
	26	GAATCGTCTGACCGATG3′

	Oligo_26,	5′GCACCATTCGCGTTACGCGTTCGCTCAT
	SEQ ID NO.: 27	CGCCGGTAGCCAGCGCGGATCATCGGTCAG
		ACGATTCATTGGCAC3′

	Oligo_27,	5′CGCGTAACGCGAATGGTGCAGCGCGATC
	SEQ ID NO.: 28	GTAATCACCCGAGTGTGATCATCTGGTCGC
		TGGGGAATGAATCAG3′

	Oligo_28,	5′GGATCGACAGATTTGATCCAGCGATACA
	SEQ ID NO.: 29	GCGCGTCGTGATTAGCGCCGTGGCCTGATT
		CATTCCCCAGCGACCAGATG3′

	Oligo_29,	5′GTATCGCTGGATCAAATCTGTCGATCCT
	SEQ ID NO.: 30	TCCCGCCCGGTGCAGTATGAAGGCGGCGGA
		GCCGACACCACGGC3′

	Oligo_30,	5′CGGGAAGGGCTGGTCTTCATCCACGCGC
	SEQ ID NO.: 31	GCGTACATCGGGCAAATAATATCGGTGGCC
		GTGGTGTCGGCTC3′

	Oligo_31,	5′TGGATGAAGACCAGCCCTTCCCGGCTGT
	SEQ ID NO.: 32	GCCGAAATGGTCCATCAAAAAATGGCTTTC
		GCTACCTGGAGAGAC3′

	Oligo_32,	5′CCAAGACTGTTACCCATCGCGTGGGCGT
	SEQ ID NO.: 33	ATTCGCAAAGGATCAGCGGGCGCGTCTCTC
		CAGGTAGCGAAAGCC3′

	Oligo_33,	5′CGCGATGGGTAACAGTCTTGGCGGTTTC
	SEQ ID NO.: 34	GCTAAATACTGGCAGGCGTTTCGTCAGTAT
		CCCCGTTTACAGGGC3′

	Oligo_34,	5′GCCGTTTTCATCATATTTAATCAGCGAC
	SEQ ID NO.: 35	TGATCCACCCAGTCCCAGACGAAGCCGCCC
		TGTAAACGGGGATACTGACG3′

	Oligo_35,	5′CAGTCGCTGATTAAATATGATGAAAACG
	SEQ ID NO.: 36	GCAACCCGTGGTCGGCTTACGGCGGTGATT
		TTGGCGATACGCCGAACG3′

	Oligo_36,	5′GCGGCGTGCGGTCGGCAAAGACCAGACC
	SEQ ID NO.: 31	GTTCATACAGAACTGGCGATCGTTCGGCGT
		ATCGCCAAA3′

	Oligo_37,	5′CGACCGCACGCCGCATCCAGCGCTGACG
	SEQ ID NO.: 38	GAAGCAAAACACCAGCAGCAGTTTTTCCAG
		TTCCGTTTATCCG3′

	Oligo_38,	5′CTCGTTATCGCTATGACGGAACAGGTAT
	SEQ ID NO.: 39	TCGCTGGTCACTTCGATGGTTTGCCCGGAT
		AAACGGAACTGGAAAAACTGC3′

	Oligo_39,	5′AATACCTGTTCCGTCATAGCGATAACGA
	SEQ ID NO.: 40	GCTCCTGCACTGGATGGTGGCGCTGGATGG
		TAAGCCGCTGGCAAGCG3′

	Oligo_40,	5′GTTCAGGCAGTTCAATCAACTGTTTACC
	SEQ ID NO.: 41	TTGTGGAGCGACATCCAGAGGCACTTCACC
		GCTTGCCAGCGGCTTACC3’

