Patent Grant
12086722
Biomolecule based information storage systems, e.g., DNA-based, have a large storage capacity and stability over time. However, there is a need for scalable, automated, highly accurate and highly efficient systems for generating biomolecules for information storage.
Provided herein are devices for storing information, comprising: a solid support, wherein the solid support comprises a plurality of wells, wherein each of the wells comprises an addressable locus comprising: a synthesis surface located in a bottom region of each of the wells; a bottom electrode in addressable communication with the synthesis surface; and at least one sidewall electrode located on a sidewall of each of the wells, wherein the at least one sidewall electrode is 50 nm to 200 nm from the bottom region. Further provided herein are devices wherein the solid support comprises addressable loci at a density of at least 100×106 addressable loci per cm2. Further provided herein are devices wherein the solid support comprises addressable loci at a density of 100×106 to 100×107 addressable loci per cm2. Further provided herein are devices wherein the addressable locus comprises a diameter up to about 750 nm. Further provided herein are devices wherein each of the wells comprises a depth up to about 1000 nm. Further provided herein are devices wherein each of the wells comprises a depth of 100 nm to 1000 nm. Further provided herein are devices wherein each of the wells comprises a longest cross-sectional diameter of 100 nm to 800 nm. Further provided herein are devices wherein each of the wells is cylindrical. Further provided herein are devices wherein the bottom electrode comprises a largest cross-sectional area of 104 nm2 to 105 nm2. Further provided herein are devices wherein the at least one sidewall electrode is 50 nm to 200 nm from the bottom region. Further provided herein are devices wherein the at least one sidewall electrode comprises a height of 5 nm to 25 nm. Further provided herein are devices comprising at least two sidewall electrodes. Further provided herein are devices wherein the at least one sidewall electrode and the bottom electrode are independently addressable.
Provided herein are devices for storing information, comprising: a solid support, wherein the solid support comprises a plurality of wells, wherein each of the wells comprises an addressable locus comprising: a synthesis surface located in a bottom region of each of the wells; a bottom electrode in addressable communication with the synthesis surface; at least one sidewall electrode located on a sidewall of each of the wells, wherein the synthesis surface at each addressable locus comprises at least one polynucleotide extending from the synthesis surface, and wherein the polynucleotides comprising different sequences on the solid support are present at a density of at least 100×106 polynucleotides per cm2. Further provided herein are devices wherein the solid support comprises polynucleotides of different sequences at a density of at least 100×107 polynucleotides per cm2. Further provided herein are devices wherein the solid support comprises addressable loci at a density of 100×106 to 100×107 polynucleotides per cm2. Further provided herein are devices wherein each of the wells comprises a depth up to about 1000 nm. Further provided herein are devices wherein each of the wells comprises a depth of 100 nm to 1000 nm. Further provided herein are devices wherein the addressable locus comprises a diameter up to about 750 nm. Further provided herein are devices wherein each of the wells comprises a longest cross-sectional diameter of 100 nm to 800 nm. Further provided herein are devices wherein each of the wells is cylindrical. Further provided herein are devices wherein the bottom electrode comprises a largest cross-sectional area of 104 nm2 to 105 nm2. Further provided herein are devices wherein the at least one sidewall electrode is 50 nm to 200 nm from the bottom region. Further provided herein are devices wherein the at least one sidewall electrode comprises a height of 5 nm to 25 nm. Further provided herein are devices comprising at least two sidewall electrodes. Further provided herein are devices wherein the at least one sidewall electrode and the base electrode are independently addressable.
Provided herein are methods for storing information, comprising: (a) providing a solid support comprising a surface; (b) depositing at least one nucleoside on the surface, wherein the at least one nucleoside couples to a polynucleotide attached to the surface; and (c) repeating step b) to synthesize a plurality of polynucleotides on the surface, wherein polynucleotides having different sequences on the surface are present at a density of at least 100×106 polynucleotides per cm2. Further provided herein are methods wherein the density of addressable loci on the solid support is at least 100×107 polynucleotides per cm2. Further provided herein are methods wherein the density of addressable loci on the solid support is 100×106 to 100×107 polynucleotides per cm2. Further provided herein are methods wherein the method further comprises cleaving at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in a droplet. Further provided herein are methods wherein the method further comprises sequencing at least one polynucleotide from the surface. Further provided herein are methods wherein the nucleoside comprises a nucleoside phosphoramidite. Further provided herein are methods wherein the method further comprises drying the surface. Further provided herein are methods wherein the method further comprises washing the nucleosides away from the surface. Further provided herein are methods wherein the method further comprises a capping step. Further provided herein are methods wherein the method further comprises an oxidation step. Further provided herein are methods wherein the method further comprises a deblocking step.
Provided herein are methods for storing information, comprising: (a) providing a solid support comprising a surface; (b) depositing at droplet comprising at least one nucleoside on the surface, wherein the at least one nucleoside couples to a polynucleotide attached to the surface; and (c) repeating step b) to synthesize a plurality of polynucleotides on the surface, wherein the droplet has a volume of less than about 100 femtoliters. Further provided herein are methods wherein the droplet has a volume of less than about 50 femtoliters. Further provided herein are methods wherein the droplet has a volume of less than about 25 femtoliters to 100 femtoliters. Further provided herein are methods wherein the method further comprises cleaving at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in a droplet. Further provided herein are methods wherein the method further comprises sequencing at least one polynucleotide from the surface. Further provided herein are methods wherein the nucleoside comprises a nucleoside phosphoramidite. Further provided herein are methods wherein the method further comprises drying the surface. Further provided herein are methods wherein the method further comprises washing the nucleosides away from the surface. Further provided herein are methods wherein the method further comprises a capping step. Further provided herein are methods wherein the method further comprises an oxidation step. Further provided herein are methods wherein the method further comprises a deblocking step.
Provided herein are methods for storing information, comprising: (a) providing a solid support comprising a surface; (b) depositing at least one nucleoside on the surface, wherein the at least one nucleoside couples to a polynucleotide attached to the surface; and (c) repeating step b) to synthesize a plurality of polynucleotides on the surface, wherein the time to repeat step b) using four different nucleotides is less than about 100 milliseconds. Further provided herein are methods wherein the time to repeat step b) using four different nucleotides is less than about 50 milliseconds. Further provided herein are methods wherein the time to repeat step b) using four different nucleotides is 25 milliseconds to 100 milliseconds. Further provided herein are methods wherein the method further comprises cleaving at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in a droplet. Further provided herein are methods further comprising sequencing at least one polynucleotide from the surface. Further provided herein are methods wherein the nucleoside comprises a nucleoside phosphoramidite. Further provided herein are methods wherein the method further comprises drying the surface. Further provided herein are methods wherein the method further comprises washing the nucleosides away from the surface. Further provided herein are methods wherein the method further comprises a capping step. Further provided herein are methods wherein the method further comprises an oxidation step. Further provided herein are methods wherein the method further comprises a deblocking step.
All publications, patents, and patent applications mentioned in this specification are herein 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.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
There is a need for larger capacity storage systems as the amount of information generated and stored is increasing exponentially. Traditional storage media have a limited capacity and require specialized technology that changes with time, requiring constant transfer of data to new media, often at a great expense. A biomolecule such as a DNA molecule provides a suitable host for information storage in-part due to its stability over time and capacity for four bit information coding, as opposed to traditional binary information coding. Thus, large amounts of data are encoded in the DNA in a relatively smaller amount of physical space than used by commercially available information storage devices. Provided herein are methods to increase DNA synthesis throughput through increased sequence density and decreased turn-around time.
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, numerical features 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 embodiments. Accordingly, the description of a range should be considered to have specifically disclosed 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 disclosed 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.
The terminology used herein is for the purpose of describing particular embodiments 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.
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.
As used herein, the terms “preselected sequence”, “predefined sequence” or “predetermined sequence” are used interchangeably. The terms mean that the sequence of the polymer 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 acids molecules, the sequence of the polynucleotide being known and chosen before the synthesis or assembly of the nucleic acid molecules.
Provided herein are methods and compositions for production of synthetic (i.e. de novo synthesized or chemically synthesized) polynucleotides. Polynucleotides may also be referred to as oligonucleotides or oligos. Polynucleotide sequences described herein may be, unless stated otherwise, comprise DNA or RNA.
Solid Support Based Nucleic Acid Synthesis and Storage
Described herein are devices, compositions, systems and methods for chip based nucleic acid synthesis and storage. In some instances, polynucleotides are de novo synthesized using solid support based methods as described herein. In some instances, polynucleotides are stored on a solid support following synthesis. In some instances, solid support based methods as described herein are used for storage only.
Described herein are devices, compositions, systems and methods for solid support based nucleic acid synthesis and storage, wherein one or more nucleic acid synthesizer components are integrated into a solid support. Components or functional equivalents of components may comprise temperature control units, addressable electrodes, semiconducting surfaces, fluid reservoirs, fluidics, synthesis surfaces, power sources, or other component used to synthesize polynucleotides. Any combination of integrated components is suitable for use with the devices, compositions, systems and methods described herein. In some instances, one or more components is external (non-integrated) to the solid support.
The density of unique loci for polynucleotide synthesis on a surface is often controlled by the spatial resolution achievable by reagent deposition. Additionally, reagent deposition at unique sites requires movement of either the deposition device or the receiving surface to move the site of reagent deposition from one site to another. Alternatively, coupling of bases is locally controlled at defined sites on a synthesis surface without movement of the surface or a reagent deposition device. Local control is achieved through an array of addressable electrodes 1503, wherein each electrode controls nucleoside (nucleoside phosphoramidite) coupling through electrochemistry at a specific loci on the surface 1505. In some instances, an electrode is a base electrode (or bottom electrode), located at a bottom region in addressable communication with the synthesis surface. In some instances, electrodes are sidewall electrodes, located in the side of a well. However, the conventional process of electrochemistry on a flat array in some instances is limited by the pitch distance (See
Coupling in some instances is controlled directly by control of the nucleoside addition step, a deprotection step, or other step that affects the efficiency of a nucleoside coupling reaction. In some instances, a pattern of electrodes are charged to generate a gradient of H+ ions 1602 on defined sites near the synthesis surface (See
Described herein are devices comprising a solid support, wherein the solid support comprises a plurality of wells, wherein each of the wells comprises an addressable locus comprising: a synthesis surface located in a bottom region of each of the wells; and at least one sidewall electrode located on a sidewall of each of the wells, wherein the electrochemical generation of reagents is spatially separated from a polynucleotide attachment point to the synthesis surface. In some instances, devices described herein further comprise a bottom electrode in addressable communication with the synthesis surface. For example, sidewall electrodes 1603 can be used to control adhesion of substrates or reagents (See
Polynucleotide synthesis generally requires repeated deposition and removal of liquids (fluidics) on the synthesis surface. Bulk movement of fluids is some instances results in fluid loss (wetting, volume of transport lines or reaction wells), which results in low efficiency of reagent usage and higher cycle times for moving fluids. An alternative to bulk fluidics during synthesis is the use of digital fluidics, wherein reagents or reaction vessels are packaged as discrete droplets. Droplets in some instances are mixed, moved (merged, reacted), split, stored, added, removed or analyzed in discrete volumes by manipulation through a surface comprising insulated (or semiconductor-coated) electrodes, or electrowetting. Electrowetting allows for local control of fluid-surface interaction; for example energizing an electrode near a droplet results in splitting of the droplet. In some instances, droplets as described herein comprise a small volume. For example, the volume of a droplet is up to 10, 20, 50, 75, 100, 125, 150, 200, 300, 500, 800, or more than 1000 femtoliters. In some instances, the volume of a droplet is about 50 to about 200 femtoliters. In some instances, digital fluidics results in at least a 2, 3, 4, 7, 10 or more than 10× decrease in cycle times relative to bulk fluidics. In some instances, digital fluidics results in about 2× to 10× decrease in cycle times relative to bulk fluidics. In some instances, the time to complete one cycle (sequential coupling of 4 bases, including washes) is about 1, 2, 3, 5, 7, 10, 12, 15, 17, 20, 30, 50, 100, or about 200 milliseconds (ms). In some instances, the time to complete one cycle is up to 1, 2, 3, 5, 7, 10, 12, 15, 17, 20, 30, 50, 100, or up to 200 ms. In some instances, the time to complete one cycle is about 10 to about 50 ms.
Movement of fluids in or out of surfaces described herein may comprise modifications or conditions that prevent unwanted fluid movement or other phenomenon. For example, fluid movement in some instances results in the formation of bubbles or pockets of gas, which limits contact of fluids with components such as surfaces or polynucleotides. Various methods to control or minimize bubble formation are contemplated by the methods, systems, and compositions described herein. Such methods include control of fluid pressure, well geometry, or surface materials/coatings. Well geometry can be implemented to minimize bubbles. For example, tapering the well, channels, or other surface can reduce or eliminate bubble formation during fluid flow. Surface materials possessing specific wetting properties can be implemented to reduce or eliminate bubble formation. For example, surfaces described herein comprise hydrophobic materials. In some instances, surfaces described herein comprise hydrophilic materials. Pressure can be used to control bubble formation during fluid movement. Pressure in some instances is applied locally to a component, an area of a surface, a capillary/channel, or applied to an entire system. Pressure is in some instances applied either behind the direction of fluid movement, or in front of it. In some instances, back pressure is applied to prevent the formation of bubbles. Suitable pressures used for preventing bubble formation can range depending on fluid, the scale, flow geometry, and the materials used. For example, 5 to 10 atmospheres of pressure are maintained in the system. In some instances, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20 or more than 50 atmospheres of pressure are applied. In some instances, up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20 or up to 50 atmospheres of pressure are applied. In some instances, about 2 to about 10, about 2 to about 8, about 2 to about 5, about 4 to about 10, about 4 to about 12, about 5 to about 15, about 5 to about 7, about 7 to about 20, about 8 to about 15, or about 10 to about 20 atmospheres of pressure are applied.
Devices described herein may utilize control units for the purpose of regulating environmental conditions, such as temperature. Temperature control units are often used to prepare or maintain conditions for storing solid supports comprising polynucleotides. Storage conditions of nucleic acids can affect their long term stability, which directly influences the quality of the digital storage information that is retrieved. Polynucleotides are optionally stored at low temperature (for example, 10 degrees C., 4 degrees C., 0 degrees C., or lower) on a solid support, wherein a temperature control unit maintains this solid support temperature. The storage medium for polynucleotides on a solid support, such as solvated or dry also influences storage stability. In some instances, polynucleotides are stored in solution, such as an aqueous solution or buffer in droplets. In some instances, polynucleotides are stored lyophilized (dry). Temperature control units in some instances increase the chip temperature to facilitate drying of polynucleotides attached thereto. Temperature control units also provide for local control of heating at addressable locations on the solid support in some instances. In some instances, following addition of the droplets comprising the polynucleotides to the solid support, the solid support is dried. In some instances, the dried solid support is later resolvated. In some instances, the solid support is stored for later use. In some instances, the solid support further comprises an index map of the polynucleotides. In some instances, the solid support further comprises metadata.
Devices described herein can comprise power sources used to energize various components of the device. Synthesis components in the solid support are optionally powered by an external power source, or a power source integrated into the solid support. Power sources may comprise batteries, solar cells, thermoelectric generators, inductive (wireless) power units, kinetic energy charger, cellular telephones, tablets, or other power source suitable for use with the synthesis components or devices described herein. In some instances, synthesis components, surfaces, or devices described herein are portable.
Fluids comprising reagents, wash solvents, or other synthesis components are deposited on the synthesis surface. Unused fluid (prior to contact with the synthesis surface) or waste fluid (after contact with the synthesis surface) is in some instances stored in one or more compartments integrated into the solid support. Alternately or in combination, polynucleotides are moved in or out of the solid support for external analysis or storage. For example, synthesized polynucleotides are cleaved from loci on the solid support in a droplet, the resulting droplet moved externally to the synthesis area of the solid support. The droplet is optionally dried for storage. In some instances, fluids are stored externally from the solid support. In some instances, a device described herein comprises a solid support with a plurality of fluidics ports which allow movement of fluids in and out of the solid support. In some instances, ports are oriented on the sides of the solid support, by other configurations are also suitable for delivery of fluids to the synthesis surface. Such a device often comprises, for example, at least 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, or at least 10,000 ports per mm length of a solid support. In some instances, a device described herein comprises about 100 to about 5000 ports per mm per length of a solid support.
Described herein are addressable electrodes integrated into a solid support. Electrodes comprise without limitation conductors, insulators, or semi-conductors, and are fabricated of materials well known in the art. Materials may comprise metals, non-metals, mixed-metal oxides, nitrides, carbides, silicon-based materials, or other material. In some instances, metal oxides include TiO2, Ta2O5, Nb2O5, Al2O3, BaO, Y2O3, HfO2, SrO or other metal oxide known in the art. In some instances, metal carbides include TiC, WC, ThC2, ThC, VC, W2C, ZrC, HfC, NbC, TaC, Ta2C, or other metal carbide known in the art. In some instances, metal nitrides include GaN, InN, BN, Be3N2, Cr2N, MoN, Si3N4, TaN, Th2N2, VN, ZrN, TiN, HfN, NbC, WN, TaN, or other metal nitride known in the art. In some instances, a device disclosed herein is manufactured with a combination of materials listed herein or any other suitable material known in the art. Electrodes can possess any shape, including discs, rods, wells, posts, a substantially planar shape, or any other form suited for nucleic acid synthesis. The or cross-sectional area of each electrode varies as a function of the size of the loci for polynucleotide synthesis, but in some instances is up to 500 um2, 200 um2, 100 um2, 75 um2, 50 um2, 25 um2, 10 um2, less than 5 um2. In some instances, the cross-sectional area of each electrode is about 500 um2 to 10 um2, about 100 um2 to 25 um2, or about 150 um2 to 50 um2. In some instances, the cross-sectional area of each electrode is about 150 um2 to 50 um2. Devices provide herein include electrodes having a diameter that varies as a function of the size of the loci for polynucleotide synthesis. Exemplary electrode diameters include, without limitation, up to 500 um, 200 um, 100 um, 75 um, 50 um, 25 um, 10 um, less than 5 um. In some instances, the diameter of each electrode is about 500 um to 10 um, about 100 um to 25 um, about 100 um to about 200 um, about 50 um to about 200 um, or about 150 um to 50 um. In some instances, the diameter of each electrode is about 200 um to 50 um. In some instances, the diameter of each electrode is about 200 um to 100 um. In some instances, the diameter of each electrode is up to 500 nm, 200 nm, 100 nm, 75 nm, 50 nm, 25 nm, 10 nm, less than 5 nm. In some instances, the diameter of each electrode is about 500 nm to 10 nm, about 100 nm to 25 nm, about 100 nm to about 200 nm, about 50 nm to about 200 nm, or about 150 nm to 50 nm. In some instances, the diameter of each electrode is about 200 nm to 50 nm. In some instances, the diameter of each electrode is about 200 nm to 100 nm. The thickness of each electrode varies as a function of the size of the loci for polynucleotide synthesis, but in some instances is about 50 nm, 100 nm, 200 nm, 500 nm, 750 nm, 1000 nm, 1200 nm, 1500 nm, 2000 nm, 2500 nm, 3000 nm, or about 3500 nm. In some instances the thickness of the electrode is at least 50 nm, 100 nm, 200 nm, 500 nm, 750 nm, 1000 nm, 1200 nm, 1500 nm, 2000 nm, 2500 nm, 3000 nm, or at least 3500 nm. In some instances the thickness of the electrode is at least 1 um, 2 um, 3 um, 5 um, 10 um, 15 um, 20 um, 30 um, 50 um or at least 75 um. In some instances the thickness of the electrode is about 1 um, 2 um, 3 um, 5 um, 10 um, 15 um, 20 um, 30 um, 50 um or about 75 um. In some instances the thickness of the electrode is up to 1 um, 2 um, 3 um, 5 um, 10 um, 15 um, 20 um, 30 um, 50 um or up to 75 um. In some instances the thickness of the electrode is up to 50 nm, 100 nm, 200 nm, 500 nm, 750 nm, 1000 nm, 1200 nm, 1500 nm, 2000 nm, 2500 nm, 3000 nm, or up to 3500 nm. In some instances the thickness of the electrode is about 20 nm to 3000 nm, about 50 nm to 2500, about 100 nm to 750 nm, about 400 nm to 750 nm, about 500 nm to 3000 nm, or about 1000 nm to 3000 nm. In some instances the thickness of the electrode is about 10 um to about 20 um. In some instances the thickness of the electrode is about 5 um to about 50 um, about 10 um to about 30 um, about 15 um to about 25 um, or about 30 um to about 50 um. In some instances, electrodes are coated with additional materials such as semiconductors or insulators. In some instances, electrodes are coated with materials for polynucleotide attachment and synthesis. The size, shape, pattern, or orientation of electrodes is in some instances chosen to minimize deleterious side reactions caused by electrochemically generated reagents. In some instances combinations of electrodes are used, such as a grid of addressable electrodes and a common electrode. Electrodes are in some instances cathodes or anodes. Electrodes or arrays of electrodes can be positioned anywhere on or near the polynucleotide surface. In some instances, electrodes are placed on the bottom of loci for synthesis, such as the bottom of a well or channel. In some instances, electrodes are placed in the sidewalls of a well or channel (“sidewall electrodes”). A plurality of sidewall electrodes are in some instances present on the sides of a well. Electrodes positioned at different locations in the device can have different functions, and are independently or simultaneously addressable. In some instances, an electrode in the bottom of a well is used to cleave polynucleotides from the surface at one or more loci, and a sidewall electrode is used to generate acid to deprotect polynucleotides. In some instances, an electrode in the bottom of a well is used to cleave polynucleotides from the surface at one or more loci, and a first sidewall electrode is used to generate acid to deprotect polynucleotides, and a second sidewall electrode is used to move polynucleotides out of the well after cleavage. In exemplary configurations, sidewall electrodes are located about 10 nm, about 25 nm, about 50 nm, about 75 nm, about 100 nm, about 125 nm, or about 200 nm above the synthesis surface. In some instances, sidewall electrodes are located about 10 nm to about 100 nm, about 50 nm to about 150 nm, about 40 nm to 100 nm, about 75 nm to about 125 nm, about 100 to 300 nm above the synthesis surface. In some instances, multiple sidewall electrodes are located at different heights above the synthesis surface. For example, a locus comprises at least one sidewall electrode, at least 2 sidewall electrodes, at least 3 sidewall electrodes, or more than 3 sidewall electrodes. In exemplary configurations, sidewall electrodes have a height of about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 25 nm about 30 nm, about 40 nm, or about 50 nm. In some instances, sidewall electrodes have a height of 1 nm to 20 nm, 2 nm to 30 nm, 5 nm to 20 nm, 10 nm to 40 nm, or 5 nm to 25 nm.
Electrode surfaces can support the movement, conformation, synthesis, growth, and release of polynucleotides. In some instances, electrodes are coated with one or more layers. In some instances, the layer is a monolayer which facilities attachment of a linker. In some instances, the electrode is charged to influence the area of the monolayer to be functionalized with a linker. This allows for masking of specific areas for chemical functionalization, such as modifying the surface with hydrophobic or hydrophilic chemical groups. In some instances, the electrode is charged to influence the area of the monolayer to be extended with a nucleoside monomer. This in some instances includes generation of reagents to facilitate or prevent coupling of monomers to synthesis surfaces in the vicinity of an electrode. In some instances, the electrode is charged to influence the area of the monolayer which releases polynucleotides. Such controlled release of specific polynucleotides in a specific order in some instances is used to control the assembly of synthesized monomers into larger polynucleotides. For example, an iterative polynucleotide assembly process is optimized by exploring which various combinations of polynucleotides that are released and allowed to hybridize for overlap PCR assembly.
Each electrode can control one, or a plurality of different loci for synthesis, wherein each locus for synthesis has a density of polynucleotides. In some instances, the density is at least 1 oligo per 10 nm2, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000 or at least 1 oligo per 10,000 nm2. In some instances, the density is about 1 oligo per 10 nm2 to about 1 oligo per 5,000 nm2, about 1 oligo per 50 nm2 to about 1 oligo per 500 nm2, or about 1 oligo per 25 nm2 to about 1 oligo per 75 nm2. In some instances, the density of polynucleotides is about 1 oligo per 25 nm2 to about 1 oligo per 75 nm2.
Provided herein are devices for polynucleotide synthesis having various types of electrodes. In some instances, the device comprises a reference electrode. Reference electrodes are placed near the synthesis surface or in the case of a well or channel, for example, above a well or channel. In some instances, a reference electrode is about 1 to about 50 um above the synthesis surface, about 2 um to about 40 um, about 3 um to about 30 um, about 5 um to about 20 um, about 10 to about 20 um, about 15 to about 50 um, about 30 to about 50 um, about 5 um to about 30 um or about 7 um to about 25 um. In some instances a reference electrode is about 1, 2, 5, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26 um or more than 26 um above the synthesis surface. In some instances a reference electrode is up to 1, 2, 5, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26 um or up to 26 um above the synthesis surface. In some instances a reference electrode is at least 1, 2, 5, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26 um or at least 26 um above the synthesis surface. Reference electrodes in some instances are adjacent to synthesis surfaces, such as adjacent to a well or channel. Each locus in some instances has one corresponding reference electrode. In some instances, each locus shares a common reference electrode with one or more adjacent loci. The devices described herein may comprise any number of reference electrodes. In some instances the reference electrode is a single, uniform plate. In some instances, devices comprise a plurality of reference electrodes. A reference electrode can address a plurality of loci, for example 2, 3, 4, 5, 10 or more loci.
Described herein are devices, compositions, systems and methods for solid support based nucleic acid synthesis and storage, wherein the solid support has varying dimensions. In some instances, a size of the solid support is between about 40 and 120 mm by between about 25 and 100 mm. In some instances, a size of the solid support is about 80 mm by about 50 mm. In some instances, a width of a solid support is at least or about 10 mm, 20 mm, 40 mm, 60 mm, 80 mm, 100 mm, 150 mm, 200 mm, 300 mm, 400 mm, 500 mm, or more than 500 mm. In some instances, a height of a solid support is at least or about 10 mm, 20 mm, 40 mm, 60 mm, 80 mm, 100 mm, 150 mm, 200 mm, 300 mm, 400 mm, 500 mm, or more than 500 mm. In some instances, the solid support has a planar surface area of at least or about 100 mm2; 200 mm2; 500 mm2; 1,000 mm2; 2,000 mm2; 4,500 mm2; 5,000 mm2; 10,000 mm2; 12,000 mm2; 15,000 mm2; 20,000 mm2; 30,000 mm2; 40,000 mm2; 50,000 mm2 or more. In some instances, the thickness of the solid support 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 thickness of the solid support include 275 mm, 375 mm, 525 mm, 625 mm, 675 mm, 725 mm, 775 mm and 925 mm. In some instances, the thickness of the solid support is at least or about 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more than 4.0 mm.
Described herein are devices wherein two or more solid supports are assembled. In some instances, solid supports are interfaced together on a larger unit. Interfacing may comprise exchange of fluids, electrical signals, or other medium of exchange between solid supports. This unit is capable of interface with any number of servers, computers, or networked devices. For example, a plurality of solid support is integrated onto a rack unit, which is conveniently inserted or removed from a server rack. The rack unit may comprise any number of solid supports. In some instances the rack unit comprises at least 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000 or more than 100,000 solid supports. In some instances, two or more solid supports are not interfaced with each other. Nucleic acids (and the information stored in them) present on solid supports can be accessed from the rack unit. See e.g.,
Solid supports as described herein comprise an active area. In some instances, the active area comprises addressable regions or loci for nucleic acid synthesis. In some instances, the active area comprises addressable regions or loci for nucleic acid storage.
The active area comprises varying dimensions. For example, the dimension of the active area is between about 1 mm to about 50 mm by about 1 mm to about 50 mm. In some instances, the active area comprises a width of at least or about 0.5, 1, 1.5, 2, 2.5, 3, 5, 5, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or more than 80 mm. In some instances, the active area comprises a height of at least or about 0.5, 1, 1.5, 2, 2.5, 3, 5, 5, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or more than 80 mm. An exemplary active area within a solid support is seen in
Described herein are devices, compositions, systems and methods for solid support based nucleic acid synthesis and storage, wherein the solid support has a number of sites (e.g., spots) or positions for synthesis or storage. In some instances, the solid support comprises up to or about 10,000 by 10,000 positions in an area. In some instances, the solid support comprises between about 1000 and 20,000 by between about 1000 and 20,000 positions in an area. In some instances, the solid support comprises at least or about 10, 30, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 12,000, 14,000, 16,000, 18,000, 20,000 positions by least or about 10, 30, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 12,000, 14,000, 16,000, 18,000, 20,000 positions in an area. In some instances the area is up to 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, or 2.0 inches squared. In some instances, the solid support comprises addressable loci having a pitch of at least or about 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, or more than 10 um. In some instances, the solid support comprises addressable loci having a pitch of about 5 um. In some instances, the solid support comprises addressable loci having a pitch of about 2 um. In some instances, the solid support comprises addressable loci having a pitch of about 1 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.2 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.2 um to about 10 um, about 0.2 to about 8 um, about 0.5 to about 10 um, about 1 um to about 10 um, about 2 um to about 8 um, about 3 um to about 5 um, about 1 um to about 3 um or about 0.5 um to about 3 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.1 um to about 3 um. See e.g.
The solid support for nucleic acid synthesis or storage as described herein comprises a high capacity for storage of data. For example, the capacity of the solid support is at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 petabytes. In some instances, the capacity of the solid support is between about 1 to about 10 petabytes or between about 1 to about 100 petabytes. In some instances, the capacity of the solid support is about 100 petabytes. In some instances, the data is stored as addressable arrays of packets as droplets. In some instances, the data is stored as addressable arrays of packets as droplets on a spot. In some instances, the data is stored as addressable arrays of packets as dry wells. In some instances, the addressable arrays comprise at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or more than 200 gigabytes of data. In some instances, the addressable arrays comprise at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or more than 200 terabytes of data. In some instances, an item of information is stored in a background of data. For example, an item of information encodes for about 10 to about 100 megabytes of data and is stored in 1 petabyte of background data. In some instances, an item of information encodes for at least or about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, or more than 500 megabytes of data and is stored in 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, or more than 500 petabytes of background data.
Provided herein are devices, compositions, systems and methods for solid support based nucleic acid synthesis and storage, wherein following synthesis, the polynucleotides are collected in packets as one or more droplets. In some instances, the polynucleotides are collected in packets as one or more droplets and stored. In some instances, a number of droplets is at least or about 1, 10, 20, 50, 100, 200, 300, 500, 1000, 2500, 5000, 75000, 10,000, 25,000, 50,000, 75,000, 100,000, 1 million, 5 million, 10 million, 25 million, 50 million, 75 million, 100 million, 250 million, 500 million, 750 million, or more than 750 million droplets. In some instances, a droplet volume comprises 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100 um (micrometer) in diameter. In some instances, a droplet volume comprises 1-100 um, 10-90 um, 20-80 um, 30-70 um, or 40-50 um in diameter.
In some instances, the polynucleotides that are collected in the packets comprise a similar sequence. In some instances, the polynucleotides further comprise a non-identical sequence to be used as a tag or barcode. For example, the non-identical sequence is used to index the polynucleotides stored on the solid support and to later search for specific polynucleotides based on the non-identical sequence. Exemplary tag or barcode lengths include barcode sequences comprising, without limitation, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more bases in length. In some instances, the tag or barcode comprise at least or about 10, 50, 75, 100, 200, 300, 400, or more than 400 base pairs in length.
Provided herein are devices, compositions, systems and methods for solid support based nucleic acid synthesis and storage, wherein the polynucleotides are collected in packets comprising redundancy. For example, the packets comprise about 100 to about 1000 copies of each polynucleotide. In some instances, the packets comprise at least or about 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, or more than 2000 copies of each polynucleotide. In some instances, the packets comprise about 1000× to about 5000× synthesis redundancy. Synthesis redundancy in some instances is at least or about 500×, 1000×, 1500×, 2000×, 2500×, 3000×, 3500×, 4000×, 5000×, 6000×, 7000×, 8000×, or more than 8000×. The polynucleotides that are synthesized using solid support based methods as described herein comprise various lengths. In some instances, the polynucleotides are synthesized and further stored on the solid support. In some instances, the polynucleotide length is in between about 100 to about 1000 bases. In some instances, the polynucleotides comprise at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more than 2000 bases in length.
Nucleic Acid Based Information Storage
Provided herein are devices, compositions, systems and methods for nucleic acid-based information (data) storage. An exemplary workflow is provided in
Optionally, an early step of data storage process disclosed herein includes obtaining or receiving one or more items of information in the form of an initial code. Items of information include, without limitation, text, audio and visual information. Exemplary sources for items of information include, without limitation, books, periodicals, electronic databases, medical records, letters, forms, voice recordings, animal recordings, biological profiles, broadcasts, films, short videos, emails, bookkeeping phone logs, internet activity logs, drawings, paintings, prints, photographs, pixelated graphics, and software code. Exemplary biological profile sources for items of information include, without limitation, gene libraries, genomes, gene expression data, and protein activity data. Exemplary formats for items of information include, without limitation, .txt, .PDF, .doc, .docx, .ppt, .pptx, .xls, .xlsx, .rtf, .jpg, .gif, .psd, .bmp, .tiff, .png, and .mpeg. The amount of individual file sizes encoding for an item of information, or a plurality of files encoding for items of information, in digital format include, without limitation, up to 1024 bytes (equal to 1 KB), 1024 KB (equal to 1 MB), 1024 MB (equal to 1 GB), 1024 GB (equal to 1 TB), 1024 TB (equal to 1PB), 1 exabyte, 1 zettabyte, 1 yottabyte, 1 xenottabyte or more. In some instances, an amount of digital information is at least 1 gigabyte (GB). In some instances, the amount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than 1000 gigabytes. In some instances, the amount of digital information is at least 1 terabyte (TB). In some instances, the amount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than 1000 terabytes. In some instances, the amount of digital information is at least 1 petabyte (PB). In some instances, the amount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than 1000 petabytes.
Structures for Polynucleotide Synthesis
Provided herein are rigid or flexibles structures for polynucleotide synthesis. In the case of rigid structures, provided herein are devices having a structure for the generation of a library of polynucleotides. In some instances, the structure comprises a plate. An exemplary structure 200 is illustrated in
In the case of flexible structures, provided herein are devices wherein the flexible structure comprises a continuous loop 601 wrapped around one or more fixed structures, e.g., a pair of rollers 603 or a non-continuous flexible structure 607 wrapped around separate fixed structures, e.g., a pair reels 605. See
In some instances, a well 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 well. In some instances, a well 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 well. In some instances, a well 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. Provided herein are structures for polynucleotide synthesis comprising a plurality of discrete loci for polynucleotide synthesis. Exemplary structures for the loci include, without limitation, substantially planar regions, channels, wells or protrusions. Structures described herein are may comprise a plurality of clusters, each cluster comprising a plurality of wells, loci or channels. Alternatively, described herein are may comprise a homogenous arrangement of wells, loci or channels. Structures provided herein may comprise wells having a height or depth 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 instances, the height of a well is less than 100 um, less than 80 um, less than 60 um, less than 40 um or less than 20 um. In some instances, well height is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 um or more. In some instances, the height or depth of the well is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 nm. In some instances, the height or depth of the well is in a range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm to about 500. In some instances, the height or depth of the well is in a range of about 50 nm to about 1 um. In some instances, well height is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 800, 900 or about 1000 nm.
Structures for polynucleotide synthesis provided herein may comprise channels. The channels may have 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 channel 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 channel 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.
Described herein are structures for polynucleotide synthesis comprising a plurality of discrete loci. Structures comprise, without limitation, substantially planar regions, channels, protrusions, or wells for polynucleotide synthesis. In some instances, structures described herein are provided comprising a plurality of channels, wherein the height or depth of the channel 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 channel 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, channel height is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 um or more. In some instances, the height or depth of the channel is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 nm. In some instances, the height or depth of the channel is in a range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm to about 500. Channels described herein may be arranged on a surface in clusters or as a homogenous field.
The width of a locus on the surface of a structure for polynucleotide synthesis described herein may be from about 0.1 um to about 500 um, from about 0.5 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 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 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 width of a locus is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 nm. In some instances, the width of a locus is in a range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm to about 500. In some instances, the width of a locus is in a range of about 50 nm to about 1000 nm. In some instances, the distance between the center of two adjacent loci is from about 0.1 um to about 500 um, 0.5 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 locus is about 5 um, 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 locus is about 1 um to 100 um, 30 um to 100 um, or 50 um to 70 um. In some instances, the distance between the center of two adjacent loci is from about 0.5 um to about 2 um, 0.5 um to about 2 um, from about 0.75 um to about 2 um, from about 1 um to about 2 um, from about 0.2 um to about 1 um, from about 0.5 um to about 1.5 um, from about 0.5 um to about 0.8 um, or from about 0.5 um to about 1 um, for example, about 1 um. In some instances, the total width of a locus is about 50 nm, 0.1 um, 0.2 um, 0.3 um, 0.4 um, 0.5 um, 0.6 um, 0.7 um, 0.8 um, 0.9 um, 1 um, 1.1 um, 1.2 um, 1.3 um, 1.4 um, or 1.5 um. In some instances, the total width of a locus is about 0.5 um to 2 um, 0.75 um to 1 um, or 0.9 um to 2 um.
In some instances, each locus supports the synthesis of a population of polynucleotides having a different sequence than a population of polynucleotides 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. 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 some instances, each cluster includes 109, 121, 130 or 137 loci.
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 channels 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.
In some instances, the number of distinct polynucleotides synthesized on the surface of a structure described herein 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 mm2, 104 loci per mm2, 105 loci per mm2, 106 loci per mm2, or more. In some cases, a substrate comprises from about 10 loci per mm2 to about 500 mm2, from about 25 loci per mm2 to about 400 mm2, from about 50 loci per mm2 to about 500 mm2, from about 100 loci per mm2 to about 500 mm2, from about 150 loci per mm2 to about 500 mm2, from about 10 loci per mm2 to about 250 mm2, from about 50 loci per mm2 to about 250 mm2, from about 10 loci per mm2 to about 200 mm2, or from about 50 loci per mm2 to about 200 mm2. In some cases, a substrate comprises from about 104 loci per mm2 to about 105 mm2. In some cases, a substrate comprises from about 105 loci per mm2 to about 107 mm2. In some cases, a substrate comprises at least 105 loci per mm2. In some cases, a substrate comprises at least 106 loci per mm2. In some cases, a substrate comprises at least 107 loci per mm2. In some cases, a substrate comprises from about 104 loci per mm2 to about 105 mm2. In some instances, the density of loci within a cluster of a substrate is at least or about 1 locus per um2, 10 loci per um2, 25 loci per um2, 50 loci per um2, 65 loci per um2, 75 loci per um2, 100 loci per um2, 130 loci per um2, 150 loci per um2, 175 loci per um2, 200 loci per um2, 300 loci per um2, 400 loci per um2, 500 loci per um2, 1,000 loci per um2 or more. In some cases, a substrate comprises from about 10 loci per um2 to about 500 um2, from about 25 loci per um2 to about 400 um2, from about 50 loci per um2 to about 500 um2, from about 100 loci per um2 to about 500 um2, from about 150 loci per um2 to about 500 um2, from about 10 loci per um2 to about 250 um2, from about 50 loci per um2 to about 250 um2, from about 10 loci per um2 to about 200 um2, or from about 50 loci per um2 to about 200 um2.
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. In some cases, the distance between the centers of two adjacent loci is less than about 10000 nm, 8000 nm, 6000 nm, 4000 nm, 2000 nm 1000 nm, 800 nm, 600 nm, 400 nm, 200 nm, 150 nm, 100 nm, 80 um, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm or 10 nm. In some instances, each square meter of a structure described herein allows for at least 107, 108, 109, 1010, 1011 loci, where each locus supports one polynucleotide. In some instances, 109 polynucleotides are supported on less than about 6, 5, 4, 3, 2 or 1 m2 of a structure described herein.
In some instances, a structure 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 polynucleotides. In some cases, the structure 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 polynucleotides encoding for distinct sequences. In some instances, at least a portion of the polynucleotides have an identical sequence or are configured to be synthesized with an identical sequence. In some instances, the structure provides a surface environment for the growth of polynucleotides having at least 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 arrangements, structures for polynucleotide synthesis described herein comprise sites for polynucleotide synthesis in a uniform arrangement.
In some instances, polynucleotides are synthesized on distinct loci of a structure, wherein each locus supports the synthesis of a population of polynucleotides. In some cases, each locus supports the synthesis of a population of polynucleotides having a different sequence than a population of polynucleotides grown on another locus. In some instances, the loci of a structure are located within a plurality of clusters. In some instances, a structure 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 structure 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, polynucleotides from distinct loci within one cluster have sequences that, when assembled, encode for a contiguous longer polynucleotide of a predetermined sequence.
Structure Size
In some instances, a structure described herein is about the size of a plate (e.g., chip), for example between about 40 and 120 mm by between about 25 and 100 mm. In some instances, a structure described herein 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, 84 mm, 76 mm, 54 mm, 51 mm and 25 mm. In some instances, a substrate has a planar surface area of at least 100 mm2; 200 mm2; 500 mm2; 1,000 mm2; 2,000 mm2; 4,500 mm2; 5,000 mm2; 10,000 mm2; 12,000 mm2; 15,000 mm2; 20,000 mm2; 30,000 mm2; 40,000 mm2; 50,000 mm2 or more. In some instances, the thickness 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 thickness include 275 mm, 375 mm, 525 mm, 625 mm, 675 mm, 725 mm, 775 mm and 925 mm. In some instances, the thickness is at least or about 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more than 4.0 mm. In some cases, the thickness of varies with diameter and depends on the composition of the substrate. For example, a structure comprising materials other than silicon may have a different thickness than a silicon structure of the same diameter. Structure thickness may be determined by the mechanical strength of the material used and the structure must be thick enough to support its own weight without cracking during handling. In some instances, a structure is more than about 1, 2, 3, 4, 5, 10, 15, 30, 40, 50 feet in any one dimension.
Provided herein are devices comprising a surface, wherein the surface is modified to support polynucleotide synthesis at predetermined locations and with a resulting low error rate, a low dropout rate, a high yield, and a high oligo representation. In some instances, surfaces of devices for polynucleotide synthesis provided herein are fabricated from a variety of materials capable of modification to support a de novo polynucleotide synthesis reaction. In some cases, the devices are sufficiently conductive, e.g., are able to form uniform electric fields across all or a portion of the devices. Devices described herein may comprise a flexible material. Exemplary flexible materials include, without limitation, modified nylon, unmodified nylon, nitrocellulose, and polypropylene. Devices described herein may comprise a rigid material. Exemplary rigid materials include, without limitation, glass, fuse silica, silicon, silicon dioxide, silicon nitride, plastics (for example, polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, and metals (for example, gold, platinum). Devices disclosed herein may be fabricated from a material comprising silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), glass, or any combination thereof. In some cases, devices disclosed herein are manufactured with a combination of materials listed herein or any other suitable material known in the art.
Devices described herein may comprise material having a range of tensile strength. Exemplary materials having a range of tensile strengths include, but are not limited to, nylon (70 MPa), nitrocellulose (1.5 MPa), polypropylene (40 MPa), silicon (268 MPa), polystyrene (40 MPa), agarose (1-10 MPa), polyacrylamide (1-10 MPa), polydimethylsiloxane (PDMS) (3.9-10.8 MPa). Solid supports described herein can have a tensile strength from 1 to 300, 1 to 40, 1 to 10, 1 to 5, or 3 to 11 MPa. Solid supports described herein can have a tensile strength of about 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 25, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 270, or more MPa. In some instances, a device described herein comprises a solid support for polynucleotide synthesis that is in the form of a flexible material capable of being stored in a continuous loop or reel, such as a tape or flexible sheet.
Young's modulus measures the resistance of a material to elastic (recoverable) deformation under load. Exemplary materials having a range of Young's modulus stiffness include, but are not limited to, nylon (3 GPa), nitrocellulose (1.5 GPa), polypropylene (2 GPa), silicon (150 GPa), polystyrene (3 GPa), agarose (1-10 GPa), polyacrylamide (1-10 GPa), polydimethylsiloxane (PDMS) (1-10 GPa). Solid supports described herein can have a Young's moduli from 1 to 500, 1 to 40, 1 to 10, 1 to 5, or 3 to 11 GPa. Solid supports described herein can have a Young's moduli of about 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 25, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 400, 500 GPa, or more. As the relationship between flexibility and stiffness are inverse to each other, a flexible material has a low Young's modulus and changes its shape considerably under load. In some instances, a solid support described herein has a surface with a flexibility of at least nylon.
In some cases, devices disclosed herein comprise a silicon dioxide base and a surface layer of silicon oxide. Alternatively, the devices may have a base of silicon oxide. Surface of the devices provided here may be textured, resulting in an increase overall surface area for polynucleotide synthesis. Devices disclosed herein in some instances comprise at least 5%, 10%, 25%, 50%, 80%, 90%, 95%, or 99% silicon. Devices disclosed herein in some instances are fabricated from silicon on insulator (SOI) wafer.
The structure may be fabricated from a variety of materials, suitable for the methods and compositions of the invention described herein. In instances, the materials from which the substrates/solid supports of the comprising the invention are fabricated exhibit a low level of polynucleotide binding. In some situations, material that are transparent to visible and/or UV light can be employed. Materials that are sufficiently conductive, e.g. those that can form uniform electric fields across all or a portion of the substrates/solids support described herein, can be utilized. In some instances, such materials may be connected to an electric ground. In some cases, the substrate or solid support can be heat conductive or insulated. The materials can be chemical resistant and heat resistant to support chemical or biochemical reactions such as a series of polynucleotide synthesis reactions. For flexible materials, materials of interest can include: nylon, both modified and unmodified, nitrocellulose, polypropylene, and the like.
For rigid materials, specific materials of interest include: glass; fuse silica; silicon, plastics (for example polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, and the like); metals (for example, gold, platinum, and the like). The structure can be fabricated from a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), and glass. The substrates/solid supports or the microstructures, reactors therein may be manufactured with a combination of materials listed herein or any other suitable material known in the art.
In some instances, a substrate disclosed herein comprises a computer readable material. Computer readable materials include, without limitation, magnetic media, reel-to-reel tape, cartridge tape, cassette tape, flexible disk, paper media, film, microfiche, continuous tape (e.g., a belt) and any media suitable for storing electronic instructions. In some cases, the substrate comprises magnetic reel-to-reel tape or a magnetic belt. In some instances, the substrate comprises a flexible printed circuit board.
Structures described herein may be transparent to visible and/or UV light. In some instances, structures described herein are sufficiently conductive to form uniform electric fields across all or a portion of a structure. In some instances, structures described herein are heat conductive or insulated. In some instances, the structures are chemical resistant and heat resistant to support a chemical reaction such as a polynucleotide synthesis reaction. In some instances, the substrate is magnetic. In some instances, the structures comprise a metal or a metal alloy.
Structures for polynucleotide synthesis may be over 1, 2, 5, 10, 30, 50 or more feet long in any dimension. In the case of a flexible structure, the flexible structure is optionally stored in a wound state, e.g., in a reel. In the case of a large rigid structure, e.g., greater than 1 foot in length, the rigid structure can be stored vertically or horizontally.
Surface Preparation
Provided herein are methods to support the immobilization of a biomolecule on a substrate, where a surface of a structure described herein comprises a material and/or is coated with a material that facilitates a coupling reaction with the biomolecule for attachment. To prepare a structure for biomolecule immobilization, surface modifications may be employed that chemically and/or physically alter the substrate surface by an additive or subtractive process to change one or more chemical and/or physical properties of a substrate surface or a selected site or region of the surface. For example, surface modification involves (1) changing the wetting properties of a surface, (2) functionalizing a surface, i.e. providing, modifying or substituting surface functional groups, (3) defunctionalizing a surface, i.e. removing surface functional groups, (4) otherwise altering the chemical composition of a surface, e.g., through etching, (5) increasing or decreasing surface roughness, (6) providing a coating on a surface, e.g., a coating that exhibits wetting properties that are different from the wetting properties of the surface, and/or (7) depositing particulates on a surface. In some instances, the surface of a structure is selectively functionalized to produce 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 instances, a surface of a structure disclosed herein is modified to comprise one or more actively functionalized surfaces configured to bind to both the surface of the substrate and a biomolecule, thereby supporting a coupling reaction to the surface. In some instances, the surface is also functionalized with a passive material that does not efficiently bind the biomolecule, thereby preventing biomolecule attachment at sites where the passive functionalization agent is bound. In some cases, the surface comprises an active layer only defining distinct loci for biomolecule support.
In some instances, the surface is contacted with a mixture of functionalization groups which are 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 cases, the ratio of the at least two types of surface functionalization agents in a mixture is about 1:1, 1:2, 1:5, 1:10, 2:10, 3:10, 4:10, 5:10, 6:10, 7:10, 8:10, 9:10, or any other ratio to achieve a desired surface representation of two groups. 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 mixture of functionalization reagents comprises one or more reagents that bind to a biomolecule and one or more reagents that do not bind to a biomolecule. Therefore, modulation of the reagents allows for the control of the amount of biomolecule binding that occurs at a distinct area of functionalization.
In some instances, a method for substrate functionalization comprises deposition of a silane molecule onto a surface of a substrate. The silane molecule may be deposited on a high energy surface of the substrate. In some instances the high surface energy region includes a passive functionalization reagent. Methods described herein provide for a silane group to bind the surface, while the rest of the molecule provides a distance from the surface and a free hydroxyl group at the end to which a biomolecule attaches. In some instances, the silane is an organofunctional alkoxysilane molecule. Non-limiting examples of organofunctional alkoxysilane molecules include dimethylchloro-octodecyl-silane, methyldichloro-octodecyl-silane, trichloro-octodecyl-silane, and trimethyl-octodecyl-silane, triethyl-octodecyl-silane. In some instances, the silane is an amino silane. Examples of amino silanes include, without limitation, 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane and N-(3-triethoxysilylpropyl)-4-hydroxybutyramide. In some instances, the silane comprises 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane, N-(3-triethoxysilylpropyl)-4-hydroxybutyramide, or any combination thereof. In some instances, an active functionalization agent comprises 11-acetoxyundecyltriethoxysilane. In some instances, an active functionalization agent comprises n-decyltriethoxysilane. 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, silanization is performed on a surface through self-assembly with organofunctional alkoxysilane molecules. 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. Exemplary hydroxyalkyl siloxanes 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). In some cases, the dimeric secondary aminoalkyl siloxanes is bis (3-trimethoxysilylpropyl) amine turning into bis(silyloxylpropyl)amine.
Active functionalization areas may 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. In some instances, an active functionalization agent comprises 11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane in a ratio from about 20:80 to about 1:99, or about 10:90 to about 2:98, or about 5:95.
In some instances, functionalization comprises deposition of a functionalization agent to a structure by any deposition technique, including, but not limiting to, chemical vapor deposition (CVD), atomic layer deposition (ALD), plasma enhanced CVD (PECVD), plasma enhanced ALD (PEALD), metal organic CVD (MOCVD), hot wire CVD (HWCVD), initiated CVD (iCVD), modified CVD (MCVD), vapor axial deposition (VAD), outside vapor deposition (OVD), physical vapor deposition (e.g., sputter deposition, evaporative deposition), and molecular layer deposition (MLD).
Any step or component in the following functionalization process 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 surface of a structure 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 surface 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 surface functionalization, in various instances, comprises lithography with patterned functionalization. In some instances, photolithography is performed following resist coating. In some instances, after lithography, the surface is visually inspected for lithography defects. The process for surface 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 surface 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 some 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 surface 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. Structures may be designed to control the flow of fluids by facilitating or inhibiting wicking effects into neighboring fluidic paths. For example, a structure 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.
In some instances, a structure described herein has a surface that comprises a material having thickness of at least or at least 0.1 nm, 0.5 nm, 1 nm, 2 nm, 5 nm, 10 nm or 25 nm that comprises a reactive group capable of binding nucleosides. Exemplary include, without limitation, glass and silicon, such as silicon dioxide and silicon nitride. In some cases, exemplary surfaces include nylon and PMMA.
In some instances, electromagnetic radiation in the form of UV light is used for surface patterning. In some instances, a lamp is used for surface patterning, and a mask mediates exposure locations of the UV light to the surface. In some instances, a laser is used for surface patterning, and a shutter opened/closed state controls exposure of the UV light to the surface. The laser arrangement may be used in combination with a flexible structure that is capable of moving. In such an arrangement, the coordination of laser exposure and flexible structure movement is used to create patterns of one or more agents having differing nucleoside coupling capabilities.
Described herein are surfaces for polynucleotide synthesis that are reusable. After synthesis and/or cleavage of polynucleotides, a surface may be bathed, washed, cleaned, baked, etched, or otherwise functionally restored to a condition suitable for subsequent polynucleotide synthesis. The number of times a surface is reused and the methods for recycling/preparing the surface for reuse vary depending on subsequent applications. Surfaces prepared for reuse are in some instances reused at least 1, 2, 3, 5, 10, 20, 50, 100, 1,000 or more times. In some instances, the remaining “life” or number of times a surface is suitable for reuse is measured or predicted.