	Oligo_41,	5′CAAGGTAAACAGTTGATTGAACTGCCTG
	SEQ ID NO.: 42	AACTACCGCAGCCGGAGAGCGCCGGGCAAC
		TCTGGCTCACAGTACGCGTA3′

	Oligo_42,	5′GCGCTGATGTGCCCGGCTTCTGACCATG
	SEQ ID NO.: 43	CGGTCGCGTTCGGTTGCACTACGCGTACTG
		TGAGCCAGAGTTG3′

	Oligo_43,	5′CCGGGCACATCAGCGCCTGGCAGCAGTG
	SEQ ID NO.: 44	GCGTCTGGCGGAAAACCTCAGTGTGACGCT
		CCCCGCCGC3′

	Oligo_44,	5′CCAGCTCGATGCAAAAATCCATTTCGCT
	SEQ ID NO.: 45	GGTGGTCAGATGCGGGATGGCGTGGGACGC
		GGCGGGGAGCGTC3′

	Oligo_45,	5′CGAAATGGATTTTTGCATCGAGCTGGGT
	SEQ ID NO.: 46	AATAAGCGTTGGCAATTTAACCGCCAGTCA
		GGCTTTCTTTCACAGATGTG3′

	Oligo_46,	5′TGAACTGATCGCGCAGCGGCGTCAGCAG
	SEQ ID NO.: 47	TTGTTTTTTATCGCCAATCCACATCTGTGA
		AAGAAAGCCTGACTGG3′

	Oligo_47,	5′GCCGCTGCGCGATCAGTTCACCCGTGCA
	SEQ ID NO.: 48	CCGCTGGATAACGACATTGGCGTAAGTGAA
		GCGACCCGCATTGAC3′

	Oligo_48,	5′GGCCTGGTAATGGCCCGCCGCCTTCCAG
	SEQ ID NO.: 49	CGTTCGACCCAGGCGTTAGGGTCAATGCGG
		GTCGCTTCACTTA3′

	Oligo_49,	5′CGGGCCATTACCAGGCCGAAGCAGCGTT
	SEQ ID NO.: 50	GTTGCAGTGCACGGCAGATACACTTGCTGA
		TGCGGTGCTGAT3′

	Oligo_50,	5′TCCGGCTGATAAATAAGGTTTTCCCCTG
	SEQ ID NO.: 51	ATGCTGCCACGCGTGAGCGGTCGTAATCAG
		CACCGCATCAGCAAGTG3′

	Oligo_51,	5′GGGGAAAACCTTATTTATCAGCCGGAAA
	SEQ ID NO.: 52	ACCTACCGGATTGATGGTAGTGGTCAAATG
		GCGATTACCGTTGATGTTGA3′

	Oligo_52,	5′GGCAGTTCAGGCCAATCCGCGCCGGATG
	SEQ ID NO.: 53	CGGTGTATCGCTCGCCACTTCAACATCAAC
		GGTAATCGCCATTTGAC3′

	Oligo_53,	5′GCGGATTGGCCTGAACTGCCAGCTGGCG
	SEQ ID NO.: 54	CAGGTAGCAGAGCGGGTAAACTGGCTCGGA
		TTAGGGCCGCAAG3′

	Oligo_54,	5′GGCAGATCCCAGCGGTCAAAACAGGCGG
	SEQ ID NO.: 55	CAGTAAGGCGGTCGGGATAGTTTTCTTGCG
		GCCCTAATCCGAGC3′

	Oligo55,	5′GTTTTGACCGCTGGGATCTGCCATTGTC
	SEQ ID NO.: 56	AGACATGTATACCCCGTACGTCTTCCCGAG
		CGAAAACGGTCTGC3′

	Oligo_56,	5′GTCGCCGCGCCACTGGTGTGGGCCATAA
	SEQ ID NO.: 57	TTCAATTCGCGCGTCCCGCAGCGCAGACCG
		TTTTCGCTCGG3′

	Oligo_57,	5′ACCAGTGGCGCGGCGACTTCCAGTTCAA
	SEQ ID NO.: 58	CATCAGCCGCTACAGTCAACAGCAACTGAT
		GGAAACCAGCCATC3′