In some cases, the synthesized polynucleotides are stored on the substrate, for example a solid support. Nucleic acid reagents may be deposited on the substrate surface in a non-continuous, or drop-on-demand method. Examples of such methods include the electromechanical transfer method, electric thermal transfer method, and electrostatic attraction method. In the electromechanical transfer method, piezoelectric elements deformed by electrical pulses cause the droplets to be ejected. In the electric thermal transfer method, bubbles are generated in a chamber of the device, and the expansive force of the bubbles causes the droplets to be ejected. In the electrostatic attraction method, electrostatic force of attraction is used to eject the droplets onto the substrate. In some cases, the drop frequency is from about 5 KHz to about 500 KHz; from about 5 KHz to about 100 KHz; from about 10 KHz to about 500 KHz; from about 10 KHz to about 100 KHz; or from about 50 KHz to about 500 KHz. In some cases, the frequency is less than about 500 KHz, 200 KHz, 100 KHz, or 50 KHz.
The size of the droplets dispensed correlates to the resolution of the device. In some instances, the devices deposit droplets of reagents at sizes from about 0.01 pl to about 20 pl, from about 0.01 pl to about 10 pl, from about 0.01 pl to about 1 pl, from about 0.01 pl to about 0.5 pl, from about 0.01 pl to about 0.01 pl, or from about 0.05 pl to about 1 pl. In some instances, the droplet size is less than about 1 pl, 0.5 pl, 0.2 pl, 0.1 pl, or 0.05 pl.
In some arrangements, the configuration of a polynucleotide synthesis system allows for a continuous polynucleotide synthesis process that exploits the flexibility of a substrate for traveling in a reel-to-reel type process. This synthesis process operates in a continuous production line manner with the substrate travelling through various stages of polynucleotide synthesis using one or more reels to rotate the position of the substrate. In an exemplary instance, a polynucleotide synthesis reaction comprises rolling a substrate: through a solvent bath, beneath a deposition device for phosphoramidite deposition, through a bath of oxidizing agent, through an acetonitrile wash bath, and through a deblock bath. Optionally, the tape is also traversed through a capping bath. A reel-to-reel type process allows for the finished product of a substrate comprising synthesized polynucleotides to be easily gathered on a take-up reel, where it can be transported for further processing or storage.
In some arrangements, polynucleotide synthesis proceeds in a continuous process as a continuous flexible tape is conveyed along a conveyor belt system. Similar to the reel-to-reel type process, polynucleotide synthesis on a continuous tape operates in a production line manner, with the substrate travelling through various stages of polynucleotide synthesis during conveyance. However, in a conveyor belt process, the continuous tape revisits a polynucleotide synthesis step without rolling and unrolling of the tape, as in a reel-to-reel process. In some arrangements, polynucleotide synthesis steps are partitioned into zones and a continuous tape is conveyed through each zone one or more times in a cycle. For example, a polynucleotide synthesis reaction may comprise (1) conveying a substrate through a solvent bath, beneath a deposition device for phosphoramidite deposition, through a bath of oxidizing agent, through an acetonitrile wash bath, and through a block bath in a cycle; and then (2) repeating the cycles to achieve synthesized polynucleotides of a predetermined length. After polynucleotide synthesis, the flexible substrate is removed from the conveyor belt system and, optionally, rolled for storage. Rolling may be around a reel, for storage. In some instances, a flexible substrate comprising thermoplastic material is coated with nucleoside coupling reagent. The coating is patterned into loci such that each locus has diameter of about 10 um, with a center-to-center distance between two adjacent loci of about 21 um. In this instance, the locus size is sufficient to accommodate a sessile drop volume of 0.2 pl during a polynucleotide synthesis deposition step. In some cases, the locus density is about 2.2 billion loci per m2 (1 locus/441×10−12 m2). In some cases, a 4.5 m2 substrate comprise about 10 billion loci, each with a 10 um diameter.
In some arrangements, a device for application of one or more reagents to a substrate during a synthesis reaction is configured to deposit reagents and/or nucleoside monomers for nucleoside phosphoramidite based synthesis. Reagents for polynucleotide synthesis include reagents for polynucleotide extension and wash buffers. As non-limiting examples, the device deposits cleaning reagents, coupling reagents, capping reagents, oxidizers, de-blocking agents, acetonitrile, gases such as nitrogen gas, and any combination thereof. In addition, the device optionally deposits reagents for the preparation and/or maintenance of substrate integrity. In some instances, the polynucleotide 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 polynucleotide synthesizer deposits between about 1 and 10000, 1 and 5000, 100 and 5000, or 1000 and 5000 droplets per second.
Described herein are devices, methods, systems and compositions where reagents for polynucleotide synthesis are recycled or reused. Recycling of reagents may comprise collection, storage, and usage of unused reagents, or purification/transformation of used reagents. For example, a reagent bath is recycled and used for a polynucleotide synthesis step on the same or a different surface. Reagents described herein may be recycled 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. Alternatively or in combination, a reagent solution comprising a reaction byproduct is filtered to remove the byproduct, and the reagent solution is used for additional polynucleotide synthesis reactions.
Many integrated or non-integrated elements are often used with polynucleotide synthesis systems. In some instances, a polynucleotide synthesis system comprises one or more elements useful for downstream processing of synthesized polynucleotides. 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.
De Novo Polynucleotide Synthesis
Provided herein are systems and methods for synthesis of a high density of polynucleotides on a substrate in a short amount of time. In some instances, the substrate is a flexible substrate. In some instances, at least 1010, 1011, 1012, 1013, 1014, or 1015 bases are synthesized in one day. In some instances, at least 10×108, 10×109, 10×1010, 10×1011, or 10×1012 polynucleotides are synthesized in one day. In some cases, each polynucleotide synthesized comprises at least 20, 50, 100, 200, 300, 400 or 500 nucleobases. In some cases, these bases are synthesized with a total average error rate of less than about 1 in 100; 200; 300; 400; 500; 1000; 2000; 5000; 10000; 15000; 20000 bases. In some instances, these error rates are for at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or more of the polynucleotides synthesized. In some instances, these at least 90%, 95%, 98%, 99%, 99.5%, or more of the polynucleotides synthesized do not differ from a predetermined sequence for which they encode. In some instances, the error rate for synthesized polynucleotides on a substrate using the methods and systems described herein is less than about 1 in 200. In some instances, the error rate for synthesized polynucleotides on a substrate using the methods and systems described herein is less than about 1 in 1,000. In some instances, the error rate for synthesized polynucleotides on a substrate using the methods and systems described herein is less than about 1 in 2,000. In some instances, the error rate for synthesized polynucleotides on a substrate using the methods and systems described herein is less than about 1 in 3,000. In some instances, the error rate for synthesized polynucleotides on a substrate using the methods and systems described herein is less than about 1 in 5,000. Individual types of error rates include mismatches, deletions, insertions, and/or substitutions for the polynucleotides synthesized on the substrate. The term “error rate” refers to a comparison of the collective amount of synthesized polynucleotide to an aggregate of predetermined polynucleotide sequences. In some instances, synthesized polynucleotides disclosed herein comprise a tether of 12 to 25 bases. In some instances, the tether comprises 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.
Described herein are methods, systems, devices, and compositions wherein chemical reactions used in polynucleotide synthesis are controlled using electrochemistry. Electrochemical reactions in some instances are controlled by any source of energy, such as light, heat, radiation, or electricity. For example, electrodes are used to control chemical reactions as all or a portion of discrete loci on a surface. Electrodes in some instances are charged by applying an electrical potential to the electrode to control one or more chemical steps in polynucleotide synthesis. In some instances, these electrodes are addressable. Any number of the chemical steps described herein is in some instances controlled with one or more electrodes. Electrochemical reactions may comprise oxidations, reductions, acid/base chemistry, or other reaction that is controlled by an electrode. In some instances, electrodes generate electrons or protons that are used as reagents for chemical transformations. Electrodes in some instances directly generate a reagent such as an acid. In some instances, an acid is a proton. Electrodes in some instances directly generate a reagent such as a base. Acids or bases are often used to cleave protecting groups, or influence the kinetics of various polynucleotide synthesis reactions, for example by adjusting the pH of a reaction solution. Electrochemically controlled polynucleotide synthesis reactions in some instances comprise redox-active metals or other redox-active organic materials. In some instances, metal or organic catalysts are employed with these electrochemical reactions. In some instances, acids are generated from oxidation of quinones.
Control of chemical reactions with is not limited to the electrochemical generation of reagents; chemical reactivity may be influenced indirectly through biophysical changes to substrates or reagents through electric fields (or gradients) which are generated by electrodes. In some instances, substrates include but are not limited to nucleic acids. In some instances, electrical fields which repel or attract specific reagents or substrates towards or away from an electrode or surface are generated. Such fields in some instances are generated by application of an electrical potential to one or more electrodes. For example, negatively charged nucleic acids are repelled from negatively charged electrode surfaces. Such repulsions or attractions of polynucleotides or other reagents caused by local electric fields in some instances provides for movement of polynucleotides or other reagents in or out of region of the synthesis device or structure. In some instances, electrodes generate electric fields which repel polynucleotides away from a synthesis surface, structure, or device. In some instances, electrodes generate electric fields which attract polynucleotides towards a synthesis surface, structure, or device. In some instances, protons are repelled from a positively charged surface to limit contact of protons with substrates or portions thereof. In some instances, repulsion or attractive forces are used to allow or block entry of reagents or substrates to specific areas of the synthesis surface. In some instances, nucleoside monomers are prevented from contacting a polynucleotide chain by application of an electric field in the vicinity of one or both components. Such arrangements allow gating of specific reagents, which may obviate the need for protecting groups when the concentration or rate of contact between reagents and/or substrates is controlled. In some instances, unprotected nucleoside monomers are used for polynucleotide synthesis. Alternatively, application of the field in the vicinity of one or both components promotes contact of nucleoside monomers with a polynucleotide chain. Additionally, application of electric fields to a substrate can alter the substrates reactivity or conformation. In an exemplary application, electric fields generated by electrodes are used to prevent polynucleotides at adjacent loci from interacting. In some instances, the substrate is a polynucleotide, optionally attached to a surface. Application of an electric field in some instances alters the three-dimensional structure of a polynucleotide. Such alterations comprise folding or unfolding of various structures, such as helices, hairpins, loops, or other 3-dimensional nucleic acid structure. Such alterations are useful for manipulating nucleic acids inside of wells, channels, or other structures. In some instances, electric fields are applied to a nucleic acid substrate to prevent secondary structures. In some instances, electric fields obviate the need for linkers or attachment to a solid support during polynucleotide synthesis.
A suitable method for polynucleotide synthesis on a substrate of this disclosure is a phosphoramidite method comprising the controlled addition of a phosphoramidite building block, i.e. nucleoside phosphoramidite, to a growing polynucleotide chain in a coupling step that forms a phosphite triester linkage between the phosphoramidite building block and a nucleoside bound to the substrate. 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 and linkage of a nucleoside phosphoramidite in the coupling step, 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, a polynucleotide 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. Protecting groups may comprise any chemical group that prevents extension of the polynucleotide chain. In some instances, the protecting group is cleaved (or removed) in the presence of an acid. In some instances, the protecting group is cleaved in the presence of a base. In some instances, the protecting group is removed with electromagnetic radiation such as light, heat, or other energy source. In some instances, the protecting group is removed through an oxidation or reduction reaction. In some instances, a protecting group comprises a triarylmethyl group. In some instances, a protecting group comprises an aryl ether. In some instances, a protecting comprises a disulfide. In some instances a protecting group comprises an acid-labile silane. In some instances, a protecting group comprises an acetal. In some instances, a protecting group comprises a ketal. In some instances, a protecting group comprises an enol ether. In some instances, a protecting group comprises a methoxybenzyl group. In some instances, a protecting group comprises an azide. In some instances, a protecting group is 4,4′-dimethoxytrityl (DMT). In some instances, a protecting group is a tert-butyl carbonate. In some instances, a protecting group is a tert-butyl ester. In some instances, a protecting group comprises a base-labile group.
Following coupling, phosphoramidite polynucleotide synthesis methods optionally comprise a capping step. In a capping step, the growing polynucleotide is treated with a capping agent. A capping step generally serves to block unreacted substrate-bound 5′-OH groups after coupling from further chain elongation, preventing the formation of polynucleotides with internal base deletions. Further, phosphoramidites activated with 1H-tetrazole often 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, undergoes depurination. The apurinic sites can end up being cleaved in the course of the final deprotection of the polynucleotide 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 polynucleotide synthesis decreases the error rate as compared to synthesis without capping. As an example, the capping step comprises treating the substrate-bound polynucleotide with a mixture of acetic anhydride and 1-methylimidazole. Following a capping step, the substrate is optionally washed.
Following addition of a nucleoside phosphoramidite, and optionally after capping and one or more wash steps, a substrate described herein comprises a bound growing nucleic acid that may be oxidized. The oxidation step comprises oxidizing the phosphite triester into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester internucleoside linkage. In some instances, phosphite triesters are oxidized electrochemically. In some instances, oxidation of the growing polynucleotide is achieved by treatment with iodine and water, optionally in the presence of a weak base such as a pyridine, lutidine, or collidine. Oxidation is sometimes carried out under anhydrous conditions using 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 polynucleotide is optionally washed. In some instances, the step of oxidation is substituted with a sulfurization step to obtain polynucleotide 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).
For a subsequent cycle of nucleoside incorporation to occur through coupling, a protected 5′ end (or 3′ end, if synthesis is conducted in a 5′ to 3′ direction) of the substrate bound growing polynucleotide is 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. In some instances, the protecting group is DMT and deblocking occurs with electrochemically generated protons. Conducting detritylation for an extended time or with stronger than recommended solutions of acids may lead to increased depurination of solid support-bound polynucleotide and thus reduces the yield of the desired full-length product. Methods and compositions described herein provide for controlled deblocking conditions limiting undesired depurination reactions. In some instances, the substrate bound polynucleotide is washed after deblocking. In some cases, efficient washing after deblocking contributes to synthesized polynucleotides having a low error rate.
Methods for the synthesis of polynucleotides on a substrate described herein may involve an iterating sequence of the following steps: application of a protected monomer to a surface of a substrate feature to link with either the 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 and/or sulfurization. In some instances, one or more wash steps precede or follow one or all of the steps.
Methods for the synthesis of polynucleotides on a substrate described herein may comprise an oxidation step. For example, methods involve an iterating sequence of the following steps: application of a protected monomer to a surface of a substrate feature to link with either the 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; application of another protected monomer for linking, and oxidation and/or sulfurization. In some instances, one or more wash steps precede or follow one or all of the steps.
Methods for the synthesis of polynucleotides on a substrate described herein may further comprise an iterating sequence of the following steps: application of a protected monomer to a surface of a substrate feature to link with either the 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 oxidation and/or sulfurization. In some instances, one or more wash steps precede or follow one or all of the steps.
Methods for the synthesis of polynucleotides on a substrate described herein may further comprise an iterating sequence of the following steps: application of a protected monomer to a surface of a substrate feature to link with either the surface, a linker or with a previously deprotected monomer; and oxidation and/or sulfurization. In some instances, one or more wash steps precede or follow one or all of the steps.
Methods for the synthesis of polynucleotides on a substrate described herein may further comprise an iterating sequence of the following steps: application of a protected monomer to a surface of a substrate feature to link with either the 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 oxidation and/or sulfurization. In some instances, one or more wash steps precede or follow one or all of the steps.
In some instances, polynucleotides 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, such as 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 such as 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.
The surface of a substrate described herein that provides support for polynucleotide synthesis may be chemically modified to allow for the synthesized polynucleotide chain to be cleaved from the surface. In some instances, the polynucleotide chain is cleaved at the same time as the polynucleotide is deprotected. In some cases, the polynucleotide chain is cleaved after the polynucleotide is deprotected. In an exemplary scheme, a trialkoxysilyl amine such as (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 an amide linkage and a free OH on which the nucleic acid chain growth is supported. Cleavage includes gas cleavage with ammonia or methylamine. In some instances cleavage includes linker cleavage with electrically generated reagents such as acids or bases. In some instances, once released from the surface, polynucleotides are assembled into larger nucleic acids that are sequenced and decoded to extract stored information.
The surfaces described herein can be reused after polynucleotide cleavage to support additional cycles of polynucleotide synthesis. For example, the linker can be reused without additional treatment/chemical modifications. In some instances, a linker is non-covalently bound to a substrate surface or a polynucleotide. In some embodiments, the linker remains attached to the polynucleotide after cleavage from the surface. Linkers in some embodiments comprise reversible covalent bonds such as esters, amides, ketals, beta substituted ketones, heterocycles, or other group that is capable of being reversibly cleaved. Such reversible cleavage reactions are in some instances controlled through the addition or removal of reagents, or by electrochemical processes controlled by electrodes. Optionally, chemical linkers or surface-bound chemical groups are regenerated after a number of cycles, to restore reactivity and remove unwanted side product formation on such linkers or surface-bound chemical groups.
Polynucleotides may be designed to collectively span a large region of a predetermined sequence that encodes for information. In some instances, larger polynucleotides are generated through ligation reactions to join the synthesized polynucleotides. One example of a ligation reaction is polymerase chain assembly (PCA). In some instances, at least of a portion of the polynucleotides are designed to include an appended region that is a substrate for universal primer binding. For PCA reactions, the presynthesized polynucleotides include overlaps with each other (e.g., 4, 20, 40 or more bases with overlapping sequence). During the polymerase cycles, the polynucleotides anneal to complementary fragments and then are filled in by polymerase. Each cycle thus increases the length of various fragments randomly depending on which polynucleotides 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 adapter sequences is amplified in a polymerase chain reaction (PCR) which includes modified primers that hybridize to the adapter sequences. In some cases, the modified primers comprise one or more uracil bases. 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.
Error correction may be performed on synthesized polynucleotides 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. 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 1, 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, CEL1, and HINF1. In some cases, DNA mismatch-binding protein MutS (Thermus 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.
After extraction and/or amplification of polynucleotides from the surface of the structure, suitable sequencing technology may be employed to sequence the polynucleotides. In some cases, the DNA sequence is read on the substrate or within a feature of a structure. In some cases, the polynucleotides stored on the substrate are extracted is optionally assembled into longer nucleic acids and then sequenced.
Polynucleotides synthesized and stored on the structures described herein encode data that can be interpreted by reading the sequence of the synthesized polynucleotides and converting the sequence into binary code readable by a computer. In some cases the sequences require assembly, and the assembly step may need to be at the nucleic acid sequence stage or at the digital sequence stage.
Provided herein are detection systems comprising a device capable of sequencing stored polynucleotides, either directly on the structure and/or after removal from the main structure. In cases where the structure is a reel-to-reel tape of flexible material, the detection system comprises a device for holding and advancing the structure through a detection location and a detector disposed proximate the detection location for detecting a signal originated from a section of the tape when the section is at the detection location. In some instances, the signal is indicative of a presence of a polynucleotide. In some instances, the signal is indicative of a sequence of a polynucleotide (e.g., a fluorescent signal). In some instances, information encoded within polynucleotides on a continuous tape is read by a computer as the tape is conveyed continuously through a detector operably connected to the computer. In some instances, a detection system comprises a computer system comprising a polynucleotide sequencing device, a database for storage and retrieval of data relating to polynucleotide sequence, software for converting DNA code of a polynucleotide sequence to binary code, a computer for reading the binary code, or any combination thereof.
Provided herein are sequencing systems that can be integrated into the devices described herein. Various methods of sequencing are well known in the art, and comprise “base calling” wherein the identity of a base in the target polynucleotide is identified. In some instances, polynucleotides synthesized using the methods, devices, compositions, and systems described herein are sequenced after cleavage from the synthesis surface. In some instances, sequencing occurs during or simultaneously with polynucleotide synthesis, wherein base calling occurs immediately after or before extension of a nucleoside monomer into the growing polynucleotide chain. Methods for base calling include measurement of electrical currents generated by polymerase-catalyzed addition of bases to a template strand. In some instances, synthesis surfaces comprise enzymes, such as polymerases. In some instances, such enzymes are tethered to electrodes or to the synthesis surface.
Computer Systems
In various aspects, any of the systems described herein are operably linked to a computer and are optionally automated through a computer either locally or remotely. In various instances, the methods and systems of the invention 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. In some instances, the computer systems are 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 800 illustrated in
As illustrated in
In some instances, a system 900 can include an accelerator card 922 attached to the peripheral bus 918. 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 924 and can be loaded into RAM 910 and/or cache 904 for use by the processor. The system 900 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 embodiments of the present invention.
In this example, system 900 also includes network interface cards (NICs) 920 and 921 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.
In some example embodiments, 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 embodiments, some or all of the processors can use a shared virtual address memory space.
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 embodiments, 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 embodiments, 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 embodiments, 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 embodiments, the computer system can be implemented using software modules executing on any of the above or other computer architectures and systems. In other embodiments, the functions of the system can be implemented partially or completely in firmware, programmable logic devices such as field programmable gate arrays (FPGAs), 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.
Provided herein are methods for storing information, comprising: a) providing a structure comprising a surface; b) depositing at least one nucleotide on the surface, wherein the at least one nucleotide couples to a polynucleotide attached to the surface; and c) repeating step b) to synthesize a plurality of polynucleotides on the surface, wherein a storage density of unique polynucleotides on the surface is at least 100×106 polynucleotides per cm2. Further provided herein are methods, wherein the method further comprises cleaving at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in a droplet. Further provided herein are methods, wherein the method further comprises sequencing at least one polynucleotide from the surface. Further provided herein are methods, wherein the method further comprises drying the surface. Further provided herein are methods, wherein the method further comprises washing the nucleotides away from the surface. Further provided herein are methods, wherein the surface is a solid support. Further provided herein are methods, wherein the surface comprises glass, fuse silica, silicon, silicon dioxide, silicon nitride, plastics, metals, or combinations thereof.
Provided herein are methods for storing information, comprising: a) providing a structure comprising a surface; b) depositing at droplet comprising at least one nucleotide on the surface, wherein the at least one nucleotide couples to a polynucleotide attached to the surface; and c) repeating step b) to synthesize a plurality of polynucleotides on the surface, wherein the droplet has a volume of less than about 100 femtoliters.
Provided herein are methods for storing information, comprising: a) providing a structure comprising a surface; b) depositing at least one nucleotide on the surface, wherein the at least one nucleotide couples to a polynucleotide attached to the surface; and c) repeating step b) to synthesize a plurality of polynucleotides on the surface, wherein the time to repeat step b) using four different nucleotides is less than about 100 milliseconds. Further provided herein are methods, wherein the method further comprises one or more wash steps. Further provided herein are methods, wherein the method further comprises deblocking, oxidizing, washing, capping, or any combination thereof.
Provided herein are devices for storing information, comprising: a chip comprising an array of addressable loci, wherein one or more addressable loci comprise at least one electrode, a synthesis surface, and at least one fluid port, wherein the synthesis surface at each addressable loci comprises at least one polynucleotide extending from the surface, wherein a density of addressable loci on the chip is at least 100×106 polynucleotides per cm2. Further provided herein are devices, wherein the at least one polynucleotide is about 150 to about 500 bases in length. Further provided herein are devices, wherein the at least one polynucleotide are about 200 bases in length. Further provided herein are devices, wherein the device further comprises a reagent reservoir. Further provided herein are devices, wherein the device further comprises a heating or cooling unit. Further provided herein are devices, wherein at least one addressable locus comprises a droplet. Further provided herein are devices, wherein the droplet is less than 50 micrometers in diameter.
Provided herein are methods for storing information, the method comprising: a) converting at least one item of information in a form of at least one digital sequence to at least one nucleic acid sequence; b) synthesizing a plurality of polynucleotides having predetermined sequences collectively encoding for the at least one nucleic acid sequence; c) depositing at droplet comprising at least one nucleotide on a surface, wherein the at least one nucleotide couples to a polynucleotide attached to the surface; d) repeating step c) to synthesize the plurality of polynucleotides on the surface; and e) storing the plurality of polynucleotides, wherein the droplet has a volume of less than about 100 femtoliters.
Provided herein are methods for storing information, the method comprising: a) converting at least one item of information in a form of at least one digital sequence to at least one nucleic acid sequence; b) synthesizing a plurality of polynucleotides having predetermined sequences collectively encoding for the at least one nucleic acid sequence; c) depositing at droplet comprising at least one nucleotide on a surface, wherein the at least one nucleotide couples to a polynucleotide attached to the surface; d) repeating step c) to synthesize the plurality of polynucleotides on the surface; and e) storing the plurality of polynucleotides, wherein the time to repeat step c) using four different nucleotides is less than about 100 milliseconds.
Provided herein are methods of synthesizing polynucleotides, comprising: a) providing a structure comprising a surface, wherein the surface comprises a plurality of loci for nucleotide extension; and b) synthesizing a plurality of polynucleotides extending from the surface, wherein synthesizing comprises depositing one or more reagents by applying a potential to the surface. Further provided are methods, wherein the potential is an electric potential. Further provided are methods, wherein the surface is a solid support.
Provided herein are devices for information storage using any one of the methods described herein. Provided herein are systems for information storage using any one of the methods described herein.
The following examples are set forth to illustrate more clearly the principle and practice of embodiments disclosed herein to those skilled in the art and are not to be construed as limiting the scope of any claimed embodiments. Unless otherwise stated, all parts and percentages are on a weight basis.
A device was functionalized to support the attachment and synthesis of a library of polynucleotides. The device surface was first wet cleaned using a piranha solution comprising 90% H2SO4 and 10% H2O2 for 20 minutes. The device was rinsed in several beakers with DI water, held under a DI water gooseneck faucet for 5 min, and dried with N2. The device 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 device was then plasma cleaned by exposing the device surface to O2. A SAMCO PC-300 instrument was used to plasma etch O2 at 250 watts for 1 min in downstream mode.
The cleaned device 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 device surface was resist coated using a Brewer Science 200× spin coater. SPR™ 3612 photoresist was spin coated on the device at 2500 rpm for 40 sec. The device was pre-baked for 30 min at 90° C. on a Brewer hot plate. The device was subjected to photolithography using a Karl Suss MA6 mask aligner instrument. The device was exposed for 2.2 sec and developed for 1 min in MSF 26A. Remaining developer was rinsed with the handgun and the device soaked in water for 5 min. The device was baked for 30 min at 100° C. in the oven, followed by visual inspection for lithography defects using a Nikon L200. A cleaning process was used to remove residual resist using the SAMCO PC-300 instrument to O2 plasma etch at 250 watts for 1 min.
The device surface was passively functionalized with a 100 μL solution of perfluorooctyltrichlorosilane mixed with 10 μL light mineral oil. The device 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 device 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 device was then soaked for 5 min in 500 mL isopropanol at room temperature with ultrasonication at maximum power. The device 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 polynucleotide synthesis.
Digital information was selected in the form of binary data totaling about 0.2 GB included content for the Universal Declaration of Human Rights in more than 100 languages, the top 100 books of Project Guttenberg and a seed database. The digital information was encrypted into a nucleic acid-based sequence and divided into strings. Over 10 million non-identical polynucleotides, each corresponding to a string, were synthesized on a rigid silicon surface. Each non-identical polynucleotide was under equal or less than 200 bases in length. The synthesized polynucleotides were collected and sequenced and decoded back to digital code, with 100% accuracy for the source digital information, compared to the initial at least one digital sequence.
Polynucleotides are de novo synthesized by methods described herein. Following synthesis, the polynucleotides are collected into a single droplet and transferred to storage on a silicon solid support. The solid support has dimensions of 86 mm×54 mm and is 1-2 mm thick. The capacity of the solid support is 1-10 petabytes (PB) that is implemented as an addressable array of 10 gigabyte packets. The physical partitioning into packets is redundantly encoded as a leading sequence with each polynucleotide within a packet sharing a common initial sequence and any other sequence information for indexing or searching. Packets are implemented as aqueous droplets with dissolved polynucleotides, with physical redundancy of 100-1000 copies of each polynucleotide. The polynucleotide length is in a range of 100-1000 bases. Droplet volume is equivalent to spheres 40-50 μm in diameter. The solid support further comprises up to 10,000×10,000 positions in an area less than a square inch.
An exemplary solid support can be seen in
Following addition of the droplets to the solid support, the solid support may be dried and later resolvated for use for downstream applications. Alternatively, the solid support is dried and stored. Because the droplets within each packet comprise sequence information for indexing and searching, specific packets are retrieved from the plurality of packets based on the sequence information.
Polynucleotides are de novo synthesized by methods described herein. Following synthesis, the polynucleotides are collected into a single droplet and transferred to storage on a paper solid support. The solid support has dimensions of 3.5 inches by 2.5 inches. The capacity of the solid support is 1 petabyte that is implemented as an addressable 32×32 array comprising 1024 spots. Each spot comprises 1 terabyte pool. See e.g.
At least one item of information of 10-100 megabytes is encoded in DNA and stored in 1 petabyte of data. At a later time, the 10-100 megabytes of encoded DNA is retrieved by random access of the encoded DNA and retrieving the encoded DNA from 1 terabyte pool.
Polynucleotides of 240 bases in length are synthesized on a solid support using the methods described herein. Polynucleotides comprising dsDNA are approximately 80 nm in length, and polynucleotides comprising ssDNA are approximately 160 nm in length. The solid support comprises an array of 500 nm (depth)×400 nm (diameter) wells (volume approximately 0.628 femtoliters). Each wells comprises an addressable locus, addressable electrodes inside the sidewalls of each well (area is 50,000 nm2/electrode), and a 250 nm (diameter) surface for polynucleotide synthesis/attachment in addressable communication with a 250 nm (diameter) addressable bottom electrode. Electrodes are independently addressable.
Polynucleotides are present on the synthesis surface at a density of 1 polynucleotide per 50 nm2. Wells are separated by a pitch of 1.0 um. 10 nm thick sidewall electrodes (located about 100 nm above the polynucleotide surface) are charged to generate a gradient of H+ ions that remove protecting groups (wherein the polynucleotide is blocked with an acid-cleavable blocking group) from the 5′ OH groups on defined polynucleotides at loci on the synthesis surface. H+ ions are then removed. (See
Polynucleotides of 240 bases in length are synthesized on a solid support using the methods described herein. The solid support comprises an array of 500 nm (depth)×500 nm (diameter) wells. Each wells comprises an addressable locus, addressable electrodes inside the sidewalls of each well (area is 50,000 nm2/electrode), and a 250 nm (diameter) surface for polynucleotide synthesis/attachment in addressable communication with a 250 nm (diameter) addressable bottom electrode. Electrodes are independently addressable.
Polynucleotides are present on the synthesis surface at a density of 1 polynucleotide per 50 nm2. Wells are separated by a pitch of 1.0 um. A nucleoside phosphoramidite monomer is added, and the polynucleotides at unblocked sites and will be available for coupling to nucleosides. Cycles of deprotection and coupling are repeated to synthesis the polynucleotides. By applying a series of electrode-controlled masks to the surface before addition of each type of monomer, the desired polynucleotides are synthesized at exact locations on the surface in a controlled sequence. Bottom electrodes in the bottom of the well are activated, which induces release of the polynucleotides attached thereto. Sidewall electrodes are charged to generate an electric field which moves the polynucleotides out of the well. Nucleoside phosphoramidite monomers are then added which extend from reusable linkers attached to the surface, and synthesis is repeated.
Polynucleotides of 240 bases in length are synthesized on a solid support using the methods described herein. Polynucleotides comprising dsDNA are approximately 80 nm in length, and polynucleotides comprising ssDNA are approximately 160 nm in length. The solid support comprises an array of 100 nm diameter addressable electrodes on the surface for polynucleotide synthesis/attachment (See
Polynucleotides are present on the synthesis surface at a density of 1 polynucleotide per 39.27 nm2. Wells are separated by a pitch of 0.25 um. The electrodes are charged to generate a gradient of H+ ions that remove protecting groups (wherein the polynucleotide is blocked with an acid-cleavable blocking group) from the 5′ OH groups on defined polynucleotides at loci on the synthesis surface. H+ ions are then removed. A nucleoside phosphoramidite monomer is added, and the polynucleotides at unblocked sites and will be available for coupling to nucleosides. Cycles of deprotection and coupling are repeated to synthesis the polynucleotides. By applying a series of electrode-controlled masks to the surface before addition of each type of monomer, the desired polynucleotides are synthesized at exact locations on the surface in a controlled sequence.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments 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 invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application is a continuation of U.S. patent application Ser. No. 16/239,453, filed Jan. 3, 2019, now U.S. Pat. No. 10,936,953, issued Mar. 2, 2021, which claims the benefit of U.S. provisional patent application No. 62/613,728 filed on Jan. 4, 2018; U.S. provisional patent application No. 62/617,067 filed on Jan. 12, 2018; and U.S. provisional patent application No. 62/650,231 filed on Mar. 29, 2018, all of which are incorporated herein by reference in their entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 3549368 | Collings et al. | Dec 1970 | A |
| 3920714 | Streck | Nov 1975 | A |
| 4123661 | Wolf et al. | Oct 1978 | A |
| 4415732 | Caruthers et al. | Nov 1983 | A |
| 4613398 | Chiong et al. | Sep 1986 | A |
| 4726877 | Fryd et al. | Feb 1988 | A |
| 4808511 | Holmes | Feb 1989 | A |
| 4837401 | Hirose et al. | Jun 1989 | A |
| 4863557 | Kokaku et al. | Sep 1989 | A |
| 4981797 | Jessee et al. | Jan 1991 | A |
| 4988617 | Landegren et al. | Jan 1991 | A |
| 5102797 | Tucker et al. | Apr 1992 | A |
| 5118605 | Urdea | Jun 1992 | A |
| 5137814 | Rashtchian et al. | Aug 1992 | A |
| 5143854 | Pirrung et al. | Sep 1992 | A |
| 5242794 | Whiteley et al. | Sep 1993 | A |
| 5242974 | Holmes | Sep 1993 | A |
| 5288514 | Ellman | Feb 1994 | A |
| 5291897 | Gastrin et al. | Mar 1994 | A |
| 5299491 | Kawada | Apr 1994 | A |
| 5368823 | McGraw et al. | Nov 1994 | A |
| 5384261 | Winkler et al. | Jan 1995 | A |
| 5387541 | Hodge et al. | Feb 1995 | A |
| 5395753 | Prakash | Mar 1995 | A |
| 5431720 | Nagai et al. | Jul 1995 | A |
| 5445934 | Fodor et al. | Aug 1995 | A |
| 5449754 | Nishioka | Sep 1995 | A |
| 5459039 | Modrich et al. | Oct 1995 | A |
| 5474796 | Brennan | Dec 1995 | A |
| 5476930 | Letsinger et al. | Dec 1995 | A |
| 5487993 | Herrnstadt et al. | Jan 1996 | A |
| 5494810 | Barany et al. | Feb 1996 | A |
| 5501893 | Laermer et al. | Mar 1996 | A |
| 5508169 | Deugau et al. | Apr 1996 | A |
| 5510270 | Fodor et al. | Apr 1996 | A |
| 5514789 | Kempe | May 1996 | A |
| 5527681 | Holmes | Jun 1996 | A |
| 5530516 | Sheets | Jun 1996 | A |
| 5556750 | Modrich et al. | Sep 1996 | A |
| 5586211 | Dumitrou et al. | Dec 1996 | A |
| 5641658 | Adams et al. | Jun 1997 | A |
| 5677195 | Winkler et al. | Oct 1997 | A |
| 5679522 | Modrich et al. | Oct 1997 | A |
| 5683879 | Laney et al. | Nov 1997 | A |
| 5688642 | Chrisey et al. | Nov 1997 | A |
| 5700637 | Southern | Dec 1997 | A |
| 5700642 | Monforte et al. | Dec 1997 | A |
| 5702894 | Modrich et al. | Dec 1997 | A |
| 5707806 | Shuber | Jan 1998 | A |
| 5712124 | Walker | Jan 1998 | A |
| 5712126 | Weissman et al. | Jan 1998 | A |
| 5739386 | Holmes | Apr 1998 | A |
| 5750672 | Kempe | May 1998 | A |
| 5780613 | Letsinger et al. | Jul 1998 | A |
| 5830643 | Yamamoto et al. | Nov 1998 | A |
| 5830655 | Monforte et al. | Nov 1998 | A |
| 5830662 | Soares et al. | Nov 1998 | A |
| 5834252 | Stemmer et al. | Nov 1998 | A |
| 5843669 | Kaiser et al. | Dec 1998 | A |
| 5843767 | Beattie | Dec 1998 | A |
| 5846717 | Brow et al. | Dec 1998 | A |
| 5854033 | Lizardi | Dec 1998 | A |
| 5858754 | Modrich et al. | Jan 1999 | A |
| 5861482 | Modrich et al. | Jan 1999 | A |
| 5863801 | Southgate et al. | Jan 1999 | A |
| 5869245 | Yeung | Feb 1999 | A |
| 5877280 | Wetmur | Mar 1999 | A |
| 5882496 | Northrup et al. | Mar 1999 | A |
| 5922539 | Modrich et al. | Jul 1999 | A |
| 5922593 | Livingston | Jul 1999 | A |
| 5928907 | Woudenberg et al. | Jul 1999 | A |
| 5962272 | Chenchik et al. | Oct 1999 | A |
| 5976842 | Wurst | Nov 1999 | A |
| 5976846 | Passmore et al. | Nov 1999 | A |
| 5989872 | Luo et al. | Nov 1999 | A |
| 5994069 | Hall et al. | Nov 1999 | A |
| 6001567 | Brow et al. | Dec 1999 | A |
| 6008031 | Modrich et al. | Dec 1999 | A |
| 6013440 | Lipshutz et al. | Jan 2000 | A |
| 6015674 | Woudenberg et al. | Jan 2000 | A |
| 6017434 | Simpson et al. | Jan 2000 | A |
| 6020481 | Benson et al. | Feb 2000 | A |
| 6027898 | Gjerde et al. | Feb 2000 | A |
| 6028189 | Blanchard | Feb 2000 | A |
| 6028198 | Liu et al. | Feb 2000 | A |
| 6040138 | Lockhart et al. | Mar 2000 | A |
| 6077674 | Schleifer et al. | Jun 2000 | A |
| 6087482 | Teng et al. | Jul 2000 | A |
| 6090543 | Prudent et al. | Jul 2000 | A |
| 6090606 | Kaiser et al. | Jul 2000 | A |
| 6103474 | Dellinger et al. | Aug 2000 | A |
| 6107038 | Choudhary et al. | Aug 2000 | A |
| 6110682 | Dellinger et al. | Aug 2000 | A |
| 6114115 | Wagner, Jr. | Sep 2000 | A |
| 6130045 | Wurst et al. | Oct 2000 | A |
| 6132997 | Shannon | Oct 2000 | A |
| 6136568 | Hiatt et al. | Oct 2000 | A |
| 6171797 | Perbost | Jan 2001 | B1 |
| 6180351 | Cattell | Jan 2001 | B1 |
| 6201112 | Ach | Mar 2001 | B1 |
| 6218118 | Sampson et al. | Apr 2001 | B1 |
| 6221653 | Caren et al. | Apr 2001 | B1 |
| 6222030 | Dellinger et al. | Apr 2001 | B1 |
| 6232072 | Fisher | May 2001 | B1 |
| 6235483 | Wolber et al. | May 2001 | B1 |
| 6242266 | Schleifer et al. | Jun 2001 | B1 |
| 6251588 | Shannon et al. | Jun 2001 | B1 |
| 6251595 | Gordon et al. | Jun 2001 | B1 |
| 6251685 | Dorsel et al. | Jun 2001 | B1 |
| 6258454 | Lefkowitz et al. | Jul 2001 | B1 |
| 6262490 | Hsu et al. | Jul 2001 | B1 |
| 6274725 | Sanghvi et al. | Aug 2001 | B1 |
| 6284465 | Wolber | Sep 2001 | B1 |
| 6287776 | Hefti | Sep 2001 | B1 |
| 6287824 | Lizardi | Sep 2001 | B1 |
| 6297017 | Schmidt et al. | Oct 2001 | B1 |
| 6300137 | Earhart et al. | Oct 2001 | B1 |
| 6306599 | Perbost | Oct 2001 | B1 |
| 6309822 | Fodor et al. | Oct 2001 | B1 |
| 6309828 | Schleifer et al. | Oct 2001 | B1 |
| 6312911 | Bancroft et al. | Nov 2001 | B1 |
| 6319674 | Fulcrand et al. | Nov 2001 | B1 |
| 6323043 | Caren et al. | Nov 2001 | B1 |
| 6329210 | Schleifer | Dec 2001 | B1 |
| 6346423 | Schembri | Feb 2002 | B1 |
| 6365355 | Mccutchen-Maloney | Apr 2002 | B1 |
| 6372483 | Schleifer et al. | Apr 2002 | B2 |
| 6375903 | Cerrina et al. | Apr 2002 | B1 |
| 6376285 | Joyner et al. | Apr 2002 | B1 |
| 6384210 | Blanchard | May 2002 | B1 |
| 6387636 | Perbost et al. | May 2002 | B1 |
| 6399394 | Dahm et al. | Jun 2002 | B1 |
| 6399516 | Ayon | Jun 2002 | B1 |
| 6403314 | Lange et al. | Jun 2002 | B1 |
| 6406849 | Dorsel et al. | Jun 2002 | B1 |
| 6406851 | Bass | Jun 2002 | B1 |
| 6408308 | Maslyn et al. | Jun 2002 | B1 |
| 6419883 | Blanchard | Jul 2002 | B1 |
| 6428957 | Delenstarr | Aug 2002 | B1 |
| 6440669 | Bass et al. | Aug 2002 | B1 |
| 6444268 | Lefkowitz et al. | Sep 2002 | B2 |
| 6446642 | Caren et al. | Sep 2002 | B1 |
| 6446682 | Viken | Sep 2002 | B1 |
| 6451998 | Perbost | Sep 2002 | B1 |
| 6458526 | Schembri et al. | Oct 2002 | B1 |
| 6458535 | Hall et al. | Oct 2002 | B1 |
| 6458583 | Bruhn et al. | Oct 2002 | B1 |
| 6461812 | Barth et al. | Oct 2002 | B2 |
| 6461816 | Wolber et al. | Oct 2002 | B1 |
| 6469156 | Schafer et al. | Oct 2002 | B1 |
| 6472147 | Janda et al. | Oct 2002 | B1 |
| 6492107 | Kauffman et al. | Dec 2002 | B1 |
| 6518056 | Schembri et al. | Feb 2003 | B2 |
| 6521427 | Evans | Feb 2003 | B1 |
| 6521453 | Crameri et al. | Feb 2003 | B1 |
| 6555357 | Kaiser et al. | Apr 2003 | B1 |
| 6558908 | Wolber et al. | May 2003 | B2 |
| 6562611 | Kaiser et al. | May 2003 | B1 |
| 6566495 | Fodor et al. | May 2003 | B1 |
| 6582908 | Fodor et al. | Jun 2003 | B2 |
| 6582938 | Su et al. | Jun 2003 | B1 |
| 6586211 | Staehler et al. | Jul 2003 | B1 |
| 6587579 | Bass | Jul 2003 | B1 |
| 6589739 | Fisher | Jul 2003 | B2 |
| 6599693 | Webb | Jul 2003 | B1 |
| 6602472 | Zimmermann et al. | Aug 2003 | B1 |
| 6610978 | Yin et al. | Aug 2003 | B2 |
| 6613513 | Parce et al. | Sep 2003 | B1 |
| 6613523 | Fischer | Sep 2003 | B2 |
| 6613560 | Tso et al. | Sep 2003 | B1 |
| 6613893 | Webb | Sep 2003 | B1 |
| 6621076 | Van De Goor et al. | Sep 2003 | B1 |
| 6630581 | Dellinger et al. | Oct 2003 | B2 |
| 6632641 | Brennan et al. | Oct 2003 | B1 |
| 6635226 | Tso et al. | Oct 2003 | B1 |
| 6642373 | Manoharan et al. | Nov 2003 | B2 |
| 6649348 | Bass et al. | Nov 2003 | B2 |
| 6660338 | Hargreaves | Dec 2003 | B1 |
| 6664112 | Mulligan et al. | Dec 2003 | B2 |
| 6670127 | Evans | Dec 2003 | B2 |
| 6670461 | Wengel et al. | Dec 2003 | B1 |
| 6673552 | Frey | Jan 2004 | B2 |
| 6682702 | Barth et al. | Jan 2004 | B2 |
| 6689319 | Fisher et al. | Feb 2004 | B1 |
| 6692917 | Neri et al. | Feb 2004 | B2 |
| 6702256 | Killeen et al. | Mar 2004 | B2 |
| 6706471 | Brow et al. | Mar 2004 | B1 |
| 6706875 | Goldberg et al. | Mar 2004 | B1 |
| 6709841 | Short | Mar 2004 | B2 |
| 6709852 | Bloom et al. | Mar 2004 | B1 |
| 6709854 | Donahue et al. | Mar 2004 | B2 |
| 6713262 | Gillibolian et al. | Mar 2004 | B2 |
| 6716629 | Hess et al. | Apr 2004 | B2 |
| 6716634 | Myerson | Apr 2004 | B1 |
| 6723509 | Ach | Apr 2004 | B2 |
| 6728129 | Lindsey et al. | Apr 2004 | B2 |
| 6743585 | Dellinger et al. | Jun 2004 | B2 |
| 6753145 | Holcomb et al. | Jun 2004 | B2 |
| 6768005 | Mellor et al. | Jul 2004 | B2 |
| 6770748 | Imanishi et al. | Aug 2004 | B2 |
| 6770892 | Corson et al. | Aug 2004 | B2 |
| 6773676 | Schembri | Aug 2004 | B2 |
| 6773888 | Li et al. | Aug 2004 | B2 |
| 6780982 | Lyamichev et al. | Aug 2004 | B2 |
| 6787308 | Balasubramanian et al. | Sep 2004 | B2 |
| 6789965 | Barth et al. | Sep 2004 | B2 |
| 6790620 | Bass et al. | Sep 2004 | B2 |
| 6794499 | Wengel et al. | Sep 2004 | B2 |
| 6796634 | Caren et al. | Sep 2004 | B2 |
| 6800439 | McGall et al. | Oct 2004 | B1 |
| 6814846 | Berndt | Nov 2004 | B1 |
| 6815218 | Jacobson et al. | Nov 2004 | B1 |
| 6824866 | Glazer, I et al. | Nov 2004 | B1 |
| 6830890 | Lockhart et al. | Dec 2004 | B2 |
| 6833246 | Balasubramanian | Dec 2004 | B2 |
| 6833450 | McGall et al. | Dec 2004 | B1 |
| 6835938 | Ghosh et al. | Dec 2004 | B2 |
| 6838888 | Peck | Jan 2005 | B2 |
| 6841131 | Zimmermann et al. | Jan 2005 | B2 |
| 6845968 | Killeen et al. | Jan 2005 | B2 |
| 6846454 | Peck | Jan 2005 | B2 |
| 6846922 | Manoharan et al. | Jan 2005 | B1 |
| 6852850 | Myerson et al. | Feb 2005 | B2 |
| 6858720 | Myerson et al. | Feb 2005 | B2 |
| 6879915 | Cattell | Apr 2005 | B2 |
| 6880576 | Karp et al. | Apr 2005 | B2 |
| 6884580 | Caren et al. | Apr 2005 | B2 |
| 6887715 | Schembri | May 2005 | B2 |
| 6890723 | Perbost et al. | May 2005 | B2 |
| 6890760 | Webb | May 2005 | B1 |
| 6893816 | Beattie | May 2005 | B1 |
| 6897023 | Fu et al. | May 2005 | B2 |
| 6900047 | Bass | May 2005 | B2 |
| 6900048 | Perbost | May 2005 | B2 |
| 6911611 | Wong et al. | Jun 2005 | B2 |
| 6914229 | Corson et al. | Jul 2005 | B2 |
| 6916113 | Van De Goor et al. | Jul 2005 | B2 |
| 6916633 | Shannon | Jul 2005 | B1 |
| 6919181 | Hargreaves | Jul 2005 | B2 |
| 6927029 | Lefkowitz et al. | Aug 2005 | B2 |
| 6929951 | Corson et al. | Aug 2005 | B2 |
| 6936472 | Earhart et al. | Aug 2005 | B2 |
| 6938476 | Chesk | Sep 2005 | B2 |
| 6939673 | Bass et al. | Sep 2005 | B2 |
| 6943036 | Bass | Sep 2005 | B2 |
| 6946285 | Bass | Sep 2005 | B2 |
| 6950756 | Kincaid | Sep 2005 | B2 |
| 6951719 | Dupret et al. | Oct 2005 | B1 |
| 6958119 | Yin et al. | Oct 2005 | B2 |
| 6960464 | Jessee et al. | Nov 2005 | B2 |
| 6969449 | Maher et al. | Nov 2005 | B2 |
| 6969488 | Bridgham et al. | Nov 2005 | B2 |
| 6976384 | Hobbs et al. | Dec 2005 | B2 |
| 6977223 | George et al. | Dec 2005 | B2 |
| 6987263 | Hobbs et al. | Jan 2006 | B2 |
| 6989267 | Kim et al. | Jan 2006 | B2 |
| 6991922 | Dupret et al. | Jan 2006 | B2 |
| 7008037 | Caren et al. | Mar 2006 | B2 |
| 7025324 | Slocum et al. | Apr 2006 | B1 |
| 7026124 | Barth et al. | Apr 2006 | B2 |
| 7027930 | Cattell | Apr 2006 | B2 |
| 7028536 | Karp et al. | Apr 2006 | B2 |
| 7029854 | Collins et al. | Apr 2006 | B2 |
| 7034290 | Lu et al. | Apr 2006 | B2 |
| 7041445 | Chenchik et al. | May 2006 | B2 |
| 7045289 | Allawi et al. | May 2006 | B2 |
| 7051574 | Peck | May 2006 | B2 |
| 7052841 | Delenstarr | May 2006 | B2 |
| 7062385 | White et al. | Jun 2006 | B2 |
| 7064197 | Rabbani et al. | Jun 2006 | B1 |
| 7070932 | Leproust et al. | Jul 2006 | B2 |
| 7075161 | Barth | Jul 2006 | B2 |
| 7078167 | Delenstarr et al. | Jul 2006 | B2 |
| 7078505 | Bass et al. | Jul 2006 | B2 |
| 7094537 | Leproust et al. | Aug 2006 | B2 |
| 7097974 | Stahler et al. | Aug 2006 | B1 |
| 7101508 | Thompson et al. | Sep 2006 | B2 |
| 7101986 | Dellinger et al. | Sep 2006 | B2 |
| 7105295 | Bass et al. | Sep 2006 | B2 |
| 7115423 | Mitchell | Oct 2006 | B1 |
| 7122303 | Delenstarr et al. | Oct 2006 | B2 |
| 7122364 | Lyamichev, I et al. | Oct 2006 | B1 |
| 7125488 | Li | Oct 2006 | B2 |
| 7125523 | Sillman | Oct 2006 | B2 |
| 7128876 | Yin et al. | Oct 2006 | B2 |
| 7129075 | Gerard et al. | Oct 2006 | B2 |
| 7135565 | Dellinger et al. | Nov 2006 | B2 |
| 7138062 | Yin et al. | Nov 2006 | B2 |
| 7141368 | Fisher et al. | Nov 2006 | B2 |
| 7141807 | Joyce et al. | Nov 2006 | B2 |
| 7147362 | Caren et al. | Dec 2006 | B2 |
| 7150982 | Allawi et al. | Dec 2006 | B2 |
| 7153689 | Tolosko et al. | Dec 2006 | B2 |
| 7163660 | Lehmann | Jan 2007 | B2 |
| 7166258 | Bass et al. | Jan 2007 | B2 |
| 7179659 | Stolowitz et al. | Feb 2007 | B2 |
| 7183406 | Belshaw et al. | Feb 2007 | B2 |
| 7192710 | Gellibolian et al. | Mar 2007 | B2 |
| 7193077 | Dellinger et al. | Mar 2007 | B2 |
| 7195872 | Agrawal et al. | Mar 2007 | B2 |
| 7198939 | Dorsel et al. | Apr 2007 | B2 |
| 7202264 | Ravikumar et al. | Apr 2007 | B2 |
| 7202358 | Hargreaves | Apr 2007 | B2 |
| 7205128 | Ilsley et al. | Apr 2007 | B2 |
| 7205400 | Webb | Apr 2007 | B2 |
| 7206439 | Zhou et al. | Apr 2007 | B2 |
| 7208322 | Stolowitz et al. | Apr 2007 | B2 |
| 7217522 | Brenner | May 2007 | B2 |
| 7220573 | Shea et al. | May 2007 | B2 |
| 7221785 | Curry et al. | May 2007 | B2 |
| 7226862 | Staehler et al. | Jun 2007 | B2 |
| 7227017 | Mellor et al. | Jun 2007 | B2 |
| 7229497 | Stott et al. | Jun 2007 | B2 |
| 7247337 | Leproust et al. | Jul 2007 | B1 |
| 7247497 | Dahm et al. | Jul 2007 | B2 |
| 7252938 | Leproust et al. | Aug 2007 | B2 |
| 7269518 | Corson | Sep 2007 | B2 |
| 7271258 | Dellinger et al. | Sep 2007 | B2 |
| 7276336 | Webb et al. | Oct 2007 | B1 |
| 7276378 | Myerson | Oct 2007 | B2 |
| 7276599 | Moore et al. | Oct 2007 | B2 |
| 7282183 | Peck | Oct 2007 | B2 |
| 7282332 | Caren et al. | Oct 2007 | B2 |
| 7282705 | Brennen | Oct 2007 | B2 |
| 7291471 | Sampson et al. | Nov 2007 | B2 |
| 7302348 | Ghosh et al. | Nov 2007 | B2 |
| 7306917 | Prudent et al. | Dec 2007 | B2 |
| 7314599 | Roitman et al. | Jan 2008 | B2 |
| 7323320 | Oleinikov | Jan 2008 | B2 |
| 7344831 | Wolber et al. | Mar 2008 | B2 |
| 7348144 | Minor | Mar 2008 | B2 |
| 7351379 | Schleifer | Apr 2008 | B2 |
| 7353116 | Webb et al. | Apr 2008 | B2 |
| 7361906 | Ghosh et al. | Apr 2008 | B2 |
| 7364896 | Schembri | Apr 2008 | B2 |
| 7368550 | Dellinger et al. | May 2008 | B2 |
| 7371348 | Schleifer et al. | May 2008 | B2 |