	Oligo_58,	5′GAAACCGTCGATATTCAGCCATGTGCCT
	SEQ ID NO.: 59	TCTTCCGCGTGCAGCAGATGGCGATGGCTG
		GTTTCCATCAGTTGCTG3′

	Oligo_59,	5′CATGGCTGAATATCGACGGTTTCCATAT
	SEQ ID NO.: 60	GGGGATTGGTGGCGACGACTCCTGGAGCCC
		GTCAGTATCGGCG3′

	Oligo_60,	5′TTATTTTTGACACCAGACCAACTGGTAA
	SEQ ID NO.: 61	TGGTAGCGACCGGCGCTCAGCTGGAATTCC
		GCCGATACTGACGGGC3′

	LacZ gene-	5′ATGACCATGATTACGGATTCACTGGCCG
	SEQ ID NO: 62	TCGTTTTACAACGTCGTGACTGGGAAAACC
		CTGGCGTTACCCAACTTAATCGCCTTGCAG
		CACATCCCCCTTTCGCCAGCTGGCGTAATA
		GCGAAGAGGCCCGCACCGATCGCCCTTCCC
		AACAGTTGCGCAGCCTGAATGGCGAATGGC
		GCTTTGCCTGGTTTCCGGCACCAGAAGCGG
		TGCCGGAAAGCTGGCTGGAGTGCGATCTTC
		CTGAGGCCGATACTGTCGTCGTCCCCTCAA
		ACTGGCAGATGCACGGTTACGATGCGCCCA
		TCTACACCAACGTGACCTATCCCATTACGG
		TCAATCCGCCGTTTGTTCCCACGGAGAATC
		CGACGGGTTGTTACTCGCTCACATTTAATG
		TTGATGAAAGCTGGCTACAGGAAGGCCAGA
		CGCGAATTATTTTTGATGGCGTTAACTCGG
		CGTTTCATCTGTGGTGCAACGGGCGCTGGG
		TCGGTTACGGCCAGGACAGTCGTTTGCCGT
		CTGAATTTGACCTGAGCGCATTTTTACGCG
		CCGGAGAAAACCGCCTCGCGGTGATGGTGC
		TGCGCTGGAGTGACGGCAGTTATCTGGAAG
		ATCAGGATATGTGGCGGATGAGCGGCATTT
		TCCGTGACGTCTCGTTGCTGCATAAACCGA
		CTACACAAATCAGCGATTTCCATGTTGCCA
		CTCGCTTTAATGATGATTTCAGCCGCGCTG
		TACTGGAGGCTGAAGTTCAGATGTGCGGCG
		AGTTGCGTGACTACCTACGGGTAACAGTTT
		CTTTATGGCAGGGTGAAACGCAGGTCGCCA
		GCGGCACCGCGCCTTTCGGCGGTGAAATTA
		TCGATGAGCGTGGTGGTTATGCCGATCGCG
		TCACACTACGTCTGAACGTCGAAAACCCGA
		AACTGTGGAGCGCCGAAATCCCGAATCTCT
		ATCGTGCGGTGGTTGAACTGCACACCGCCG
		ACGGCACGCTGATTGAAGCAGAAGCCTGCG
		ATGTCGGTTTCCGCGAGGTGCGGATTGAAA
		ATGGTCTGCTGCTGCTGAACGGCAAGCCGT
		TGCTGATTCGAGGCGTTAACCGTCACGAGC
		ATCATCCTCTGCATGGTCAGGTCATGGATG
		AGCAGACGATGGTGCAGGATATCCTGCTGA
		TGAAGCAGAACAACTTTAACGCCGTGCGCT
		GTTCGCATTATCCGAACCATCCGCTGTGGT
		ACACGCTGTGCGACCGCTACGGCCTGTATG
		TGGTGGATGAAGCCAATATTGAAACCCACG
		GCATGGTGCCAATGAATCGTCTGACCGATG
		ATCCGCGCTGGCTACCGGCGATGAGCGAAC
		GCGTAACGCGAATGGTGCAGCGCGATCGTA
		ATCACCCGAGTGTGATCATCTGGTCGCTGG