| 7371519 | Wolber et al. | May 2008 | B2 |
| 7371580 | Yakhini et al. | May 2008 | B2 |
| 7372982 | Le | May 2008 | B2 |
| 7384746 | Lyamichev et al. | Jun 2008 | B2 |
| 7385050 | Dellinger et al. | Jun 2008 | B2 |
| 7390457 | Schembri | Jun 2008 | B2 |
| 7393665 | Brenner | Jul 2008 | B2 |
| 7396676 | Robotti et al. | Jul 2008 | B2 |
| 7399844 | Sampson et al. | Jul 2008 | B2 |
| 7402279 | Schembri | Jul 2008 | B2 |
| 7411061 | Myerson et al. | Aug 2008 | B2 |
| 7413709 | Roitman et al. | Aug 2008 | B2 |
| 7417139 | Dellinger et al. | Aug 2008 | B2 |
| 7422911 | Schembri | Sep 2008 | B2 |
| 7427679 | Dellinger et al. | Sep 2008 | B2 |
| 7432048 | Neri et al. | Oct 2008 | B2 |
| 7435810 | Myerson et al. | Oct 2008 | B2 |
| 7439272 | Xu | Oct 2008 | B2 |
| 7476709 | Moody et al. | Jan 2009 | B2 |
| 7482118 | Allawi et al. | Jan 2009 | B2 |
| 7488607 | Tom-Moy et al. | Feb 2009 | B2 |
| 7504213 | Sana et al. | Mar 2009 | B2 |
| 7514369 | Li et al. | Apr 2009 | B2 |
| 7517979 | Wolber | Apr 2009 | B2 |
| 7524942 | Wang et al. | Apr 2009 | B2 |
| 7524950 | Dellinger et al. | Apr 2009 | B2 |
| 7527928 | Neri et al. | May 2009 | B2 |
| 7531303 | Dorsel et al. | May 2009 | B2 |
| 7534561 | Sana et al. | May 2009 | B2 |
| 7534563 | Hargreaves | May 2009 | B2 |
| 7537936 | Dahm et al. | May 2009 | B2 |
| 7541145 | Prudent et al. | Jun 2009 | B2 |
| 7544473 | Brenner | Jun 2009 | B2 |
| 7556919 | Chenchik et al. | Jul 2009 | B2 |
| 7563600 | Oleinikov | Jul 2009 | B2 |
| 7572585 | Wang | Aug 2009 | B2 |
| 7572907 | Dellinger et al. | Aug 2009 | B2 |
| 7572908 | Dellinger et al. | Aug 2009 | B2 |
| 7585970 | Dellinger et al. | Sep 2009 | B2 |
| 7588889 | Wolber et al. | Sep 2009 | B2 |
| 7595350 | Xu | Sep 2009 | B2 |
| 7604941 | Jacobson | Oct 2009 | B2 |
| 7604996 | Stuelpnagel et al. | Oct 2009 | B1 |
| 7608396 | Delenstarr | Oct 2009 | B2 |
| 7618777 | Myerson et al. | Nov 2009 | B2 |
| 7629120 | Bennett et al. | Dec 2009 | B2 |
| 7635772 | McCormac | Dec 2009 | B2 |
| 7648832 | Jessee et al. | Jan 2010 | B2 |
| 7651762 | Xu et al. | Jan 2010 | B2 |
| 7659069 | Belyaev et al. | Feb 2010 | B2 |
| 7678542 | Lyamichev et al. | Mar 2010 | B2 |
| 7682809 | Sampson | Mar 2010 | B2 |
| 7709197 | Drmanac | May 2010 | B2 |
| 7718365 | Wang | May 2010 | B2 |
| 7718786 | Dupret et al. | May 2010 | B2 |
| 7723077 | Young et al. | May 2010 | B2 |
| 7737088 | Stahler et al. | Jun 2010 | B1 |
| 7737089 | Guimil et al. | Jun 2010 | B2 |
| 7741463 | Gormley et al. | Jun 2010 | B2 |
| 7749701 | Leproust et al. | Jul 2010 | B2 |
| 7759471 | Dellinger et al. | Jul 2010 | B2 |
| 7776021 | Borenstein et al. | Aug 2010 | B2 |
| 7776532 | Gibson et al. | Aug 2010 | B2 |
| 7790369 | Stahler et al. | Sep 2010 | B2 |
| 7790387 | Dellinger et al. | Sep 2010 | B2 |
| 7807356 | Sampson et al. | Oct 2010 | B2 |
| 7807806 | Allawi et al. | Oct 2010 | B2 |
| 7811753 | Eshoo | Oct 2010 | B2 |
| 7816079 | Fischer | Oct 2010 | B2 |
| 7820387 | Neri et al. | Oct 2010 | B2 |
| 7829314 | Prudent et al. | Nov 2010 | B2 |
| 7855281 | Dellinger et al. | Dec 2010 | B2 |
| 7862999 | Zheng et al. | Jan 2011 | B2 |
| 7867782 | Barth | Jan 2011 | B2 |
| 7875463 | Adaskin et al. | Jan 2011 | B2 |
| 7879541 | Kincaid | Feb 2011 | B2 |
| 7879580 | Carr et al. | Feb 2011 | B2 |
| 7894998 | Kincaid | Feb 2011 | B2 |
| 7919239 | Wang | Apr 2011 | B2 |
| 7919308 | Schleifer | Apr 2011 | B2 |
| 7927797 | Nobile et al. | Apr 2011 | B2 |
| 7927838 | Shannon | Apr 2011 | B2 |
| 7932025 | Carr et al. | Apr 2011 | B2 |
| 7932070 | Hogrefe et al. | Apr 2011 | B2 |
| 7935800 | Allawi et al. | May 2011 | B2 |
| 7939645 | Borns | May 2011 | B2 |
| 7943046 | Martosella et al. | May 2011 | B2 |
| 7943358 | Hogrefe et al. | May 2011 | B2 |
| 7960157 | Borns | Jun 2011 | B2 |
| 7977119 | Kronick et al. | Jul 2011 | B2 |
| 7979215 | Sampas | Jul 2011 | B2 |
| 7998437 | Berndt et al. | Aug 2011 | B2 |
| 7999087 | Dellinger et al. | Aug 2011 | B2 |
| 8021842 | Brenner | Sep 2011 | B2 |
| 8021844 | Wang | Sep 2011 | B2 |
| 8034917 | Yamada | Oct 2011 | B2 |
| 8036835 | Sampas et al. | Oct 2011 | B2 |
| 8048664 | Guan et al. | Nov 2011 | B2 |
| 8053191 | Blake | Nov 2011 | B2 |
| 8058001 | Crameri et al. | Nov 2011 | B2 |
| 8058004 | Oleinikov | Nov 2011 | B2 |
| 8058055 | Barrett et al. | Nov 2011 | B2 |
| 8063184 | Allawi et al. | Nov 2011 | B2 |
| 8067556 | Hogrefe et al. | Nov 2011 | B2 |
| 8073626 | Troup et al. | Dec 2011 | B2 |
| 8076064 | Wang | Dec 2011 | B2 |
| 8076152 | Robotti | Dec 2011 | B2 |
| 8097711 | Timar et al. | Jan 2012 | B2 |
| 8137936 | Macevicz | Mar 2012 | B2 |
| 8148068 | Brenner | Apr 2012 | B2 |
| 8154729 | Baldo et al. | Apr 2012 | B2 |
| 8168385 | Brenner | May 2012 | B2 |
| 8168388 | Gormley et al. | May 2012 | B2 |
| 8173368 | Staehler et al. | May 2012 | B2 |
| 8182991 | Kaiser et al. | May 2012 | B1 |
| 8194244 | Wang et al. | Jun 2012 | B2 |
| 8198071 | Goshoo et al. | Jun 2012 | B2 |
| 8202983 | Dellinger et al. | Jun 2012 | B2 |
| 8202985 | Dellinger et al. | Jun 2012 | B2 |
| 8206952 | Carr et al. | Jun 2012 | B2 |
| 8213015 | Kraiczek et al. | Jul 2012 | B2 |
| 8242258 | Dellinger et al. | Aug 2012 | B2 |
| 8247221 | Fawcett et al. | Aug 2012 | B2 |
| 8263335 | Carr et al. | Sep 2012 | B2 |
| 8268605 | Sorge et al. | Sep 2012 | B2 |
| 8283148 | Sorge et al. | Oct 2012 | B2 |
| 8288093 | Hall et al. | Oct 2012 | B2 |
| 8298767 | Brenner et al. | Oct 2012 | B2 |
| 8304273 | Stellacci et al. | Nov 2012 | B2 |
| 8309307 | Barrett et al. | Nov 2012 | B2 |
| 8309706 | Dellinger et al. | Nov 2012 | B2 |
| 8309710 | Sierzchala et al. | Nov 2012 | B2 |
| 8314220 | Mullinax et al. | Nov 2012 | B2 |
| 8318433 | Brenner | Nov 2012 | B2 |
| 8318479 | Domansky et al. | Nov 2012 | B2 |
| 8357489 | Chua et al. | Jan 2013 | B2 |
| 8357490 | Froehlich et al. | Jan 2013 | B2 |
| 8367016 | Quan et al. | Feb 2013 | B2 |
| 8367335 | Staehler et al. | Feb 2013 | B2 |
| 8380441 | Webb et al. | Feb 2013 | B2 |
| 8383338 | Kitzman et al. | Feb 2013 | B2 |
| 8415138 | Leproust | Apr 2013 | B2 |
| 8435736 | Gibson et al. | May 2013 | B2 |
| 8445205 | Brenner | May 2013 | B2 |
| 8445206 | Bergmann et al. | May 2013 | B2 |
| 8470996 | Brenner | Jun 2013 | B2 |
| 8476018 | Brenner | Jul 2013 | B2 |
| 8476598 | Pralle et al. | Jul 2013 | B1 |
| 8481292 | Casbon et al. | Jul 2013 | B2 |
| 8481309 | Zhang et al. | Jul 2013 | B2 |
| 8491561 | Borenstein et al. | Jul 2013 | B2 |
| 8497069 | Hutchison et al. | Jul 2013 | B2 |
| 8500979 | Elibol et al. | Aug 2013 | B2 |
| 8501454 | Liu et al. | Aug 2013 | B2 |
| 8507226 | Carr et al. | Aug 2013 | B2 |
| 8507239 | Lubys et al. | Aug 2013 | B2 |
| 8507272 | Zhang et al. | Aug 2013 | B2 |
| 8530197 | Li et al. | Sep 2013 | B2 |
| 8552174 | Dellinger et al. | Oct 2013 | B2 |
| 8563478 | Gormley et al. | Oct 2013 | B2 |
| 8569046 | Love et al. | Oct 2013 | B2 |
| 8577621 | Troup et al. | Nov 2013 | B2 |
| 8586310 | Mitra et al. | Nov 2013 | B2 |
| 8614092 | Zhang et al. | Dec 2013 | B2 |
| 8642755 | Sierzchala et al. | Feb 2014 | B2 |
| 8664164 | Ericsson et al. | Mar 2014 | B2 |
| 8669053 | Stuelpnagel et al. | Mar 2014 | B2 |
| 8679756 | Brenner et al. | Mar 2014 | B1 |
| 8685642 | Sampas | Apr 2014 | B2 |
| 8685676 | Hogrefe et al. | Apr 2014 | B2 |
| 8685678 | Casbon et al. | Apr 2014 | B2 |
| 8715933 | Oliver | May 2014 | B2 |
| 8715967 | Casbon et al. | May 2014 | B2 |
| 8716467 | Jacobson | May 2014 | B2 |
| 8722368 | Casbon et al. | May 2014 | B2 |
| 8722585 | Wang | May 2014 | B2 |
| 8728766 | Casbon et al. | May 2014 | B2 |
| 8741606 | Casbon et al. | Jun 2014 | B2 |
| 8808896 | Choo et al. | Aug 2014 | B2 |
| 8808986 | Jacobson et al. | Aug 2014 | B2 |
| 8815600 | Liu et al. | Aug 2014 | B2 |
| 8889851 | Leproust et al. | Nov 2014 | B2 |
| 8932994 | Gormley et al. | Jan 2015 | B2 |
| 8962532 | Shapiro et al. | Feb 2015 | B2 |
| 8968999 | Gibson et al. | Mar 2015 | B2 |
| 8980563 | Zheng et al. | Mar 2015 | B2 |
| 9018365 | Brenner | Apr 2015 | B2 |
| 9023601 | Oleinikov | May 2015 | B2 |
| 9051666 | Oleinikov | Jun 2015 | B2 |
| 9073962 | Fracchia et al. | Jul 2015 | B2 |
| 9074204 | Anderson et al. | Jul 2015 | B2 |
| 9085797 | Gebeyehu et al. | Jul 2015 | B2 |
| 9133510 | Andersen et al. | Sep 2015 | B2 |
| 9139874 | Myers et al. | Sep 2015 | B2 |
| 9150853 | Hudson et al. | Oct 2015 | B2 |
| 9187777 | Jacobson et al. | Nov 2015 | B2 |
| 9194001 | Brenner | Nov 2015 | B2 |
| 9216414 | Chu | Dec 2015 | B2 |
| 9217144 | Jacobson et al. | Dec 2015 | B2 |
| 9279149 | Efcavitch et al. | Mar 2016 | B2 |
| 9286439 | Shapiro et al. | Mar 2016 | B2 |
| 9295965 | Jacobson et al. | Mar 2016 | B2 |
| 9315861 | Hendricks et al. | Apr 2016 | B2 |
| 9328378 | Earnshaw et al. | May 2016 | B2 |
| 9347091 | Bergmann et al. | May 2016 | B2 |
| 9375748 | Harumoto et al. | Jun 2016 | B2 |
| 9376677 | Mir | Jun 2016 | B2 |
| 9376678 | Gormley et al. | Jun 2016 | B2 |
| 9384320 | Church | Jul 2016 | B2 |
| 9384920 | Bakulich | Jul 2016 | B1 |
| 9388407 | Jacobson | Jul 2016 | B2 |
| 9394333 | Wada et al. | Jul 2016 | B2 |
| 9403141 | Banyai et al. | Aug 2016 | B2 |
| 9409139 | Banyai et al. | Aug 2016 | B2 |
| 9410149 | Brenner et al. | Aug 2016 | B2 |
| 9410173 | Betts et al. | Aug 2016 | B2 |
| 9416411 | Stuelpnagel et al. | Aug 2016 | B2 |
| 9422600 | Ramu et al. | Aug 2016 | B2 |
| 9487824 | Kutyavin | Nov 2016 | B2 |
| 9499848 | Carr et al. | Nov 2016 | B2 |
| 9523122 | Zheng et al. | Dec 2016 | B2 |
| 9528148 | Zheng et al. | Dec 2016 | B2 |
| 9534251 | Young et al. | Jan 2017 | B2 |
| 9555388 | Banyai et al. | Jan 2017 | B2 |
| 9568839 | Stahler et al. | Feb 2017 | B2 |
| 9580746 | Leproust et al. | Feb 2017 | B2 |
| 9670529 | Osborne et al. | Jun 2017 | B2 |
| 9670536 | Casbon et al. | Jun 2017 | B2 |
| 9677067 | Toro et al. | Jun 2017 | B2 |
| 9695211 | Wada et al. | Jul 2017 | B2 |
| 9718060 | Venter et al. | Aug 2017 | B2 |
| 9745573 | Stuelpnagel et al. | Aug 2017 | B2 |
| 9745619 | Rabbani et al. | Aug 2017 | B2 |
| 9765387 | Rabbani et al. | Sep 2017 | B2 |
| 9771576 | Gibson et al. | Sep 2017 | B2 |
| 9833761 | Banyai et al. | Dec 2017 | B2 |
| 9834774 | Carstens | Dec 2017 | B2 |
| 9839894 | Banyai et al. | Dec 2017 | B2 |
| 9879283 | Ravinder et al. | Jan 2018 | B2 |
| 9889423 | Banyai et al. | Feb 2018 | B2 |
| 9895673 | Peck et al. | Feb 2018 | B2 |
| 9925510 | Jacobson et al. | Mar 2018 | B2 |
| 9932576 | Raymond et al. | Apr 2018 | B2 |
| 9981239 | Banyai et al. | May 2018 | B2 |
| 10053688 | Cox | Aug 2018 | B2 |
| 10272410 | Banyai et al. | Apr 2019 | B2 |
| 10384188 | Banyai et al. | Aug 2019 | B2 |
| 10384189 | Peck | Aug 2019 | B2 |
| 10417457 | Peck | Sep 2019 | B2 |
| 10583415 | Banyai et al. | Mar 2020 | B2 |
| 10618024 | Banyai et al. | Apr 2020 | B2 |
| 10632445 | Banyai et al. | Apr 2020 | B2 |
| 10639609 | Banyai et al. | May 2020 | B2 |
| 10669304 | Indermuhle et al. | Jun 2020 | B2 |
| 10744477 | Banyai et al. | Aug 2020 | B2 |
| 10754994 | Peck | Aug 2020 | B2 |
| 10773232 | Banyai et al. | Sep 2020 | B2 |
| 10844373 | Cox et al. | Nov 2020 | B2 |
| 10894242 | Marsh et al. | Jan 2021 | B2 |
| 10894959 | Cox et al. | Jan 2021 | B2 |
| 10907274 | Cox | Feb 2021 | B2 |
| 10936953 | Peck | Mar 2021 | B2 |
| 10969965 | Malina et al. | Apr 2021 | B2 |
| 20010018512 | Blanchard | Aug 2001 | A1 |
| 20010039014 | Bass et al. | Nov 2001 | A1 |
| 20010055761 | Kanemoto et al. | Dec 2001 | A1 |
| 20020012930 | Rothberg et al. | Jan 2002 | A1 |
| 20020025561 | Hodgson | Feb 2002 | A1 |
| 20020076716 | Sabanayagam et al. | Jun 2002 | A1 |
| 20020081582 | Gao et al. | Jun 2002 | A1 |
| 20020094533 | Hess et al. | Jul 2002 | A1 |
| 20020095073 | Jacobs et al. | Jul 2002 | A1 |
| 20020119459 | Griffiths | Aug 2002 | A1 |
| 20020132308 | Liu et al. | Sep 2002 | A1 |
| 20020155439 | Rodriguez et al. | Oct 2002 | A1 |
| 20020160536 | Regnier et al. | Oct 2002 | A1 |
| 20020164824 | Xiao et al. | Nov 2002 | A1 |
| 20030008411 | Van Dam et al. | Jan 2003 | A1 |
| 20030022207 | Balasubramanian et al. | Jan 2003 | A1 |
| 20030022240 | Luo et al. | Jan 2003 | A1 |
| 20030022317 | Jack et al. | Jan 2003 | A1 |
| 20030044781 | Korlach et al. | Mar 2003 | A1 |
| 20030058629 | Hirai et al. | Mar 2003 | A1 |
| 20030064398 | Barnes | Apr 2003 | A1 |
| 20030068633 | Belshaw et al. | Apr 2003 | A1 |
| 20030082719 | Schumacher et al. | May 2003 | A1 |
| 20030100102 | Rothberg et al. | May 2003 | A1 |
| 20030108903 | Wang et al. | Jun 2003 | A1 |
| 20030120035 | Gao et al. | Jun 2003 | A1 |
| 20030130827 | Bentzien et al. | Jul 2003 | A1 |
| 20030138782 | Evans | Jul 2003 | A1 |
| 20030143605 | Lok et al. | Jul 2003 | A1 |
| 20030148291 | Robotti | Aug 2003 | A1 |
| 20030148344 | Rothberg et al. | Aug 2003 | A1 |
| 20030169618 | Lindsey et al. | Sep 2003 | A1 |
| 20030171325 | Gascoyne et al. | Sep 2003 | A1 |
| 20030186226 | Brennan et al. | Oct 2003 | A1 |
| 20030228602 | Parker et al. | Dec 2003 | A1 |
| 20030228620 | Du | Dec 2003 | A1 |
| 20040009498 | Short | Jan 2004 | A1 |
| 20040043509 | Stahler et al. | Mar 2004 | A1 |
| 20040053362 | De et al. | Mar 2004 | A1 |
| 20040086892 | Crothers et al. | May 2004 | A1 |
| 20040087008 | Schembri | May 2004 | A1 |
| 20040106130 | Besemer et al. | Jun 2004 | A1 |
| 20040106728 | McGall et al. | Jun 2004 | A1 |
| 20040110133 | Xu et al. | Jun 2004 | A1 |
| 20040175710 | Haushalter | Sep 2004 | A1 |
| 20040175734 | Stahler et al. | Sep 2004 | A1 |
| 20040191810 | Yamamoto | Sep 2004 | A1 |
| 20040213795 | Collins et al. | Oct 2004 | A1 |
| 20040219663 | Page et al. | Nov 2004 | A1 |
| 20040236027 | Maeji et al. | Nov 2004 | A1 |
| 20040248161 | Rothberg et al. | Dec 2004 | A1 |
| 20040259146 | Friend et al. | Dec 2004 | A1 |
| 20050022895 | Barth et al. | Feb 2005 | A1 |
| 20050049402 | Babcook et al. | Mar 2005 | A1 |
| 20050049796 | Webb et al. | Mar 2005 | A1 |
| 20050053968 | Bharadwaj et al. | Mar 2005 | A1 |
| 20050079510 | Berka et al. | Apr 2005 | A1 |
| 20050100932 | Lapidus et al. | May 2005 | A1 |
| 20050112608 | Grossman et al. | May 2005 | A1 |
| 20050112636 | Hurt et al. | May 2005 | A1 |
| 20050112679 | Myerson et al. | May 2005 | A1 |
| 20050124022 | Srinivasan et al. | Jun 2005 | A1 |
| 20050137805 | Lewin et al. | Jun 2005 | A1 |
| 20050208513 | Agbo et al. | Sep 2005 | A1 |
| 20050214778 | Peck et al. | Sep 2005 | A1 |
| 20050227235 | Carr et al. | Oct 2005 | A1 |
| 20050255477 | Carr et al. | Nov 2005 | A1 |
| 20050266045 | Canham et al. | Dec 2005 | A1 |
| 20050277125 | Benn et al. | Dec 2005 | A1 |
| 20050282158 | Landegren | Dec 2005 | A1 |
| 20050287585 | Oleinikov | Dec 2005 | A1 |
| 20060003381 | Gilmore et al. | Jan 2006 | A1 |
| 20060003958 | Melville et al. | Jan 2006 | A1 |
| 20060012784 | Ulmer | Jan 2006 | A1 |
| 20060012793 | Harris | Jan 2006 | A1 |
| 20060019084 | Pearson | Jan 2006 | A1 |
| 20060024678 | Buzby | Feb 2006 | A1 |
| 20060024711 | Lapidus et al. | Feb 2006 | A1 |
| 20060024721 | Pedersen | Feb 2006 | A1 |
| 20060076482 | Hobbs et al. | Apr 2006 | A1 |
| 20060078909 | Srinivasan et al. | Apr 2006 | A1 |
| 20060078927 | Peck et al. | Apr 2006 | A1 |
| 20060078937 | Korlach et al. | Apr 2006 | A1 |
| 20060127920 | Church et al. | Jun 2006 | A1 |
| 20060134638 | Mulligan et al. | Jun 2006 | A1 |
| 20060160138 | Church | Jul 2006 | A1 |
| 20060171855 | Yin et al. | Aug 2006 | A1 |
| 20060202330 | Reinhardt et al. | Sep 2006 | A1 |
| 20060203236 | Ji et al. | Sep 2006 | A1 |
| 20060203237 | Ji et al. | Sep 2006 | A1 |
| 20060207923 | Li | Sep 2006 | A1 |
| 20060219637 | Killeen et al. | Oct 2006 | A1 |
| 20070031857 | Makarov et al. | Feb 2007 | A1 |
| 20070031877 | Stahler et al. | Feb 2007 | A1 |
| 20070043516 | Gustafsson et al. | Feb 2007 | A1 |
| 20070054127 | Hergenrother et al. | Mar 2007 | A1 |
| 20070059692 | Gao et al. | Mar 2007 | A1 |
| 20070087349 | Staehler et al. | Apr 2007 | A1 |
| 20070099208 | Drmanac et al. | May 2007 | A1 |
| 20070122817 | Church et al. | May 2007 | A1 |
| 20070128635 | Macevicz | Jun 2007 | A1 |
| 20070141557 | Raab et al. | Jun 2007 | A1 |
| 20070196834 | Cerrina et al. | Aug 2007 | A1 |
| 20070196854 | Stahler et al. | Aug 2007 | A1 |
| 20070207482 | Church et al. | Sep 2007 | A1 |
| 20070207487 | Emig et al. | Sep 2007 | A1 |
| 20070231800 | Roberts et al. | Oct 2007 | A1 |
| 20070238104 | Barrett et al. | Oct 2007 | A1 |
| 20070238106 | Barrett et al. | Oct 2007 | A1 |
| 20070238108 | Barrett et al. | Oct 2007 | A1 |
| 20070259344 | Leproust et al. | Nov 2007 | A1 |
| 20070259345 | Sampas | Nov 2007 | A1 |
| 20070259346 | Gordon et al. | Nov 2007 | A1 |
| 20070259347 | Gordon et al. | Nov 2007 | A1 |
| 20070269870 | Church et al. | Nov 2007 | A1 |
| 20080085511 | Peck et al. | Apr 2008 | A1 |
| 20080085514 | Peck et al. | Apr 2008 | A1 |
| 20080087545 | Jensen et al. | Apr 2008 | A1 |
| 20080161200 | Yu et al. | Jul 2008 | A1 |
| 20080182296 | Chanda et al. | Jul 2008 | A1 |
| 20080214412 | Stahler et al. | Sep 2008 | A1 |
| 20080227160 | Kool | Sep 2008 | A1 |
| 20080233616 | Liss | Sep 2008 | A1 |
| 20080287320 | Baynes et al. | Nov 2008 | A1 |
| 20080300842 | Govindarajan et al. | Dec 2008 | A1 |
| 20080308884 | Kalvesten | Dec 2008 | A1 |
| 20080311628 | Shoemaker | Dec 2008 | A1 |
| 20090036664 | Peter | Feb 2009 | A1 |
| 20090053704 | Novoradovskaya et al. | Feb 2009 | A1 |
| 20090062129 | McKernan et al. | Mar 2009 | A1 |
| 20090074771 | Koenig et al. | Mar 2009 | A1 |
| 20090087840 | Baynes et al. | Apr 2009 | A1 |
| 20090088679 | Wood et al. | Apr 2009 | A1 |
| 20090105094 | Heiner et al. | Apr 2009 | A1 |
| 20090170802 | Stahler et al. | Jul 2009 | A1 |
| 20090176280 | Hutchison, III et al. | Jul 2009 | A1 |
| 20090181861 | Li et al. | Jul 2009 | A1 |
| 20090194483 | Robotti et al. | Aug 2009 | A1 |
| 20090230044 | Bek | Sep 2009 | A1 |
| 20090238722 | Mora-Fillat et al. | Sep 2009 | A1 |
| 20090239759 | Balch | Sep 2009 | A1 |
| 20090246788 | Albert et al. | Oct 2009 | A1 |
| 20090263802 | Drmanac | Oct 2009 | A1 |
| 20090285825 | Kini et al. | Nov 2009 | A1 |
| 20090324546 | Notka et al. | Dec 2009 | A1 |
| 20100004143 | Shibahara | Jan 2010 | A1 |
| 20100008851 | Nicolaides et al. | Jan 2010 | A1 |
| 20100009872 | Eid et al. | Jan 2010 | A1 |
| 20100047805 | Wang | Feb 2010 | A1 |
| 20100051967 | Bradley et al. | Mar 2010 | A1 |
| 20100069250 | White, III et al. | Mar 2010 | A1 |
| 20100090341 | Wan et al. | Apr 2010 | A1 |
| 20100099103 | Hsieh et al. | Apr 2010 | A1 |
| 20100111768 | Banerjee et al. | May 2010 | A1 |
| 20100160463 | Wang et al. | Jun 2010 | A1 |
| 20100167950 | Juang et al. | Jul 2010 | A1 |
| 20100173364 | Evans, Jr. et al. | Jul 2010 | A1 |
| 20100216648 | Staehler et al. | Aug 2010 | A1 |
| 20100256017 | Larman et al. | Oct 2010 | A1 |
| 20100258487 | Zelechonok et al. | Oct 2010 | A1 |
| 20100272711 | Feldman et al. | Oct 2010 | A1 |
| 20100286290 | Lohmann et al. | Nov 2010 | A1 |
| 20100292102 | Nouri | Nov 2010 | A1 |
| 20100300882 | Zhang et al. | Dec 2010 | A1 |
| 20110009607 | Komiyama et al. | Jan 2011 | A1 |
| 20110082055 | Fox et al. | Apr 2011 | A1 |
| 20110114244 | Yoo et al. | May 2011 | A1 |
| 20110114549 | Yin et al. | May 2011 | A1 |
| 20110124049 | Li et al. | May 2011 | A1 |
| 20110124055 | Carr et al. | May 2011 | A1 |
| 20110126929 | Velasquez-Garcia et al. | Jun 2011 | A1 |
| 20110171651 | Richmond | Jul 2011 | A1 |
| 20110172127 | Jacobson et al. | Jul 2011 | A1 |
| 20110201057 | Carr et al. | Aug 2011 | A1 |
| 20110217738 | Jacobson | Sep 2011 | A1 |
| 20110230653 | Novoradovskaya et al. | Sep 2011 | A1 |
| 20110254107 | Bulovic et al. | Oct 2011 | A1 |
| 20110287435 | Grunenwald et al. | Nov 2011 | A1 |
| 20120003713 | Hansen et al. | Jan 2012 | A1 |
| 20120021932 | Mershin et al. | Jan 2012 | A1 |
| 20120027786 | Gupta et al. | Feb 2012 | A1 |
| 20120028843 | Ramu et al. | Feb 2012 | A1 |
| 20120032366 | Ivniski et al. | Feb 2012 | A1 |
| 20120046175 | Rodesch et al. | Feb 2012 | A1 |
| 20120050411 | Mabritto et al. | Mar 2012 | A1 |
| 20120094847 | Warthmann et al. | Apr 2012 | A1 |
| 20120128548 | West et al. | May 2012 | A1 |
| 20120129704 | Gunderson et al. | May 2012 | A1 |
| 20120149602 | Friend et al. | Jun 2012 | A1 |
| 20120164127 | Short et al. | Jun 2012 | A1 |
| 20120164633 | Laffler | Jun 2012 | A1 |
| 20120164691 | Eshoo et al. | Jun 2012 | A1 |
| 20120184724 | Sierzchala et al. | Jul 2012 | A1 |
| 20120220497 | Jacobson et al. | Aug 2012 | A1 |
| 20120231968 | Bruhn et al. | Sep 2012 | A1 |
| 20120238737 | Dellinger et al. | Sep 2012 | A1 |
| 20120258487 | Chang et al. | Oct 2012 | A1 |
| 20120264653 | Carr et al. | Oct 2012 | A1 |
| 20120270750 | Oleinikov | Oct 2012 | A1 |
| 20120270754 | Blake | Oct 2012 | A1 |
| 20120283140 | Chu | Nov 2012 | A1 |
| 20120288476 | Hartmann et al. | Nov 2012 | A1 |
| 20120289691 | Dellinger et al. | Nov 2012 | A1 |
| 20120315670 | Jacobson et al. | Dec 2012 | A1 |
| 20120322681 | Kung et al. | Dec 2012 | A1 |
| 20130005585 | Anderson et al. | Jan 2013 | A1 |
| 20130005612 | Carr et al. | Jan 2013 | A1 |
| 20130017642 | Milgrew et al. | Jan 2013 | A1 |
| 20130017977 | Oleinikov | Jan 2013 | A1 |
| 20130017978 | Kavanagh et al. | Jan 2013 | A1 |
| 20130035261 | Sierzchala et al. | Feb 2013 | A1 |
| 20130040836 | Himmler et al. | Feb 2013 | A1 |
| 20130045483 | Treusch et al. | Feb 2013 | A1 |
| 20130053252 | Xie et al. | Feb 2013 | A1 |
| 20130059296 | Jacobson et al. | Mar 2013 | A1 |
| 20130059761 | Jacobson et al. | Mar 2013 | A1 |
| 20130065017 | Sieber | Mar 2013 | A1 |
| 20130109595 | Routenberg | May 2013 | A1 |
| 20130109596 | Peterson et al. | May 2013 | A1 |
| 20130123129 | Zeiner et al. | May 2013 | A1 |
| 20130130321 | Staehler et al. | May 2013 | A1 |
| 20130137161 | Zhang et al. | May 2013 | A1 |
| 20130137173 | Zhang et al. | May 2013 | A1 |
| 20130137174 | Zhang et al. | May 2013 | A1 |
| 20130137861 | Leproust et al. | May 2013 | A1 |
| 20130164308 | Foletti et al. | Jun 2013 | A1 |
| 20130165328 | Previte et al. | Jun 2013 | A1 |
| 20130196864 | Govindarajan et al. | Aug 2013 | A1 |
| 20130225421 | Li et al. | Aug 2013 | A1 |
| 20130244884 | Jacobson et al. | Sep 2013 | A1 |
| 20130252849 | Hudson et al. | Sep 2013 | A1 |
| 20130261027 | Li et al. | Oct 2013 | A1 |
| 20130281308 | Kung et al. | Oct 2013 | A1 |
| 20130289246 | Crowe et al. | Oct 2013 | A1 |
| 20130296192 | Jacobson et al. | Nov 2013 | A1 |
| 20130296194 | Jacobson et al. | Nov 2013 | A1 |
| 20130298265 | Cunnac et al. | Nov 2013 | A1 |
| 20130309725 | Jacobson et al. | Nov 2013 | A1 |
| 20130323725 | Peter et al. | Dec 2013 | A1 |
| 20130330778 | Zeiner et al. | Dec 2013 | A1 |
| 20140011226 | Bernick et al. | Jan 2014 | A1 |
| 20140018441 | Fracchia et al. | Jan 2014 | A1 |
| 20140031240 | Behlke et al. | Jan 2014 | A1 |
| 20140038240 | Temme et al. | Feb 2014 | A1 |
| 20140106394 | Ko et al. | Apr 2014 | A1 |
| 20140141982 | Jacobson et al. | May 2014 | A1 |
| 20140170665 | Hiddessen et al. | Jun 2014 | A1 |
| 20140178992 | Nakashima et al. | Jun 2014 | A1 |
| 20140221250 | Vasquez et al. | Aug 2014 | A1 |
| 20140274729 | Kurn et al. | Sep 2014 | A1 |
| 20140274741 | Hunter et al. | Sep 2014 | A1 |
| 20140303000 | Armour et al. | Oct 2014 | A1 |
| 20140309119 | Jacobson et al. | Oct 2014 | A1 |
| 20140309142 | Tian | Oct 2014 | A1 |
| 20150010953 | Lindstrom et al. | Jan 2015 | A1 |
| 20150012723 | Park et al. | Jan 2015 | A1 |
| 20150031089 | Lindstrom | Jan 2015 | A1 |
| 20150038373 | Banyai et al. | Feb 2015 | A1 |
| 20150056609 | Daum et al. | Feb 2015 | A1 |
| 20150057625 | Coulthard | Feb 2015 | A1 |
| 20150065357 | Fox | Mar 2015 | A1 |
| 20150065393 | Jacobson | Mar 2015 | A1 |
| 20150099870 | Bennett et al. | Apr 2015 | A1 |
| 20150119293 | Short | Apr 2015 | A1 |
| 20150120265 | Amirav-Drory et al. | Apr 2015 | A1 |
| 20150159152 | Allen et al. | Jun 2015 | A1 |
| 20150183853 | Sharma et al. | Jul 2015 | A1 |
| 20150191524 | Smith et al. | Jul 2015 | A1 |
| 20150191719 | Hudson et al. | Jul 2015 | A1 |
| 20150196917 | Kay et al. | Jul 2015 | A1 |
| 20150203839 | Jacobson et al. | Jul 2015 | A1 |
| 20150211047 | Borns | Jul 2015 | A1 |
| 20150225782 | Walder et al. | Aug 2015 | A1 |
| 20150240232 | Zamore et al. | Aug 2015 | A1 |
| 20150240280 | Gibson et al. | Aug 2015 | A1 |
| 20150261664 | Goldman et al. | Sep 2015 | A1 |
| 20150269313 | Church | Sep 2015 | A1 |
| 20150293102 | Shim | Oct 2015 | A1 |
| 20150307875 | Happe et al. | Oct 2015 | A1 |
| 20150321191 | Kendall et al. | Nov 2015 | A1 |
| 20150322504 | Lao et al. | Nov 2015 | A1 |
| 20150344927 | Sampson et al. | Dec 2015 | A1 |
| 20150353921 | Tian | Dec 2015 | A9 |
| 20150353994 | Myers et al. | Dec 2015 | A1 |
| 20150361420 | Hudson et al. | Dec 2015 | A1 |
| 20150361422 | Sampson et al. | Dec 2015 | A1 |
| 20150361423 | Sampson et al. | Dec 2015 | A1 |
| 20150368687 | Saaem et al. | Dec 2015 | A1 |
| 20150376602 | Jacobson et al. | Dec 2015 | A1 |
| 20160001247 | Oleinikov | Jan 2016 | A1 |
| 20160002621 | Nelson et al. | Jan 2016 | A1 |
| 20160002622 | Nelson et al. | Jan 2016 | A1 |
| 20160010045 | Cohen et al. | Jan 2016 | A1 |
| 20160017394 | Liang et al. | Jan 2016 | A1 |
| 20160017425 | Ruvolo et al. | Jan 2016 | A1 |
| 20160019341 | Harris et al. | Jan 2016 | A1 |
| 20160024138 | Gebeyehu et al. | Jan 2016 | A1 |
| 20160024576 | Chee | Jan 2016 | A1 |
| 20160026753 | Krishnaswami et al. | Jan 2016 | A1 |
| 20160026758 | Jabara et al. | Jan 2016 | A1 |
| 20160032396 | Diehn et al. | Feb 2016 | A1 |
| 20160046973 | Efcavitch et al. | Feb 2016 | A1 |
| 20160046974 | Efcavitch et al. | Feb 2016 | A1 |
| 20160082472 | Perego et al. | Mar 2016 | A1 |
| 20160089651 | Banyai | Mar 2016 | A1 |
| 20160090422 | Reif et al. | Mar 2016 | A1 |
| 20160090592 | Banyai et al. | Mar 2016 | A1 |
| 20160096160 | Banyai et al. | Apr 2016 | A1 |
| 20160097051 | Jacobson et al. | Apr 2016 | A1 |
| 20160102322 | Ravinder et al. | Apr 2016 | A1 |
| 20160108466 | Nazarenko et al. | Apr 2016 | A1 |
| 20160122755 | Hall et al. | May 2016 | A1 |
| 20160122800 | Bernick et al. | May 2016 | A1 |
| 20160152972 | Stapleton et al. | Jun 2016 | A1 |
| 20160168611 | Efcavitch et al. | Jun 2016 | A1 |
| 20160184788 | Hall et al. | Jun 2016 | A1 |
| 20160200759 | Srivastava et al. | Jul 2016 | A1 |
| 20160215283 | Braman et al. | Jul 2016 | A1 |
| 20160229884 | Indermuhle et al. | Aug 2016 | A1 |
| 20160230175 | Carstens | Aug 2016 | A1 |
| 20160230221 | Bergmann et al. | Aug 2016 | A1 |
| 20160251651 | Banyai et al. | Sep 2016 | A1 |
| 20160256846 | Smith et al. | Sep 2016 | A1 |
| 20160264958 | Toro et al. | Sep 2016 | A1 |
| 20160289758 | Akeson et al. | Oct 2016 | A1 |
| 20160289839 | Harumoto et al. | Oct 2016 | A1 |
| 20160303535 | Banyai et al. | Oct 2016 | A1 |
| 20160304862 | Igawa et al. | Oct 2016 | A1 |
| 20160304946 | Betts et al. | Oct 2016 | A1 |
| 20160310426 | Wu | Oct 2016 | A1 |
| 20160310927 | Banyai et al. | Oct 2016 | A1 |
| 20160318016 | Hou et al. | Nov 2016 | A1 |
| 20160333340 | Wu | Nov 2016 | A1 |
| 20160339409 | Banyai et al. | Nov 2016 | A1 |
| 20160340672 | Banyai et al. | Nov 2016 | A1 |
| 20160348098 | Stuelpnagel et al. | Dec 2016 | A1 |
| 20160354752 | Banyai et al. | Dec 2016 | A1 |
| 20160355880 | Gormley et al. | Dec 2016 | A1 |
| 20170017436 | Church | Jan 2017 | A1 |
| 20170066844 | Glanville | Mar 2017 | A1 |
| 20170067099 | Zheng et al. | Mar 2017 | A1 |
| 20170073664 | McCafferty et al. | Mar 2017 | A1 |
| 20170073731 | Zheng et al. | Mar 2017 | A1 |
| 20170081660 | Cox et al. | Mar 2017 | A1 |
| 20170081716 | Peck | Mar 2017 | A1 |
| 20170088887 | Makarov et al. | Mar 2017 | A1 |
| 20170095785 | Banyai et al. | Apr 2017 | A1 |
| 20170096706 | Behlke et al. | Apr 2017 | A1 |
| 20170114404 | Behlke et al. | Apr 2017 | A1 |
| 20170141793 | Strauss et al. | May 2017 | A1 |
| 20170147748 | Staehler et al. | May 2017 | A1 |
| 20170151546 | Peck et al. | Jun 2017 | A1 |
| 20170159044 | Toro et al. | Jun 2017 | A1 |
| 20170175110 | Jacobson et al. | Jun 2017 | A1 |
| 20170218537 | Olivares | Aug 2017 | A1 |
| 20170233764 | Young et al. | Aug 2017 | A1 |
| 20170247473 | Short | Aug 2017 | A1 |
| 20170249345 | Malik et al. | Aug 2017 | A1 |
| 20170253644 | Steyaert et al. | Sep 2017 | A1 |
| 20170298432 | Holt | Oct 2017 | A1 |
| 20170320061 | Venter et al. | Nov 2017 | A1 |
| 20170327819 | Banyai et al. | Nov 2017 | A1 |
| 20170355984 | Evans et al. | Dec 2017 | A1 |
| 20170357752 | Diggans | Dec 2017 | A1 |
| 20170362589 | Banyai et al. | Dec 2017 | A1 |
| 20180029001 | Banyai et al. | Feb 2018 | A1 |
| 20180051278 | Cox et al. | Feb 2018 | A1 |
| 20180051280 | Gibson et al. | Feb 2018 | A1 |
| 20180068060 | Ceze et al. | Mar 2018 | A1 |
| 20180101487 | Peck | Apr 2018 | A1 |
| 20180104664 | Fernandez | Apr 2018 | A1 |
| 20180126355 | Peck et al. | May 2018 | A1 |
| 20180142289 | Zeitoun et al. | May 2018 | A1 |
| 20180171509 | Cox et al. | Jun 2018 | A1 |
| 20180236425 | Banyai et al. | Aug 2018 | A1 |
| 20180253563 | Peck et al. | Sep 2018 | A1 |
| 20180264428 | Banyai et al. | Sep 2018 | A1 |
| 20180273936 | Cox et al. | Sep 2018 | A1 |
| 20180282721 | Cox et al. | Oct 2018 | A1 |
| 20180291445 | Betts et al. | Oct 2018 | A1 |
| 20180312834 | Cox et al. | Nov 2018 | A1 |
| 20180326388 | Banyai et al. | Nov 2018 | A1 |
| 20180334712 | Singer et al. | Nov 2018 | A1 |
| 20180346585 | Zhang et al. | Dec 2018 | A1 |
| 20180355351 | Nugent et al. | Dec 2018 | A1 |
| 20190060345 | Harrison et al. | Feb 2019 | A1 |
| 20190083596 | Orentas et al. | Mar 2019 | A1 |
| 20190118154 | Marsh et al. | Apr 2019 | A1 |
| 20190135926 | Glanville | May 2019 | A1 |
| 20190224711 | Demeris, Jr. | Jul 2019 | A1 |
| 20190240636 | Peck et al. | Aug 2019 | A1 |
| 20190244109 | Bramlett et al. | Aug 2019 | A1 |
| 20190314783 | Banyai et al. | Oct 2019 | A1 |
| 20190352635 | Toro et al. | Nov 2019 | A1 |
| 20190366293 | Banyai et al. | Dec 2019 | A1 |
| 20190366294 | Banyai et al. | Dec 2019 | A1 |
| 20190370164 | Goldman et al. | Dec 2019 | A1 |
| 20200017907 | Zeitoun et al. | Jan 2020 | A1 |
| 20200102611 | Zeitoun et al. | Apr 2020 | A1 |
| 20200156037 | Banyai et al. | May 2020 | A1 |
| 20200181667 | Wu et al. | Jun 2020 | A1 |
| 20200222875 | Peck et al. | Jul 2020 | A1 |
| 20200283760 | Nugent et al. | Sep 2020 | A1 |
| 20200299322 | Indermuhle et al. | Sep 2020 | A1 |
| 20200299684 | Toro et al. | Sep 2020 | A1 |
| 20200308575 | Sato | Oct 2020 | A1 |
| 20200325235 | Tabibiazar et al. | Oct 2020 | A1 |
| 20200342143 | Peck | Oct 2020 | A1 |
| 20210002710 | Gantt et al. | Jan 2021 | A1 |
| 20210040476 | Cox et al. | Feb 2021 | A1 |
| 20210071168 | Nugent et al. | Mar 2021 | A1 |
| 20210102192 | Tabibiazar et al. | Apr 2021 | A1 |
| 20210102195 | Sato et al. | Apr 2021 | A1 |
| 20210102198 | Cox et al. | Apr 2021 | A1 |
| 20210115594 | Cox et al. | Apr 2021 | A1 |
| 20210129108 | Marsh et al. | May 2021 | A1 |
| 20210170356 | Peck et al. | Jun 2021 | A1 |
| 20210179724 | Sato et al. | Jun 2021 | A1 |
| Number | Date | Country |
|---|---|---|
| 3157000 | Sep 2000 | AU |
| 2362939 | Aug 2000 | CA |
| 2792676 | Sep 2011 | CA |
| 1771336 | May 2006 | CN |
| 101277758 | Oct 2008 | CN |
| 102159726 | Aug 2011 | CN |
| 103907117 | Jul 2014 | CN |
| 104520864 | Apr 2015 | CN |
| 104562213 | Apr 2015 | CN |
| 104734848 | Jun 2015 | CN |
| 105637097 | Jun 2016 | CN |
| 10260805 | Jul 2004 | DE |
| 0090789 | Oct 1983 | EP |
| 0126621 | Aug 1990 | EP |
| 0753057 | Jan 1997 | EP |
| 1314783 | May 2003 | EP |
| 1363125 | Nov 2003 | EP |
| 1546387 | Jun 2005 | EP |
| 1153127 | Jul 2006 | EP |
| 1728860 | Dec 2006 | EP |
| 1072010 | Apr 2010 | EP |
| 2175021 | Apr 2010 | EP |
| 2330216 | Jun 2011 | EP |
| 1343802 | May 2012 | EP |
| 2504449 | Oct 2012 | EP |
| 2751729 | Jul 2014 | EP |
| 2872629 | May 2015 | EP |
| 2928500 | Oct 2015 | EP |
| 2971034 | Jan 2016 | EP |
| 3030682 | Jun 2016 | EP |
| 3044228 | Apr 2017 | EP |
| 2994509 | Jun 2017 | EP |
| 3204518 | Aug 2017 | EP |
| H07505530 | Jun 1995 | JP |
| H09504910 | May 1997 | JP |
| 2001518086 | Oct 2001 | JP |
| 2002511276 | Apr 2002 | JP |
| 2002536977 | Nov 2002 | JP |
| 2002538790 | Nov 2002 | JP |
| 2006503586 | Feb 2006 | JP |
| 2006238724 | Sep 2006 | JP |
| 2008505642 | Feb 2008 | JP |
| 2008097189 | Apr 2008 | JP |
| 2008523786 | Jul 2008 | JP |
| 2008214343 | Sep 2008 | JP |
| 2008218579 | Sep 2008 | JP |
| 2009294195 | Dec 2009 | JP |
| 2016527313 | Sep 2016 | JP |
| 2019512480 | May 2019 | JP |
| WO-9015070 | Dec 1990 | WO |
| WO-9210092 | Jun 1992 | WO |
| WO-9210588 | Jun 1992 | WO |
| WO-9309668 | May 1993 | WO |
| WO-9320242 | Oct 1993 | WO |
| WO-9525116 | Sep 1995 | WO |
| WO-9526397 | Oct 1995 | WO |
| WO-9615861 | May 1996 | WO |
| WO-9710365 | Mar 1997 | WO |
| WO-9822541 | May 1998 | WO |
| WO-9841531 | Sep 1998 | WO |
| WO-9942813 | Aug 1999 | WO |
| WO-9953101 | Oct 1999 | WO |
| WO-0013017 | Mar 2000 | WO |
| WO-0018957 | Apr 2000 | WO |
| WO-0042559 | Jul 2000 | WO |
| WO-0042560 | Jul 2000 | WO |
| WO-0042561 | Jul 2000 | WO |
| WO-0049142 | Aug 2000 | WO |
| WO-0053617 | Sep 2000 | WO |
| WO-0156216 | Aug 2001 | WO |
| WO-0210443 | Feb 2002 | WO |
| WO-0156216 | Mar 2002 | WO |
| WO-0220537 | Mar 2002 | WO |
| WO-0224597 | Mar 2002 | WO |
| 0233669 | Apr 2002 | WO |
| WO-0227638 | Apr 2002 | WO |
| WO-02072791 | Sep 2002 | WO |
| WO-03040410 | May 2003 | WO |
| WO-03046223 | Jun 2003 | WO |
| WO-03054232 | Jul 2003 | WO |
| WO-03060084 | Jul 2003 | WO |
| WO-03064026 | Aug 2003 | WO |
| WO-03064027 | Aug 2003 | WO |
| WO-03064699 | Aug 2003 | WO |
| WO-03065038 | Aug 2003 | WO |
| WO-03066212 | Aug 2003 | WO |
| WO-03089605 | Oct 2003 | WO |
| WO-03093504 | Nov 2003 | WO |
| WO-03100012 | Dec 2003 | WO |
| WO-2004024886 | Mar 2004 | WO |
| WO-2004029220 | Apr 2004 | WO |
| WO-2004029586 | Apr 2004 | WO |
| WO-2004031351 | Apr 2004 | WO |
| WO-2004031399 | Apr 2004 | WO |
| WO-2004059556 | Jul 2004 | WO |
| WO-03060084 | Aug 2004 | WO |
| WO-2005014850 | Feb 2005 | WO |
| WO-2005051970 | Jun 2005 | WO |
| WO-2005059096 | Jun 2005 | WO |
| WO-2005059097 | Jun 2005 | WO |
| WO-2005093092 | Oct 2005 | WO |
| WO-2006023144 | Mar 2006 | WO |
| WO-2006044956 | Apr 2006 | WO |
| WO-2006076679 | Jul 2006 | WO |
| WO-2006116476 | Nov 2006 | WO |
| WO-2007109221 | Sep 2007 | WO |
| WO-2007118214 | Oct 2007 | WO |
| WO-2007120627 | Oct 2007 | WO |
| WO-2007137242 | Nov 2007 | WO |
| WO-2008003116 | Jan 2008 | WO |
| WO-2008006078 | Jan 2008 | WO |
| WO-2008027558 | Mar 2008 | WO |
| WO-2008045380 | Apr 2008 | WO |
| WO-2008054543 | May 2008 | WO |
| WO-2008063134 | May 2008 | WO |
| WO-2008063135 | May 2008 | WO |
| WO-2008068280 | Jun 2008 | WO |
| WO-2008103474 | Aug 2008 | WO |
| WO-2008109176 | Sep 2008 | WO |
| WO-2009132876 | Nov 2009 | WO |
| WO-2010001251 | Jan 2010 | WO |
| WO-2010025310 | Mar 2010 | WO |
| WO-2010025566 | Mar 2010 | WO |
| WO-2010027512 | Mar 2010 | WO |
| WO-2010089412 | Aug 2010 | WO |
| WO-2010141249 | Dec 2010 | WO |
| WO-2010141433 | Dec 2010 | WO |
| WO-2011020529 | Feb 2011 | WO |
| WO-2010141433 | Apr 2011 | WO |
| WO-2011053957 | May 2011 | WO |
| WO-2011056644 | May 2011 | WO |
| WO-2011056872 | May 2011 | WO |
| WO-2011066185 | Jun 2011 | WO |
| WO-2011066186 | Jun 2011 | WO |
| WO-2011085075 | Jul 2011 | WO |
| WO-2011103468 | Aug 2011 | WO |
| WO-2011109031 | Sep 2011 | WO |
| WO-2011143556 | Nov 2011 | WO |
| WO-2011150168 | Dec 2011 | WO |
| WO-2011161413 | Dec 2011 | WO |
| WO-2012013913 | Feb 2012 | WO |
| WO-2012061832 | May 2012 | WO |
| WO-2012078312 | Jun 2012 | WO |
| WO-2012149171 | Nov 2012 | WO |
| WO-2012154201 | Nov 2012 | WO |
| WO-2013030827 | Mar 2013 | WO |
| WO-2013032850 | Mar 2013 | WO |
| WO-2013036668 | Mar 2013 | WO |
| 2013049227 | Apr 2013 | WO |
| WO-2013101896 | Jul 2013 | WO |
| WO-2013134881 | Sep 2013 | WO |
| WO-2013154770 | Oct 2013 | WO |
| WO-2013170168 | Nov 2013 | WO |
| WO-2013177220 | Nov 2013 | WO |
| WO-2014004393 | Jan 2014 | WO |
| WO-2014008447 | Jan 2014 | WO |
| WO-2014021938 | Feb 2014 | WO |
| WO-2014035693 | Mar 2014 | WO |
| WO-2014088693 | Jun 2014 | WO |
| WO-2014089160 | Jun 2014 | WO |
| WO-2014093330 | Jun 2014 | WO |
| WO-2014093694 | Jun 2014 | WO |
| WO-2014151117 | Sep 2014 | WO |
| WO-2014151696 | Sep 2014 | WO |
| WO-2014160004 | Oct 2014 | WO |
| WO-2014160059 | Oct 2014 | WO |
| WO-2014206304 | Dec 2014 | WO |
| WO-2015017527 | Feb 2015 | WO |
| WO-2015021080 | Feb 2015 | WO |
| WO-2015021280 | Feb 2015 | WO |
| WO-2015031689 | Mar 2015 | WO |
| WO-2015040075 | Mar 2015 | WO |
| WO-2015054292 | Apr 2015 | WO |
| WO-2015066174 | May 2015 | WO |
| WO-2015081114 | Jun 2015 | WO |
| WO-2015081142 | Jun 2015 | WO |
| WO-2015081440 | Jun 2015 | WO |
| WO-2015090879 | Jun 2015 | WO |
| WO-2015095404 | Jun 2015 | WO |
| WO-2015120403 | Aug 2015 | WO |
| WO-2015136072 | Sep 2015 | WO |
| 2015160004 | Oct 2015 | WO |
| WO-2015175832 | Nov 2015 | WO |
| WO-2016007604 | Jan 2016 | WO |
| WO-2016011080 | Jan 2016 | WO |
| WO-2016022557 | Feb 2016 | WO |
| WO-2016053883 | Apr 2016 | WO |
| WO-2016055956 | Apr 2016 | WO |
| WO-2016065056 | Apr 2016 | WO |
| WO-2016126882 | Aug 2016 | WO |
| WO-2016126987 | Aug 2016 | WO |
| WO-2016130868 | Aug 2016 | WO |
| WO-2016161244 | Oct 2016 | WO |
| WO-2016162127 | Oct 2016 | WO |
| WO-2016164779 | Oct 2016 | WO |
| WO-2016172377 | Oct 2016 | WO |
| WO-2016173719 | Nov 2016 | WO |
| WO-2016183100 | Nov 2016 | WO |
| 2017017423 | Feb 2017 | WO |
| WO-2017049231 | Mar 2017 | WO |
| WO-2017053450 | Mar 2017 | WO |
| WO-2017059399 | Apr 2017 | WO |
| WO-2017095958 | Jun 2017 | WO |
| WO-2017100441 | Jun 2017 | WO |
| WO-2017118761 | Jul 2017 | WO |
| 2017151680 | Sep 2017 | WO |
| WO-2017158103 | Sep 2017 | WO |
| WO-2017214574 | Dec 2017 | WO |
| WO-2018026920 | Feb 2018 | WO |
| WO-2018038772 | Mar 2018 | WO |
| WO-2018057526 | Mar 2018 | WO |
| WO-2018094263 | May 2018 | WO |
| WO-2018112426 | Jun 2018 | WO |
| WO-2018119246 | Jun 2018 | WO |
| WO-2018156792 | Aug 2018 | WO |
| WO-2018170164 | Sep 2018 | WO |
| WO-2018170169 | Sep 2018 | WO |
| WO-2018170559 | Sep 2018 | WO |
| WO-2018200380 | Nov 2018 | WO |
| WO-2018231872 | Dec 2018 | WO |
| WO-2019014781 | Jan 2019 | WO |
| WO-2019051501 | Mar 2019 | WO |
| WO-2019079769 | Apr 2019 | WO |
| WO-2019084500 | May 2019 | WO |
| WO-2019136175 | Jul 2019 | WO |
| WO-2019222706 | Nov 2019 | WO |
| WO-2020139871 | Jul 2020 | WO |
| WO-2020176362 | Sep 2020 | WO |
| WO-2020176678 | Sep 2020 | WO |
| WO-2020176680 | Sep 2020 | WO |
| WO-2020257612 | Dec 2020 | WO |
| WO-2021119193 | Jun 2021 | WO |
| Entry |
|---|
| Abudayyeh et al.: C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science, available on line, Jun. 13, 2016, at: http://zlab.mit.edu/assets/reprints/Abudayyeh_OO_Science_2016.pdf 17 pages. |
| Acevedo-Rocha et al.: Directed evolution of stereoselective enzymes based on genetic selection as opposed to screening systems. J. Biotechnol. 191:3-10 (2014). |
| Adessi et al.: Solid phase DNA amplification: characterisation of primer attachment and amplification mechanisms. Nucleic Acids Res. 28(20):E87, 2000. |
| Alberts et al.: Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. The Generation of Antibody Diversity. https://www.ncbi.nlm.nih.gov/books/NBK26860/. |
| Alexeyev et al.: Gene synthesis, bacterial expression and purification of the Rickettsia prowazekii ATP/ADP translocase, Biochimica et Biophysics Acta, 1419:299-306, 1999. |
| Al-Housseiny et al.: Control of interfacial instabilities using flow geometry Nature Physics, 8:747-750, 2012. |
| Almagro et al.: Progress and Challenges in the Design and Clinical Development of Antibodies for Cancer Therapy. Frontiers in immunology; 8, 1751 (2018) doi:10.3389/fimmu.2017.01751 https://www.frontiersin.org/articles/10.3389/fimmu.2017.01751/full. |
| Amblard et al.: A magnetic manipulator for studying local rheology and micromechanical properties of biological systems, Rev. Sci. Instrum., 67(3):18-827, 1996. |
| Andoni and Indyk. Near-Optimal Hashing Algorithms for Approximate Nearest Neighbor in High Dimensions, Communications of the ACM, 51(1):117-122, 2008. |
| Arand et al.: Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site. EMBO J. 22:2583-2592 (2003). |
| Arkles et al.: The Role of Polarity in the Structure of Silanes Employed in Surface Modification. Silanes and Other Coupling Agents. 5:51-64, 2009. |
| Arkles. Hydrophobicity, Hydrophilicity Reprinted with permission from the Oct. 2006 issue of Paint & Coatings Industry magazine, Retrieved on Mar. 19, 2016, 10 pages. |
| Assembly manual for the POSaM: The ISB Piezoelectric Oligonucleotide Synthesizer and Microarrayer, The Institute for Systems Biology, May 28, 2004 (50 pages). |
| Assi et al.: Massive-parallel adhesion and reactivity-measurements using simple and inexpensive magnetic tweezers. J. Appl. Phys. 92(9):5584-5586 (2002). |
| ATDBio. Nucleic Acid Structure, Nucleic Acids Book, 9 pages, published on Jan. 22, 2005. from: http://www.atdbio.com/content/5/Nucleic-acid-structure. |
| ATDBio. Solid-Phase Oligonucleotide Synthesis, Nucleic Acids Book, 20 pages, Published on Jul. 31, 2011. from: http://www.atdbio.com/content/17/Solid-phase-oligonucleotide-synthesis. |
| Au et al.: Gene synthesis by a LCR-based approach: high level production of Leptin-L54 using synthetic gene in Escherichia coli. Biochemical and Biophysical Research Communications 248:200-203 (1998). |
| Baedeker et al.: Overexpression of a designed 2.2kb gene of eukaryotic phenylalanine ammonialyase in Escherichia coli·. FEBS Letters, 457:57-60, 1999. |
| Barbee et al.: Magnetic Assembly of High-Density DNA Arrays for Genomic Analyses. Anal Chem. 80(6):2149-2154, 2008. |
| Barton et al.: A desk electrohydrodynamic jet printing system. Mechatronics, 20:611-616, 2010. |
| Beaucage et al.: Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron. 48:2223-2311, 1992. |
| Beaucage et al.: Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22(20):1859-1862, 1981. |