		GGAATGAATCAGGCCACGGCGCTAATCACG
		ACGCGCTGTATCGCTGGATCAAATCTGTCG
		ATCCTTCCCGCCCGGTGCAGTATGAAGGCG
		GCGGAGCCGACACCACGGCCACCGATATTA
		TTTGCCCGATGTACGCGCGCGTGGATGAAG
		ACCAGCCCTTCCCGGCTGTGCCGAAATGGT
		CCATCAAAAAATGGCTTTCGCTACCTGGAG
		AGACGCGCCCGCTGATCCTTTGCGAATACG
		CCCACGCGATGGGTAACAGTCTTGGCGGTT
		TCGCTAAATACTGGCAGGCGTTTCGTCAGT
		ATCCCCGTTTACAGGGCGGCTTCGTCTGGG
		ACTGGGTGGATCAGTCGCTGATTAAATATG
		ATGAAAACGGCAACCCGTGGTCGGCTTACG
		GCGGTGATTTTGGCGATACGCCGAACGATC
		GCCAGTTCTGTATGAACGGTCTGGTCTTTG
		CCGACCGCACGCCGCATCCAGCGCTGACGG
		AAGCAAAACACCAGCAGCAGTTTTTCCAGT
		TCCGTTTATCCGGGCAAACCATCGAAGTGA
		CCAGCGAATACCTGTTCCGTCATAGCGATA
		ACGAGCTCCTGCACTGGATGGTGGCGCTGG
		ATGGTAAGCCGCTGGCAAGCGGTGAAGTGC
		CTCTGGATGTCGCTCCACAAGGTAAACAGT
		TGATTGAACTGCCTGAACTACCGCAGCCGG
		AGAGCGCCGGGCAACTCTGGCTCACAGTAC
		GCGTAGTGCAACCGAACGCGACCGCATGGT
		CAGAAGCCGGGCACATCAGCGCCTGGCAGC
		AGTGGCGTCTGGCGGAAAACCTCAGTGTGA
		CGCTCCCCGCCGCGTCCCACGCCATCCCGC
		ATCTGACCACCAGCGAAATGGATTTTTGCA
		TCGAGCTGGGTAATAAGCGTTGGCAATTTA
		ACCGCCAGTCAGGCTTTCTTTCACAGATGT
		GGATTGGCGATAAAAAACAACTGCTGACGC
		CGCTGCGCGATCAGTTCACCCGTGCACCGC
		TGGATAACGACATTGGCGTAAGTGAAGCGA
		CCCGCATTGACCCTAACGCCTGGGTCGAAC
		GCTGGAAGGCGGCGGGCCATTACCAGGCCG
		AAGCAGCGTTGTTGCAGTGCACGGCAGATA
		CACTTGCTGATGCGGTGCTGATTACGACCG
		CTCACGCGTGGCAGCATCAGGGGAAAACCT
		TATTTATCAGCCGGAAAACCTACCGGATTG
		ATGGTAGTGGTCAAATGGCGATTACCGTTG
		ATGTTGAAGTGGCGAGCGATACACCGCATC
		CGGCGCGGATTGGCCTGAACTGCCAGCTGG
		CGCAGGTAGCAGAGCGGGTAAACTGGCTCG
		GATTAGGGCCGCAAGAAAACTATCCCGACC
		GCCTTACTGCCGCCTGTTTTGACCGCTGGG
		ATCTGCCATTGTCAGACATGTATACCCCGT
		ACGTCTTCCCGAGCGAAAACGGTCTGCGCT
		GCGGGACGCGCGAATTGAATTATGGCCCAC
		ACCAGTGGCGCGGCGACTTCCAGTTCAACA
		TCAGCCGCTACAGTCAACAGCAACTGATGG
		AAACCAGCCATCGCCATCTGCTGCACGCGG
		AAGAAGGCACATGGCTGAATATCGACGGTT
		TCCATATGGGGATTGGTGGCGACGACTCCT
		GGAGCCCGTCAGTATCGGCGGAATTCCAGC
		TGAGCGCCGGTCGCTACCATTACCAGTTGG
		TCTGGTGTCAAAAATAA3′