| Beaucage et al.: The Chemical synthesis of DNA/RNA Chapter 2 in: Encyclopedia of Cell Biology, 1:36-53, 2016. |
| Beaulieu et al.: PCR candidate region mismatch scanning adaptation to quantitative, high-throughput genotyping, Nucleic Acids Research, 29(5):1114-1124, 2001. |
| Beigelman et al.: Base-modified phosphoramidite analogs of pyrimidine ribonucleosides for RNA structure-activity studies. Methods Enzymol. 317:39-65, 2000. |
| Bethge et al.: Reverse synthesis and 3′-modification of RNA. Jan. 1, 2011, pp. 64-64, XP055353420. Retrieved from the Internet: URL:http://www.is3na.org/assets/events/Category%202-Medicinal %20Chemistry%20of%2001igonucleotides%20%2864-108%29.pdf. |
| Binkowski et al.: Correcting errors in synthetic DNA through consensus shuffling. Nucleic Acids Research, 33(6):e55, 8 pages, 2005. |
| Biswas et al.: Identification and characterization of a thermostable MutS homolog from Thennus aquaticus, The Journal of Biological Chemistry, 271(9):5040-5048, 1996. |
| Biswas et al.: Interaction of MutS protein with the major and minor grooves of a heteroduplex DNA, The Journal of Biological Chemistry, 272(20):13355-13364, 1997. |
| Bjornson et al.: Differential and simultaneous adenosine Di- and Triphosphate binding by MutS, The Journal of Biological Chemistry, 278(20):18557-18562, 2003. |
| Blanchard et al.: High-Density Oligonucleotide Arrays, Biosensors & Bioelectronics, 11(6/7):687-690, 1996. |
| Blanchard: Genetic Engineering, Principles and Methods, vol. 20, Ed. J. Sedlow, New York: Plenum Press, p. 111-124, 1979. |
| Blawat et al.: Forward error correction for DNA data storage. Procedia Computer Science, 80:1011-1022, 2016. |
| Bonini and Mondino. Adoptive T-cell therapy for cancer: The era of engineered T cells. European Journal of Immunology, 45:2457-2469, 2015 . |
| Bornholt et al.: A DNA-Based Archival Storage System, in International Conference on Architectural Support for Programming Languages and Operating Systems (ASPLOS), Apr. 2-6, 2016, Atlanta, GA, 2016, 637-649. |
| Borovkov et al.: High-quality gene assembly directly from unpurified mixtures of microassay-synthesized oligonucleotides. Nucleic Acid Research, 38(19):e180, 10 pages, 2010. |
| Brunet: Aims and methods of biosteganography. Journal of Biotechnology, 226:56-64, 2016. |
| Buermans et al.: Next Generation sequencing technology: Advances and applications, Biochimica et Biophysica Acta (BBA)—Molecular Basis of Disease, 1842:1931-1941, 2014. |
| Butler et al.: In situ synthesis of oligonucleotide arrays by using surface tension. J Am Chem Soc. 123(37):8887-94, 2001. |
| Calvert. Lithographically patterned self-assembled films. In: Organic Thin Films and Surfaces: Directions for The Nineties, vol. 20, p. 109, ed. By Abraham Ulman, San Diego: Academic Press, 1995. |
| Cardelli. Two-Domain DNA Strand Displacement, Electron. Proc. Theor. Comput. Sci., 26:47-61, 2010. |
| Carlson. Time for New DNA Synthesis and Sequencing Cost Curves, 2014. [Online]. Available: http://www.synthesis.cc/synthesis/2014/02/time_for_new_cost_curves_2014. 10 pages. |
| Carr et al.: Protein-mediated error correction for de novo DNA synthesis. Nucleic Acids Res. 32(20):e162, 9 pages, 2004. |
| Carter and Friedman. DNA synthesis and Biosecurity: Lessons learned and options for the future. J. Craig Venter Institute, La Jolla, CA, 28 pages, Oct. 2015. |
| Caruthers. Chemical synthesis of deoxyoligonucleotides by the phosphoramidite method. In Methods in Enzymology, Chapter 15, 154:287-313, 1987. |
| Caruthers. Gene synthesis machines: DNA chemistry and its uses. Science 230(4723):281-285 (1985). |
| Caruthers. The Chemical Synthesis of DNA/RNA: Our Gift to Science. J. Biol. Chem., 288(2):1420-1427, 2013. |
| Casmiro et al.: PCR-based gene synthesis and protein NMR spectroscopy, Structure, 5(11):1407-1412, 1997. |
| CeGaT. Tech Note available at https://www.cegat.de/web/wp-content/uploads/2018/06/Twist-Exome-Tech-Note.pdf (4 pgs.) (2018). |
| Cello et al.: Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science. 297(5583):1016-8, 2000. |
| Chalmers et al.: Scaling up the ligase chain reaction-based approach to gene synthesis. Biotechniques. 30(2):249-52, 2001. |
| Chan et al.: Natural and engineered nicking endonucleases—from cleavage mechanism to engineering of strand-specificity. Nucleic Acids Res. 39(1):1-18, 2011. |
| Chen et al.: Chemical modification of gene silencing oligonucleotides for drug discovery and development. Drug Discov Today. 10(8):587-93 2005. |
| Chen et al.: Programmable chemical controllers made from DNA, Nat. Nanotechnol., 8(10):755-762, 2013. |
| Cheng et al.: High throughput parallel synthesis of oligonucleotides with 1536 channel synthesizer. Nucleic Acids Res. 30(18):e93, 2002. |
| Chervin et al.: Design of T-cell receptor libraries with diverse binding properties to examine adoptive T-cell responses. Gene Therapy. 20(6):634-644 (2012). |
| Chilamakuri et al.: Performance comparison of four exome capture systems for deep sequencing. BMC Genomics 15(1):449 (2014). |
| Cho et al.: Capillary passive valve in microfluidic systems. NSTI-Nanotech. 2004; 1:263-266. |
| Chrisey et al.: Fabrication of patterned DNA surfaces Nucleic Acids Research, 24(15):3040-3047 (1996). |
| Chung et al.: One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A. Apr. 1989;86(7):2172-2175. |
| Church et al.: Next-generation digital information storage in DNA. Science, 337:6102, 1628-1629, 2012. |
| Cleary et al.: Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis. Nat Methods 1(3):241-248 (2004). |
| Cohen et al.: Human population: The next half century. Science, 302:1172-1175, 2003. |
| Crick. On protein synthesis. Symp Soc Exp Biol12:138-163, 1958. |
| Cruse et al.: Atlas of Immunology, Third Edition. Boca Raton:CRC Press (pp. 282-283) (2010). |
| Cui et al.: Information Security Technology Based on DNA Computing. International Workshop on Anti-Counterfeiting, Security and Identification (ASID); IEEE Xplore 4 pages (2007). |
| Cutler et al.: High-throughput variation detection and genotyping using microarrays, Genome Research, vol. 11, 1913-19 (2001). |
| Dahl et al.: Circle-to-circle amplification for precise and sensitive DNA analysis. Proc Natl Acad Sci U S A. Mar. 30, 2004;101(13):4548-53. Epub Mar. 15, 2004. |
| De Mesmaeker et al.: Backbone modifications in oligonucleotides and peptide nucleic acid systems. Curr Opin Struct Biol. Jun. 1995;5(3):343-55. |
| De Silva et al.: New Trends of Digital Data Storage in DNA. BioMed Res Int. 2016:8072463 (2016). |
| Deamer et al.: Characterization of nucleic acids by nanopore analysis, Ace. Cham. Res., vol. 35, No. 10, 817-825 (2002). |
| Deaven. The Human Genome Project: Recombinant clones for mapping and sequencing DNA. Los Alamos Science, 20:218-249, 1992. |
| Deng et al.: Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming Nature Biotechnology, 27:352-360 (2009). |
| Dietrich et al.: Gene assembly based on blunt-ended double-stranded DNA-modules, Biotechnology Techniques, vol. 12, No. 1, 49-54 (Jan. 1998). |
| Dillon et al.: Exome sequencing has higher diagnostic yield compared to simulated disease-specific panels in children with suspected monogenic disorders. Eur J Hum Genet 26(5):644-651 (2018). |
| Dormitzer et al.: Synthetic generation of influenza vaccine viruses for rapid response to pandemics. Sci Translational Medicine, 5(185):185ra68, 14 pages, 2013. |
| Doudna et al.: Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1258096-1-1258096-9, 2014. |
| Douthwaite et al.: Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCF formyl-peptide receptor 1; mAbs, vol. 7, Iss. 1, pp. 152-166 (Jan. 1, 2015). |
| Dower et al.: High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16(13):6127-45 (1988). |
| Dressman et al.: Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc Natl Acad Sci U S A. Jul. 22, 2003;100(15):8817-22. Epub Jul. 11, 2003. |
| Drmanac et al.: Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays. Science. Jan. 1, 2010;327(5961):78-81. doi: 10.1126/science. 1181498. Epub Nov. 5, 2009. |
| Droege and Hill. The Genome Sequencer FLXTM System-Longer reads, more applications, straight forward bioinformatics and more complete data sets Journal of Biotechnology, 136:3-10, 2008. |
| Duffy et al.: Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane). Anal Chem. Dec. 1, 1998;70(23):4974-84. doi: 10.1021/ac980656z. |
| Duggan et al.: Expression profiling using cDNA microarrays. Nat Genet. Jan. 1999;21(1 Suppl):10-4. |
| Dvorsky. Living Bacteria Can Now Store Data. GIZMODO internet publication. Retrieved from https://gizmodo.com/living-bacteria-can-now-store-data-1781773517 (4 pgs) (Jun. 10, 2016). |
| Eadie et al.: Guanine modification during chemical DNA synthesis. Nucleic Acids Res. Oct. 26, 1987;15(20):8333-49. |
| Eisen. A phylogenomic study of the MutS family of proteins, Nucleic Acids Research, vol. 26, No. 18, 4291-4300 (1998). |
| Ellis et al.: DNA assembly for synthetic biology: from parts to pathways and beyond. Integr Biol (Camb). Feb. 2011;3(2):109-18. doi: 10.1039/c0ib00070a. Epub Jan. 19, 2011. |
| El-Sagheer et al.: Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli. Proc Natl Acad Sci U S A. Jul. 12, 2011;108(28):11338-43. doi: 10.1073/pnas.1101519108. Epub Jun. 27, 2011. |
| Elsik et al.: The Genome sequence of taurine cattle: A window of ruminant biology and evolution. Science, 324:522-528, 2009. |
| Elsner et al.: 172 nm excimer VUV-triggered photodegradation and micropatterning of aminosilane films, Thin Solid Films, 517:6772-6776 (2009). |
| Engler et al.: A one pot, one step, precision cloning method with high throughput capability. PLoS One. 2008;3(11):e3647. doi: 10.1371/journal.pone.0003647. Epub Nov. 5, 2008. |
| Engler et al.: Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One. 2009;4(5):e5553. doi: 10.1371/journal.pone.0005553. Epub May 14, 2009. |
| Erlich and Zielinski. DNA fountain enables a robust and efficient storage architecture. Science, 355(6328):950-054, 2017. |
| Eroshenko et al.: Gene Assembly from Chip-Synthesized Oligonucleotides; Current Protocols in Chemical biology 4: 1-17 (2012). |
| Evans et al.: DNA Repair Enzymes. Current Protocols in Molecular Biology 84:III:3.9:3.9.1-3.9.12 http://www.ncbi.nlm.nih.gov/pubmed/18972391 (Published online Oct. 1, 2008 Abstract only provided). |
| Fahy et al.: Self-sustained sequence replication (3SR): an isothermal transcription-based amplification system alternative to PCR. PCR Methods Appl. Aug. 1991;1(1):25-33. |
| Fedoryak et al.: Brominated hydroxyquinoline as a photolabile protecting group with sensitivity to multiphoton excitation, Org. Lett., vol. 4, No. 2 , 3419-3422 (2002). |
| Ferretti et al.: Total synthesis of a gene for bovine rhodopsin. PNAS, 83:599-603 (1986). |
| Finger et al.: The wonders of Flap Endonucleases: Structure, function, mechanism and regulation. Subcell Biochem., 62:301-326, 2012. |
| Fodor et al.: Light-directed, spatially addressable parallel chemical synthesis. Science. 251(4995):767-773 (1991). |
| Fogg et al.: Structural basis for uracil recognition by archaeal family B DNA polymerases. Nature Structural Biology, 9(12):922-927, 2002. |
| Foldesi et al.: The synthesis of deuterionucleosides. Nucleosides Nucleotides Nucleic Acids. Oct.-Dec. 2000;19(10-12):1615-56. |
| Frandsen et al.: Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi. BMC Molecular Biology 2008, 9:70. |
| FRANDSEN. Experimental setup. Dec. 7, 2010, 3 pages. http://www.rasmusfrandsen.dk/experimental_setup.htm. |
| Frandsen. The USER Friendly technology. USER cloning. Oct. 7, 2010, 2 pages. http://www.rasmusfrandsen.dk/user_cloning.htm. |
| Fullwood et al.: Next-generation DNA sequencing of paired-end tags [PET] for transcriptome and genome analysis Genome Research, 19:521-532, 2009. |
| Galka et al.: QuickLib, a method for building fully synthetic plasmid libraries by seamless cloning of degenerate oligonucleotides. PLOS ONE, 12, e0175146:1-9 (2017). |
| Galka et al.: QuickLib, a method for building fully synthetic plasmid libraries by seamless cloning of degenerate oligonucleotides. PLOS ONE, 12, e0175146:S1 figure (2017). |
| Galka et al.: QuickLib, a method for building fully synthetic plasmid libraries by seamless cloning of degenerate oligonucleotides. PLOS ONE, 12, e0175146:S1 Table (2017). |
| Galka et al.: QuickLib, a method for building fully synthetic plasmid libraries by seamless cloning of degenerate oligonucleotides. PLOS ONE, 12, e0175146:S2 figure (2017). |
| Galneder et al.: Microelectrophoresis of a bilayer-coated silica bead in an optical trap: application to enzymology. Biophysical Journal, vol. 80, No. 5, 2298-2309 (May 2001). |
| Gao et al.: A flexible light-directed DNA chip synthesis gated by deprotection using solution photogenerated acids. Nucleic Acids Res. Nov. 15, 2001;29(22):4744-50. |
| Gao et al.: A method for the generation of combinatorial antibody libraries using pIX phage display. PNAS 99(20):12612-12616 (2002). |
| Gao et al.: Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences. Nucleic Acids Res. Nov. 15, 2003;31(22):e143. |
| Garaj et al.: Graphene as a subnanometre trans-electrode membrane. Nature. Sep. 9, 2010;467(7312):190-3. doi: 10.1038/nature09379. |
| Garbow et al.: Optical tweezing electrophoresis of isolated, highly charged colloidal spheres, Colloids and Surfaces A: Physiochem. Eng. Aspects, vol. 195, 227-241 (2001). |
| Geetha et al.: Survey on Security Mechanisms for Public Cloud Data. 2016 International Conference on Emerging Trends in Engineering, Technology and Science (ICETETS). 8 pages (2016). |
| GeneArt Seamless Cloning and Assembly Kits. Life Technologies Synthetic Biology. 8 pages, available online Jun. 15, 2012. |
| Genomics 101. An Introduction to the Genomic Workflow. 2016 edition, 64 pages. Available at: http://www.frontlinegenomics.com/magazine/6757/genomics-101/. |
| Geu-Flores et al.: USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res. 2007;35(7):e55. Epub Mar. 27, 2007. |
| Gibson Assembly. Product Listing. Application Overview. 2 pages, available online Dec. 16, 2014. |
| Gibson et al.: Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science. Feb. 29, 2008;319(5867):1215-20. doi: 10.1126/science.1151721. Epub Jan. 24, 2008. |
| Gibson et al.: Creation of a Bacterial Cell Controlled by A Chemically Synthesized Genome. Science 329(5989):52-56 (2010). |
| Goldfeder et al.: Medical implications of technical accuracy in genome sequencing. Genome Med 8(1):24 (2016). |
| Goldman et al.: Towards practical, high-capacity, low-maintenance information storage in synthesized DNA, Nature, 494(7435):77-80, 2013. |
| Goodwin et al.: immunoglobulin heavy chain variable region, partial [Homo sapiens]. Genbank entry (online). National Institute of Biotechnology Information. (2018) https://www.ncbi.nim.nih.gov/protein/AXA12486.1. |
| Gosse et al.: Magnetic tweezers: micromanipulation and force measurement at the molecular level, Biophysical Journal, vol. 8, 3314-3329 (Jun. 2002). |
| Grass et al.: Robust chemical preservation of digital information on DNA in silica with error-correcting codes, Angew. Chemie—Int. Ed., 54(8):2552-2555, 2015. |
| Greagg et al.: A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil. Proc. Nat. Acad. Sci. USA, 96:9045-9050, 1999. |
| Grovenor. Microelectronic materials. Graduate Student Series in Materials Science and Engineering. Bristol, England: Adam Hilger, 1989; p. 113-123. |
| Gu et al.: Depletion of abundant sequences by hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications. Genome Biology, 17:41, 13 pages, 2016. |
| Haber et al.: Magnetic tweezers for DNA micromanipulation, Rev. Sci. Instrum., vol. 71, No. 12, 4561-4570 (Dec. 2000). |
| Han et al.: Linking T-cell receptor sequence to functional phenotype at the single-cell level. Nat Biotechnol 32(7):684-692 (2014). |
| Hanahan and Cold Spring Harbor Laboratory. Studies on transformation of Escherichia coli with plasmids J. Mol. Biol. 166:557-580 (1983). |
| Hanahan et al.: Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol, vol. 204, p. 63-113 (1991). |
| Harada et al.: Unexpected substrate specificity of T4 DNA ligase revealed by in vitro selection. Nucleic Acids Res. May 25, 1993;21(10):2287-91. |
| Hauser et al.: Trends in GPCR drug discovery: new agents, targets and indications. Nature Reviews Drug Discovery, 16, 829-842 (2017). doi:10.1038/nrd.2017.178 https://www.nature.com/articles/nrd.2017.178. |
| Heckers et al.: Error analysis of chemically synthesized polynucleotides, BioTechniques, vol. 24, No. 2, 256-260 (1998). |
| Herzer et al.: Fabrication of patterned silane based self-assembled monolayers by photolithography and surface reactions on silicon-oxide substrates Chem. Commun., 46:5634-5652 (2010). |
| Hoover et al.: DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis, Nucleic Acids Research, vol. 30, No. 10, e43, 7 pages (2002). |
| Hopcroft et al.: What is the Young's Modulus of Silicon?. Journal of Microelectromechanical Systems. 19(2):229-238 (2010). |
| Hosu et al.: Magnetic tweezers for intracellular applications⋅, Rev. Sci. Instrum., vol. 74, No. 9, 4158-4163 (Sep. 2003). |
| Hötzel et al.: A strategy for risk mitigation of antibodies with fast clearance. mAbs, 4(6), 753-760 (2012). doi: 10.4161/mabs.22189 https://www.ncbi.nlm.nih.gov/pubmed/23778268. |
| Huang et al.: Three-dimensional cellular deformation analysis with a two-photon magnetic manipulator workstation, Biophysical Journal, vol. 82, No. 4, 2211·2223 (Apr. 2002). |
| Hughes et al.: Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer Nat Biotech 4:342-347 (2001). |
| Hughes et al.: Principles of early drug discovery. Br J Pharmacol 162(2):1239-1249, 2011. |
| Hutchison et al.: Cell-free cloning using phi29 DNA polymerase. Proc Natl Acad Sci U S A. Nov. 29, 2005;102(48):17332-6. Epub Nov. 14, 2005. |
| Imgur: The magic of the internet. Uploaded May 10, 2012, 2 pages, retrieved from: https://imgur.com/mEWuW. |
| In-Fusion Cloning: Accuracy, Not Background. Cloning & Competent Cells, ClonTech Laboratories, 3 pages, available online Jul. 6, 2014. |
| Jackson et al.: Recognition of DNA base mismatches by a rhodium intercalator, J. Am. Chem. Soc., vol. 19, 12986·12987 (1997). |
| Jacobs et al. DNA glycosylases: In DNA repair and beyond. Chromosoma 121:1-20 (2012)—http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3260424/. |
| Jacobus et al.: Optimal cloning of PCR fragments by homologous recombination in Escherichia soli. PLoS One 10(3):e0119221 (2015). |
| Jager et al.: Simultaneous Humoral and Cellular: Immune Response against Cancer-Testis Antigen NY-ES0-1: Definition of Human Histocompatibility Leukocyte Antigen (HLA)-A2-binding Peptide Epitopes. J. Exp. Med. 187(2):265-270 (1998). |
| Jaiswal et al.: An architecture for creating collaborative semantically capable scientific data sharing infrastructures. Proceeding WIDM '06 Proceedings of the 8th annual ACM international workshop on Web information and data management. ACM Digital Library pp. 75-82 (2006). |
| Jang et al.: Characterization of T cell repertoire of blood, tumor, and ascites in ovarian cancer patients using next generation sequencing. Oncoimmunology, 4(11):e1030561:1-10 (2015). |
| Jinek et al.: A Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337:816-821, 2012. |
| Jo et al.: Engineering therapeutic antibodies targeting G-protein-coupled receptors; Experimental & Molecular Medicine; 48; 9 pages (2016). |
| Karagiannis and El-Osta. RNA interference and potential therapeutic applications of short interfering RNAs Cancer Gene Therapy, 12:787-795, 2005. |
| Ke et al.: Influence of neighboring base pairs on the stability of single base bulges and base pairs in a DNA fragment, Biochemistry, Vo. 34, 4593-4600 (1995). |
| Kelley et al.: Single-base mismatch detection based on charge transduction through DNA, Nucleic Acids Research, vol. 27, No. 24, 4830-4837 (1999). |
| Kim et al.: Chimeric restriction endonuclease, Proc. Natl. Acad. Sci. USA, vol. 91, 883-887 (Feb. 1994). |
| Kim et al.: High-resolution patterns of quantum dots formed by electrohydrodynamic jet printing for light-emitting diodes. Nano Letters, 15:969-973, 2015. |
| Kim et al.: Site-specific cleavage of DNA-RNA hybrids by zinc finger/Fok I cleavage domain fusions Gene, vol. 203, 43-49 (1997). |
| Kim. The interaction between Z-ONA and the Zab domain of double-stranded RNA adenosine deaminase characterized using fusion nucleases, The Journal of Biological Chemistry, vol. 274, No. 27, 19081-19086 (1999). |
| Kinde et al.: Detection and quantification of rare mutations with massively parallel sequencing. Proc Natl Acad Sci U S A. Jun. 7, 2011;108(23):9530-5. Epub May 17, 2011. |
| Kodumal et al.: Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster. Proc Natl Acad Sci U S A. Nov. 2, 2004;101(44):15573-8. Epub Oct. 20, 2004. |