TABLE 9

PCA reaction mixture	1 (×100 ul)	Final conc.

	H₂O	62.00
	5x Q5 buffer	20.00	1x
	10 mM dNTP	1.00	100 uM
	BSA
20 mg/ml	5.00	1 mg/ml
	Oligonucleic acid mix (50 nM each)	10.00	5 nM
	Q5 polymerase 2U/ul	2.00	2 U/50 ul

PCA reaction mixture drops of about 400 nL were dispensed using a Mantis dispenser (Formulatrix, MA) on the top of channels of a device side of a three-dimensional substrate having a plurality of loci microchannels in fluid communication with a single main channel of a cluster. A nanoreactor chip was manually mated with the substrate to pick up the droplets having the PCA reaction mixture and oligonucleic acids from each channel. The droplets were picked up into individual nanoreactors in the nanoreactor chip by releasing the nanoreactor from the substrate immediately after pick up. The nanoreactors were sealed with a heat sealing film, placed in a thermocycler for PCA. PCA thermocycling conditions are shown in Table 10. An aliquot of 0.5 ul was collected from 1-10 individual wells and the aliquots were amplified in plastic PCR tubes using forward primer (5′ATGACCATGATTACGGATTCACTGGCC3′ SEQ ID NO.: 63) and reverse primer (5′TTATTTTTGACACCAGACCAACTGGTAATGG3′ SEQ ID NO.: 64). Thermocycling conditions for PCR are shown in Table 11 and PCR reaction components are shown in Table 12. The amplification products were ran on a BioAnalyzer instrument and on a gel. The gel showed products 1-10 having a size slightly larger than 3000 bp (data not shown). A PCA reaction performed in plastic tube was also ran on the gel as a positive control (panel 11), which shows a product having a similar size to the products from wells 1-10. A negative control (panel 12) was also run on the gel, which corresponds to a PCR reaction ran without a PCA template. The BioAnalyzer data is not shown.

TABLE 10

No. of cycles	Temperature (° C.)	Time

1	98	45 seconds
40	98	15 seconds
	63	45 seconds
	72	60 seconds
1	72	5 minutes
1	4	Hold

TABLE 11

No. of cycles	Temperature (° C.)	Time

1	98	30 seconds
30	98	7 seconds
	63	30 seconds
	72	90 seconds
1	72	5 minutes
1	4	Hold

TABLE 12

PCR	1 (×25 ul)	Final conc.

	H2O	17.50
	5x Q5 buffer	5.00	1x
	10 mM dNTP	0.50	200 uM
	F-primer 20 uM	0.63	0.5 uM
	R-primer 20 uM	0.63	0.5 uM
	BSA
20 mg/ml	0.00
	Q5 pol 2U/ul	0.25	1U/50 ul
	Template (PCA assembly)	0.50	1 ul/50 ul rxn

Example 6: Error Correction of Assembled Nucleic Acids

A gene of about 1 kbp (SEQ ID.: 67; Table 13) was assembled using 6 purchased Ultramer oligonucleotides (SEQ ID NO.: 68-73; Table 13) and assembled in a PCA reaction. Ultramer oligonucleotides are expected to have error rates of at least 1 in 500 to 1 in 200 nucleotides. The assembled gene was amplified by PCR using a forward primer (5′ ATGACCATGATTACGGATTCACTGGCC3′ SEQ ID NO.: 65) and a reverse primer (5′GATAGAGATTCGGGATTTCGGCGCTCC3′ SEQ ID NO.: 66). The amplified assembled gene was analyzed in a BioAnalyzer and cloned. DNA preparations from 24 colonies were Sanger sequenced. The BioAnalyzer analysis provided a broad peak and a tail for the uncorrected gene, indicating a high error rate. The sequencing indicated an error rate of 1/789 bases. Two rounds of error correction were followed using CorectASE (Life Technologies) according to the manufacturer's instructions. The resulting gene samples were analyzed in a BioAnalyzer after round one and round two and cloned. Twenty-four colonies were picked for sequencing. The sequencing results indicated an error rate of 1/5190 bases and 1/6315 bases after the first and second rounds of error correction, respectively.

	TABLE 13

	Nucleic Acid	Sequence

	Assembled	5′ATGACCATGATTACGGATTCACTGGCCG
	Gene, SEQ ID	TCGTTTTACAACGTCGTGACTGGGAAAACC
	NO.: 67	CTGGCGTTACCCAACTTAATCGCCTTGCAG
		CACATCCCCCTTTCGCCAGCTGGCGTAATA
		GCGAAGAGGCCCGCACCGATCGCCCTTCCC
		AACAGTTGCGCAGCCTGAATGGCGAATGGC
		GCTTTGCCTGGTTTCCGGCACCAGAAGCGG
		TGCCGGAAAGCTGGCTGGAGTGCGATCTTC
		CTGAGGCCGATACTGTCGTCGTCCCCTCAA
		ACTGGCAGATGCACGGTTACGATGCGCCCA
		TCTACACCAACGTGACCTATCCCATTACGG
		TCAATCCGCCGTTTGTTCCCACGGAGAATC
		CGACGGGTTGTTACTCGCTCACATTTAATG
		TTGATGAAAGCTGGCTACAGGAAGGCCAGA
		CGCGAATTATTTTTGATGGCGTTAACTCGG
		CGTTTCATCTGTGGTGCAACGGGCGCTGGG
		TCGGTTACGGCCAGGACAGTCGTTTGCCGT
		CTGAATTTGACCTGAGCGCATTTTTACGCG
		CCGGAGAAAACCGCCTCGCGGTGATGGTGC
		TGCGCTGGAGTGACGGCAGTTATCTGGAAG
		ATCAGGATATGTGGCGGATGAGCGGCATTT
		TCCGTGACGTCTCGTTGCTGCATAAACCGA
		CTACACAAATCAGCGATTTCCATGTTGCCA
		CTCGCTTTAATGATGATTTCAGCCGCGCTG
		TACTGGAGGCTGAAGTTCAGATGTGCGGCG
		AGTTGCGTGACTACCTACGGGTAACAGTTT
		CTTTATGGCAGGGTGAAACGCAGGTCGCCA
		GCGGCACCGCGCCTTTCGGCGGTGAAATTA
		TCGATGAGCGTGGTGGTTATGCCGATCGCG
		TCACACTACGTCTGAACGTCGAAAACCCGA
		AACTGTGGAGCGCCGAAATCCCGAATCTCT
		ATC3′

	Assembly
	5′ATGACCATGATTACGGATTCACTGGCCG
	Oligonucleotide	TCGTTTTACAACGTCGTGACTGGGAAAACC

	1, SEQ ID	CTGGCGTTACCCAACTTAATCGCCTTGCAG
	NO.: 68	CACATCCCCCTTTCGCCAGCTGGCGTAATA
		GCGAAGAGGCCCGCACCGATCGCCCTTCCC
		AACAGTTGCGCAGCC
3′

	Assembly	5′GATAGGTCACGTTGGTGTAGATGGGCGC
	Oligonucleotide	ATCGTAACCGTGCATCTGCCAGTTTGAGGG

	2, SEQ ID	GACGACGACAGTATCGGCCTCAGGAAGATC
	NO.: 69	GCACTCCAGCCAGCTTTCCGGCACCGCTTC
		TGGTGCCGGAAACCAGGCAAAGCGCCATTC
		GCCATTCAGGCTGCGCAACTGTTGGGA3′

	Assembly
	5′CCCATCTACACCAACGTGACCTATCCCA
	Oligonucleotide	TTACGGTCAATCCGCCGTTTGTTCCCACGG

	3, SEQ ID	AGAATCCGACGGGTTGTTACTCGCTCACAT
	NO.: 70	TTAATGTTGATGAAAGCTGGCTACAGGAAG
		GCCAGACGCGAATTATTTTTGATGGCGTTA
		ACTCGGCGTTTCATCTGTGGTGCAACGG3′

	Assembly
	5′GCCGCTCATCCGCCACATATCCTGATCT
	Oligonucleotide	TCCAGATAACTGCCGTCACTCCAGCGCAGC

	4, SEQ ID	ACCATCACCGCGAGGCGGTTTTCTCCGGCG
	NO.: 71	CGTAAAAATGCGCTCAGGTCAAATTCAGAC
		GGCAAACGACTGTCCTGGCCGTAACCGACC
		CAGCGCCCGTTGCACCACAGATGAAACG3′

	Assembly
	5′AGGATATGTGGCGGATGAGCGGCATTTT
	Oligonucleotide	CCGTGACGTCTCGTTGCTGCATAAACCGAC

	5, SEQ ID	TACACAAATCAGCGATTTCCATGTTGCCAC
	NO.: 72	TCGCTTTAATGATGATTTCAGCCGCGCTGT
		ACTGGAGGCTGAAGTTCAGATGTGCGGCGA
		GTTGCGTGACTACCTACGGGTAACAGTTT

		3′

	Assembly	5′GATAGAGATTCGGGATTTCGGCGCTCCA
	Oligonucleotide	CAGTTTCGGGTTTTCGACGTTCAGACGTAG
	6, SEQ ID	TGTGACGCGATCGGCATAACCACCACGCTC
	NO.: 73	ATCGATAATTTCACCGCCGAAAGGCGCGGT
		GCCGCTGGCGACCTGCGTTTCACCCTGCCA
		TAAAGAAACTGTTACCCGTAGGTAGTCACG

		3′

Example 7: Parallel Assembly of 240 Genes on Flat Plate

A substrate 1700 comprising 256 clusters each comprising 121 loci on a flat silicon plate was manufactured as shown in FIG. 17 . An expanded view of a cluster is shown in 1705 with 121 loci. Loci from 240 of the 256 clusters provided an attachment and support for the synthesis of oligonucleic acids having distinct sequences. Oligonucleic acid synthesis was performed by phosphoramidite chemistry using general methods from Table 5 in Example 4. Loci from 16 of the 256 clusters were control clusters. The distribution of the 29,040 unique oligonucleic acids synthesized (240×121) is shown in FIG. 18 . The distribution of unique oligonucleic acids synthesized in 4 representative clusters is shown in FIG. 19 . The error rate for each oligonucleic acid was determined using an Illumina MiSeq gene sequencer. The error rate distribution for the 29,040 unique oligonucleic acids is shown in FIG. 20 and averages around 1 in 500 bases, with some error rates as low as 1 in 800 bases. The error rate distribution for unique oligonucleic acids in four representative clusters is shown in FIG. 21 . The library of 29,040 unique oligonucleic acids was synthesized in less than 20 hours.
Oligonucleic acids synthesized within the loci of a cluster had overlapping sequences with one another so that when all oligonucleic acids synthesized within one cluster are pooled, they are ready for assembly into a larger nucleic acid or gene using PCA. Oligonucleic acids within a cluster were pooled and assembled using PCA reaction conditions similar to those described in Example 5. The 240 unique genes were synthesized in 3 business days, however, with 24 hour a day operation, the 240 unique genes could be synthesized in less time. The assembled genes from each of the 240 clusters were sequenced using an Illumina MiSeq gene sequencer. The read counts for the assembled genes are represented in FIG. 22 . The assembled gene products were visualized on a DNA gel as shown in FIGS. 23-26 . The genes synthesized ranged in size from 838 base pairs to 1470 base pairs. All 240 gene products were generated with their expected size. An output from the sequencing run is shown in FIG. 27 .

Example 8: Oligonucleic Acid Library Synthesis with Low Error Rate

A substrate comprising three-dimensional features with high surface area loci was manufactured according to the methods similar to that of Example 3. Each locus was manufactured to have a single comb shape. The substrate has a plurality of clusters corresponding to a plurality of wells, wherein each channel is 1.150 mm in diameter and includes 109 loci in the form of microchannels. FIG. 10C provides a depiction for a collection of microchannels/loci extending from a main channel. Microchannels of the substrate were functionalized and used as an attachment and support for the synthesis of distinct oligonucleic acids. A library of oligonucleic acids was synthesized on the substrate and subsequently gas cleaved from the surface for sequence analysis using an Illumina MiSeq.
Error mismatch rates for oligonucleic acids synthesized within each cluster were calculated (data not shown). Error rates were from 1 in 7,000 bases to as high as 1 in 100 bases. The average mismatch error rate was 1 in 586.82 bases.
Error deletion rates for oligonucleic acids synthesized within each cluster were calculated (data not shown). Error rates were from around 1 in 1,000 bases to around 1 in 2,000 bases. The average deletion error rate was 1 in 1,966.86 bases.
Insertion rates for oligonucleic acids synthesized within each cluster were calculated (data not shown). Error rates were from 1 in 2,700 bases to around 1 in 10,000 bases. The average insertion error rate was 1 in 4,740.03 bases.
The percentage of perfect oligonucleic acids synthesized (a perfect sequence being 100% identical to a preselected nucleic acid sequence) within each cluster were calculated (data not shown). The percentage of perfect sequences ranged from about 70% to about 93.54% perfect sequences. Overall more than 90% of the oligonucleic acids had perfect sequences.

Example 9: Linker Length Analysis

An oligonucleic acid library was synthesized as described in Example 8. Each oligonucleic acid synthesized on a locus of a cluster was tethered to the locus by a linker. Error rate as a function of base distance from substrate surface was analyzed and graphed as depicted in FIG. 28 . The lowest error rates correspond to oligonucleic acids with tether between about 12 and 25 bases from the surface.

Example 10: Parallel Synthesis of Distinct Oligonucleic Acids

Oligonucleic acids of various sequences and lengths were synthesized by phosphoramidite chemistry using methods as generally described in Table 5 of Example 4. Oligonucleic acids having lengths from 25 bases to 200 bases were synthesized within different clusters of a substrate. The synthesized oligonucleic acids were released from the surface, collected, and visualized by gel electrophoresis. FIG. 29 provides a captured image of the electrophoresis gel loaded with representative synthesized oligonucleic acids having lengths of 25, 50, 75, 100, 125, 150, 175 and 200 nucleotides.
As exemplified in FIG. 29 , the methods and substrates described herein allow for the simultaneous synthesis of a plurality of oligonucleic acids each having different sequences and, in some cases, different sequence lengths. In particular, oligonucleic acids having 200 bases were synthesized on, and removed from a substrate. These synthesized oligonucleic acids were released from the substrate and used in downstream processes, such as visualization by gel electrophoresis. Representative quantities of synthesized oligonucleic acids extracted from each cluster in this example ranged from 113 fmol to 344 fmol. Representative yields from each cluster ranged from 48 pmol/cm²to 145 pmol/cm².
While specific instances have been shown and described herein, it will be apparent to those skilled in the art that such instances are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosed instances. It should be understood that various alternatives to the instances described herein may be employed in practicing the invention.

Claims

What is claimed is:

1. A method for de novo oligonucleotide synthesis, comprising:

(a) providing predetermined sequences for a plurality of non-identical oligonucleotides;

(b) providing a device for synthesizing oligonucleotides, comprising:

(i) a solid substrate;

(ii) a main channel, wherein the main channel extends vertically into the solid substrate from an opening on a top side of the solid substrate; and

(iii) a plurality of microchannels, wherein each microchannel of the plurality of microchannels extends vertically from an opening on a bottom side of the solid substrate into the main channel, and wherein each microchannel of the plurality of microchannels comprises a fluid path having at least one turn of 45 degrees to 180 degrees in total, when viewed from a top view, and

wherein the device comprises more than 20,000 of the microchannels in total;

(c) adding a droplet of fluid comprising an extension reaction reagent specific to a microchannel;

(d) allowing sufficient time for an extension reaction step to occur; and

(e) repeating steps (c) and (d) until the plurality of non-identical oligonucleotides are synthesized.

2. The method of claim 1, wherein the fluid path comprises up to 10 turns in total.

3. The method of claim 1, wherein the at least one turn is 45, 90 or 180 degrees in total.

4. The method of claim 1, wherein the device comprises a total of at least 700,000 microchannels.

5. The method of claim 1, wherein the plurality of microchannels comprises 100 to 150 microchannels.

6. The method of claim 1, wherein the plurality of microchannels comprises up to 500 microchannels.

7. The method of claim 1, wherein a ratio of width to depth of a narrowest segment of each microchannel is from 0.5 to 0.01.

8. The method of claim 1, wherein each microchannel of the plurality of microchannels has a width of 30 um to 100 um.

9. The method of claim 1, wherein each microchannel of the plurality of microchannels has a depth of 10 um to 500 um.

10. The method of claim 1, wherein the plurality of microchannels has a higher surface energy than the main channel.

11. The method of claim 1, wherein the plurality of microchannels has a higher hydrophobicity than the main channel.

12. The method of claim 1, wherein the main channel has a width of 0.5 to 2 mm.

13. The method of claim 1, wherein the device comprises at least 50 main channels.

14. The method of claim 1, wherein the device comprises at least 500 main channels.

15. The method of claim 1, wherein the device comprises at least 5000 main channels.

16. The method of claim 1, wherein the solid substrate is in a form of a plate or a tape.

17. The method of claim 1, wherein the solid substrate comprises silicon, silicon dioxide, silicon nitride, nylon, nitrocellulose, polypropylene, or polydimethylsiloxane (PDMS).