| Koike-Yusa et al.: Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nature Biotechnology, 32:267-273, 2014 (with three pages of supplemental Online Methods). |
| Kong et al.: Parallel gene synthesis in a microfluidic device. Nucleic Acids Res., 35(8):e61 (2007). |
| Kong. Microfluidic Gene Synthesis. MIT Thesis. Submitted to the program in Media Arts and Sciences, School of Architecture and Planning, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Media Arts and Sciences at the Massachusetts Institute of Technology. 143 pages Jun. 2008. |
| Kopp et al.: Chemical amplification: continuous-flow PCR on a chip, Science, vol. 280, 1046-1048 (May 15, 1998). |
| Kosuri and Church. Large-scale de novo DNA synthesis: technologies and applications, Nature Methods, 11:499-507, 2014. Available at: http://www.nature.com/nmeth/journal/v11/n5/full/nmeth.2918.html. |
| Kosuri et al.: A scalable gene synthesis platform using high-fidelity DNA microchips Nat.Biotechnol. 28(12):1295-1299 (2010). |
| Kosuri et al.: A scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nature Biotechnology. 2010; 28:1295-1299. |
| Krayden, Inc.: A Guide to Silane Solutions. Silane coupling agents. 7 pages. Published on May 31, 2005 at: http://krayden.com/pdf/xia_silane_chemistry.pdf. |
| Lagally et al.: Single-Molecule DNA Amplification and Analysis in an Integrated Microfluidic Device. Analytical Chemistry. 2001;73(3): 565-570. |
| Lahue et al.: DNA mismatch correction in a defined system, Science, vol. 425; No. 4914, 160-164 (Jul. 14, 1989). |
| Lambrinakos et al.: Reactivity of potassium permanganate and tetraethylammonium chloride with mismatched bases and a simple mutation detection protocol. Nucleic Acids Research, vol. 27, No. 8, 1866-1874 (1999). |
| Landegren et al.: A ligase-mediated gene detection technique. Science. Aug. 26, 1988;241(4869):1077-80. |
| Lang et al.: An automated two-dimensional optical force clamp for single molecule studies, Biophysical Journal, vol. 83, 491-501 (Jul. 2002). |
| Lashkari et al.: An automated multiplex oligonucleotide synthesizer: development of high-throughput, low-cost DNA synthesis. Proc Natl Acad Sci U S A. Aug. 15, 1995;92(17):7912-5. |
| Lausted et al.: POSaM: a fast, flexible, open-source, inkjet oligonucleotide synthesizer and microarrayer, Genome Biology, 5:R58, 17 pages, 2004. available at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC507883/. |
| Leamon et al.: A massively parallel PicoTiterPlate based platform for discrete picoliter-scale polymerase chain reactions. Electrophoresis. Nov. 2003;24(21):3769-77. |
| Lee et al.: A microfluidic oligonucleotide synthesizer. Nucleic Acids Research 2010 vol. 38(8):2514-2521. DOI: 10.1093/nar/gkq092. |
| Lee et al.: Microelectromagnets for the control of magnetic nanoparticles, Appl. Phys. Lett., vol. 79, No. 20, 3308-3310 (Nov. 12, 2001). |
| Lee: Covalent End-Immobilization of Oligonucleotides onto Solid Surfaces; Thesis, Massachusetts Institute of Technology, Aug. 2001 (315 pages). |
| Leproust et al.: Agilent's Microarray Platform: How High-Fidelity DNA Synthesis Maximizes the Dynamic Range of Gene Expression Measurements. 2008; 1-12. http://www.miltenyibiotec.com/˜/media/Files/Navigation/Genomic%20Services/Agilent_DNA_Microarray_Platform.ashx. |
| Leproust et al.: Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process. Nucleic Acids Research. 2010; 38(8):2522-2540. |
| Lesnikowski et al.: Nucleic acids and nucleosides containing carboranes. J. Organometallic Chem. 1999; 581:156-169. |
| Leumann. DNA analogues: from supramolecular principles to biological properties. Bioorg Med Chem. Apr. 2002;10(4):841-54. |
| Levene et al.: Zero-mode waveguides for single-molecule analysis at high concentrations. Science. Jan. 31, 2003;299(5607):682-6. |
| Lewontin and Harti. Population genetics in forensic DNA typing. Science, 254:1745-1750, 1991. |
| Li et al.: Beating Bias in the Directed Evolution of Proteins: Combining High-Fidelity on-Chip Solid-Phase Gene Synthesis with Efficient Gene Assembly for Combinatorial Library Construction. ChemBioChem 19:221-228 (2018). |
| Li et al.: Beating bias in the directed evolution of proteins: Combining high-fidelity on-chip solid-phase gene synthesis with efficient gene assembly for combinatorial library construction. First published Nov. 24, 2017, 2 pages. retrieved from: https://doi.org/10.1002/cbic.201700540. |
| Light source unit for printable patterning VUV-Aligner / USHIO Inc., Link here: https://www.ushio.co.jp/en/products/1005.html, published Apr. 25, 2016, printed from the internet on Aug. 2, 2016, 3 pages. |
| Limbachiya et al.: Natural data storage: A review on sending information from now to then via Nature. ACM Journal on Emerging Technologies in Computing Systems, V(N):Article A, May 19, 2015, 17 pages. |
| Link Technologies. Product Guide 2010. Nov. 27, 2009, 136 pages. XP055353191. Retrieved from the Internet: URL:http://www.linktech.co.uk/documents/517/517.pdf. |
| Lipshutz et al.: High density synthetic oligonucleotide arrays, Nature Genetics Supplement, vol. 21, 20-24 (Jan. 1999). |
| Lishanski et al.: Mutation detection by mismatch binding protein, MutS, in amplified DNA: application to the cystic fibrosis gene, Proc. Natl. Acad. Sci. USA, vol. 91, 2674-2678 (Mar. 1994). |
| Liu et al.: Comparison of Next-Generation Sequencing Systems. J Biomed Biotechnol 2012: 251364 (2012). |
| Liu et al.: Enhanced Signals and Fast Nucleic Acid Hybridization By Microfluidic Chaotic Mixing. Angew. Chem. Int. Ed. 2006; 45:3618-3623. |
| Liu et al.: Rational design of CXCR4 specific antibodies with elongated CDRs. JACS, 136:10557-10560, 2014. |
| Lizardi et al.: Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat Genet. Jul. 1998;19(3):225-32. |
| Li et al.: Functional domains in Fok I restriction endonuclease, Proc. Natl. Acad. Sci. USA, 89:4275-4279, 1992. |
| Lu et al.: Methyl-directed repair of DNA base-pair mismatches in vitro, Proc. Natl. Acad. Sci. USA, 80:4639-4643, 1983. |
| Lund et al.: A validated system for ligation-free uracilexcision based assembly of expression vectors for mammalian cell engineering. DTU Systems of Biology. 2011. 1 page. http://www.lepublicsystemepco.com/files/modules/gestion_rubriques/REF-B036-Lund_Anne%20Mathilde.pdf. |
| Ma et al.: DNA synthesis, assembly and application in synthetic biology. Current Opinion in Chemical Biology. 16:260-267, 2012. |
| Ma et al.: Versatile surface functionalization of cyclic olefin copolymer (COC) with sputtered SiO2 thin film for potential BioMEMS applications. Journal of Materials Chemistry, 11 pages, 2009. |
| Mahato et al.: Modulation of gene expression by antisense and antigene oligodeoxynucleotides and small interfering RNA Expert Opin. Drug Delivery, 2(1):3-28, 2005. |
| Malecek et al.: Engineering improved T cell receptors using an alanine-scan guided T cell display selection system. Journal of Immunological Methods. Elsevier Science Publishers. 392(1):1-11 (2013). |
| Margulies et al.: Genome sequencing in open microfabricated high-density picolitre reactors. Nature. 437(7057):376-80, 2005. |
| Martinez-Torrecuadrada et al.: Targeting the Extracellular Domain of Fibroblast Growth Factor Receptor 3 with Human Single-Chain Fv Antibodies Inhibits Bladder Carcinoma Cell Line Proliferation; Clinical Cancer Research; vol. 11; pp. 6282-6290 (2005). |
| Matteucci et al.: Synthesis of deoxyoligonucleotides on a polymer support. J. Am. Chem. Soc. 103(11):3185-3191, 1981. |
| Matzas et al.: Next generation gene synthesis by targeted retrieval of bead-immobilized, sequence verified DNA clones from a high throughput pyrosequencing device. Nat. Biotechnol., 28(12):1291-1294, 2010. |
| Mazor et al.: Isolation of Full-Length IgG Antibodies from Combinatorial Libraries Expressed in Escherichia coli; Antony S. Dimitrov (ed.), Therapeutic Antibodies: Methods and Protocols, vol. 525, Chapter 11, pp. 217-239 (2009). |
| McBride & Caruthers. An investigation of several deoxynucleoside phosphoramidites useful for synthesizing deoxyoligonucleotides. Tetrahedron Lett. 24: 245-248, 1983. |
| McGall et al.: Light-directed synthesis of high-density oligonucleotide arrays using semiconductor photoresists. Proc Natl Acad Sci U S A. 93(24):13555-60, 1996. |
| McGall et al.: The Efficiency of Light-Directed Synthesis of DNA Arrays on Glass Substrates. J. Am. Chem. Soc. 119(22):5081-5090, 1997. |
| Mei et al.: Cell-free protein synthesis in microfluidic array devices Biotechnol. Prog., 23(6):1305-1311, 2007. |
| Mendel-Hartvig. Padlock probes and rolling circle amplification. New possibilities for sensitive gene detection. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1175. Uppsala University. 2002, 39 pages. http://www.diva-portal.org/smash/get/diva2:161926/FULLTEXT01.pdf. |
| Meyers and Friedland. Knowledge-based simulation of genetic regulation in bacteriophage lambda. Nucl. Acids Research, 12(1):1-16, 1984. |
| Meynert et al.: Quantifying single nucleotide variant detection sensitivity in exome sequencing. BMC Bioinformatics 14:195 (2013). |
| Meynert et al.: Variant detection sensitivity and biases in whole genome and exome sequencing. BMC Bioinformatics 15:247 (2014). |
| Milo and Phillips. Numbers here reflect the No. of protein coding genes and excludes tRNA and non-coding RNA. Cell Biology by the Numbers, p. 286, 2015. |
| Mitra et al.: In situ localized amplification and contact replication of many individual DNA molecules. Nucleic Acids Res. 27(24):e34, 1999. |
| MLAB 2321 Molecular Diagnostics for Clinical Laboratory Science. Mar. 6, 2015. |
| Morin et al.: Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing. Biotechniques, 45:81-94, 2008. |
| Morris and Stauss. Optimizing T-cell receptor gene therapy for hematologic malignancies. Blood, 127(26):3305-3311, 2016. |
| Muller et al.: Protection and labelling of thymidine by a fluorescent photolabile group, Helvetica Chimica Acta, vol. 84, 3735-3741 (2001). |
| Mulligan. Commercial Gene Synthesis Technology PowerPoint presentation. BlueHeron® Biotechnology. Apr. 5, 2006 (48 pgs). |
| Nakatani et al.: Recognition of a single guanine bulge by 2-Acylamino-1 ,8-naphthyridine, J. Am. Chem. Soc., vol. 122, 2172-2177 (2000). |
| Neiman M.S.: Negentropy principle in information processing systems. Radiotekhnika, 1966, No. 11, p. 2-9. |
| Neiman M.S.: On the bases of the theory of information retrieval. Radiotekhnika, 1967, No. 5, p. 2-10. |
| Neiman M.S.: On the molecular memory systems and the directed mutations. Radiotekhnika, 1965, No. 6, pp. 1-8. |
| Neiman M.S.: On the relationships between the reliability, performance and degree of microminiaturization at the molecular-atomic level. Radiotekhnika, 1965, No. 1, pp. 1-9. |
| Neiman M.S.: Some fundamental issues of microminiaturization. Radiotekhnika, 1964, No. 1, pp. 3-12. |
| Nishikura. A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst Cell, 107:415-418, 2001. |
| Nour-Eldin et al.: USER Cloning and USER Fusion: The Ideal Cloning Techniques for Small and Big Laboratories. Plant Secondary Metabolism Engineering. Methods in Molecular Biology vol. 643, 2010, pp. 185-200. |
| Novartis Institutes for Biomedical Research. Immunoglobulin Heavy Chain [Homo sapiens]. National Center for Biotechnology Information. Genbank Entry. pp. 1-2 (2018) https://www.ncbi.nlm.nih.gov/nuccore/MH975524.1ttps://https://www.ncbi.nlm.nih.gov/nuccore/MH975524.1. |
| Novartis Institutes for Biomedical Research. Immunoglobulin Lambda Chain [Homo sapiens]. National Center for Biotechnology Information. Genbank Entry. pp. 1-2 (2018) https://www.ncbi.nlm.nih.gov/nuccore/MH975524.1. |
| Nucleic acid thermodynamics. Wikipedia. Feb. 4, 2021. |
| Ochman et al.: Genetic applications of an inverse polymerase chain reaction. Genetics. Nov. 1988;120(3):621-3. |
| O'Driscoll et al.: Synthetic DNA: The next generation of big data storage. Bioengineered. 4(3):123-125 (2013). |
| Organick et al.: Random access in large-scale DNA data storage. Nature Biotechnology, Advance Online Publication, 8 pages, 2018. |
| Organick et al.: Scaling up DNA data storage and random access retrieval, bioRxiv, preprint first posted online Mar. 7, 2017, 14 pages. |
| Pan et al.: An approach for global scanning of single nucleotide variations. Proc Natl Acad Sci U S A. Jul. 9, 2002;99(14):9346-51. |
| Pankiewicz. Fluorinated nucleosides. Carbohydr Res. Jul. 10, 2000;327(1-2):87-105. |
| Paul et al.: Acid binding and detritylation during oligonucleotide synthesis. Nucleic Acids Research. 15. pp. 3048-3052 (1996). |
| PCT/IL2012/000326 International Preliminary Report on Patentability dated Dec. 5, 2013. |
| PCT/IL2012/000326 International Search Report dated Jan. 29, 2013. |
| PCT/US2014/049834 International Preliminary Report on Patentability dated Feb. 18, 2016. |
| PCT/US2014/049834 International Search Report and Written Opinion mailed Mar. 19, 2015. |
| PCT/US2014/049834, Invitation to Pay Additional Fees and, where applicable, protest fee, mailed Jan. 5, 2015. |
| PCT/US2015/043605 International Preliminary Report on Patentability dated Feb. 16, 2017. |
| PCT/US2015/043605 International Search Report and Written Opinion dated Jan. 6, 2016. |
| PCT/US2015/043605 Invitation To Pay Additional Fees dated Oct. 28, 2015. |
| PCT/US2016/016459 International Preliminary Report on Patentability dated Aug. 17, 2017. |
| PCT/US2016/016459 International Search Report and Written Opinion dated Apr. 13, 2016. |
| PCT/US2016/016636 International Preliminary Report on Patentability dated Aug. 17, 2017. |
| PCT/US2016/016636 International Search Report and Written Opinion dated May 2, 2016. |
| PCT/US2016/028699 International Preliminary Report on Patentability dated Nov. 2, 2017. |
| PCT/US2016/028699 International Search Report and Written Opinion dated Jul. 29, 2016. |
| PCT/US2016/031674 International Preliminary Report on Patentability dated Nov. 23, 2017. |
| PCT/US2016/031674 International Search Report and Written Opinion dated Aug. 11, 2016. |
| PCT/US2016/052336 International Preliminary Report on Patentability dated Mar. 29, 2018. |
| PCT/US2016/052336 International Search Report and Written Opinion dated Dec. 7, 2016. |
| PCT/US2016/052916 International Preliminary Report on Patentability dated Apr. 5, 2018. |
| PCT/US2016/052916 International Search Report and Written Opinion dated Dec. 30, 2016. |
| PCT/US2016/064270 International Preliminary Report on Patentability dated Jun. 14, 2018. |
| PCT/US2016/064270 International Search Report and Written Opinion dated Apr. 28, 2017. |
| PCT/US2017/026232 International Preliminary Report on Patentability dated Feb. 26, 2019. |
| PCT/US2017/026232 International Search Report and Written Opinion dated Aug. 28, 2017. |
| PCT/US2017/036868 International Search Report and Written Opinion dated Aug. 11, 2017. |
| PCT/US2017/045105 International Preliminary Report on Patentability dated Feb. 5, 2019. |
| PCT/US2017/045105 International Search Report and Written Opinion dated Oct. 20, 2017. |
| PCT/US2017/052305 International Preliminary Report on Patentability dated Apr. 30, 2019. |
| PCT/US2017/052305 International Search Report and Written Opinion dated Feb. 2, 2018. |
| PCT/US2017/062391 International Preliminary Report on Patentability dated May 21, 2019. |
| PCT/US2017/062391 International Search Report and Written Opinion dated Mar. 28, 2018. |
| PCT/US2017/066847 International Search Report and Written Opinion dated May 4, 2018. |
| PCT/US2018/019268 International Preliminary Report on Patentability dated Aug. 27, 2019. |
| PCT/US2018/022487 International Search Report and Written Opinion dated Aug. 1, 2018. |
| PCT/US2018/022493 International Search Report and Written Opinion dated Aug. 1, 2018. |
| PCT/US2018/037152 International Preliminary Report on Patentability dated Dec. 17, 2019. |
| PCT/US2018/037152 International Search Report and Written Opinion dated Aug. 28, 2018. |
| PCT/US2018/037161 International Preliminary Report on Patentability dated Dec. 17, 2019. |
| PCT/US2018/037161 International Search Report and Written Opinion dated Oct. 22, 2018. |
| PCT/US2018/037161 Invitation to Pay Additional Fees dated Aug. 27, 2018. |
| PCT/US2018/050511 International Preliminary Report on Patentability dated Mar. 17, 2020. |
| PCT/US2018/050511 International Search Report and Written Opinion dated Jan. 11, 2019. |
| PCT/US2018/056783 International Preliminary Report on Patentability dated Apr. 30, 2020. |
| PCT/US2018/056783 International Search Report and Written Opinion of the International Searching Authority dated Dec. 20, 2018. |
| PCT/US2018/057857 International Preliminary Report on Patentability dated Apr. 28, 2020. |
| PCT/US2018/057857 International Search Report and Written Opinion dated Mar. 18, 2019. |
| PCT/US2018/19268 International Search Report and Written Opinion dated Jun. 26, 2018. |
| PCT/US2018/19268 Invitation to Pay Additional Fees and, where applicable, protest fee dated May 2, 2018. |
| PCT/US2018/22487 Invitation to Pay Additional Fees and, where applicable, protest fee dated May 31, 2018. |
| PCT/US2018/22493 Invitation to Pay Additional Fees and, where applicable, protest fee dated May 31, 2018. |
| PCT/US2019/012218 International Preliminary Report on Patentability dated Jul. 16, 2020. |
| PCT/US2019/012218 International Search Report and Written Opinion dated Mar. 21, 2019. |
| PCT/US2019/032992 International Preliminary Report on Patentability dated Nov. 24, 2020. |
| PCT/US2019/032992 International Search Report and Written Opinion dated Oct. 28, 2019. |
| PCT/US2019/032992 Invitation to Pay Additional Fees dated Sep. 6, 2019. |
| PCT/US2019/068435 International Search Report and Written Opinion dated Apr. 23, 2020. |
| PCT/US2020/019371 International Search Report and Written Opinion dated Jun. 25, 2020. |
| PCT/US2020/019986 International Search Report and Written Opinion dated Jul. 29, 2020. |
| PCT/US2020/019986 Invitation to Pay Additional Fees dated Jun. 5, 2020. |
| PCT/US2020/019988 International Search Report and Written Opinion dated Jul. 29, 2020. |
| PCT/US2020/019988 Invitation to Pay Additional Fees dated Jun. 8, 2020. |
| PCT/US2020/038679 International Search Report and Written Opinion dated Oct. 28, 2020. |
| PCT/US2020/052306 Invitation to Pay Additional Fees dated Dec. 18, 2020. |
| PCT/US2020/052291 Invitation to Pay Additional Fees dated Dec. 31, 2020. |
| Pease et al.: Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc Natl Acad Sci U S A. May 24, 1994;91(11):5022-6. |
| Peisajovich et al.: BBF RFC 28: A method for combinatorial multi-part assembly based on the type-lis restriction enzyme aarl. Sep. 16, 2009, 7 pages. |
| Pellois et al.: Individually addressable parallel peptide synthesis on microchips, Nature Biotechnology, vol. 20, 922-926 (Sep. 2002). |
| Petersen et al.: LNA: a versatile tool for therapeutics and genomics. Trends Biotechnol. Feb. 2003;21(2):74-81. |
| Pierce and Wangh. Linear-after-the-exponential polymerase chain reaction and allied technologies Real-time detection strategies for rapid, reliable diagnosis from single cells Methods Mol. Med. 132:65-85 (2007) (Abstract only). |
| Pierce et al.: Linear-after-the-exponential polymerase chain reaction and allied technologies. Real-time detection strategies for rapid, reliable diagnosis from single cells. Methods Mol Med. 132:65-85 (2007). |
| Pirrung. How to make a DNA chip. Angew. Chem. Int. Ed., 41:1276-1289, 2002. |
| Plesa et al.: Multiplexed gene synthesis in emulsions for exploring protein functional landscapes. Science, 10.1126/science.aao5167, 10 pages, 2018. |
| Pon. Solid-phase supports for oligonucleotide synthesis. Methods Mol Biol. 1993;20:465-96. |
| Poster. Reimagine Genome Scale Research. 2016, 1 page. Available at http://www2.twistbioscience.com/Oligo_Pools_CRISPR_poster. |
| Powers et al.: Optimal strategies for the chemical and enzymatic synthesis of bihelical deoxyribonucleic acids. J Am Chem Soc., 97(4):875-884, 1975. |
| Pray. Discovery of DNA Structure and Function: Watson and Crick, Nature Education, 2008, 6 pages. available at: http://www.nature.com/scitable/topicpage/discovery-of-dna-structure-and-function-watson-397. |
| Prodromou et al.: Recursive PCR: a novel technique for total gene synthesis. Protein Eng. Dec. 1992;5(8):827-9. |
| PubChem Data Sheet Acetonitrile. Printed from website https://pubchem.ncbi.nlm.nig.gov/ pp. 1-124 (2020). |
| PubChem Data Sheet Dichloromethane. Printed from website https://pubchem.ncbi.nlm.nih.gov/compound/Dichloromethane (2020). |
| PubChem Data Sheet Methylene Chloride. Printed from website https://pubchem.ncbi.nlm.nih.gov/ pp. 1-140 (2020). |
| Puigbo. Optimizer: a web server for optimizing the codon usage of DNA sequences. Nucleic Acid Research, 35(14):126-131, 2007. |
| Qian and Winfree. Scaling up digital circuit computation with DNA strand displacement cascades. Science, 332(6034):196-1201, 2011 . |
| Qian et al.: Neural network computation with DNA strand displacement cascades, Nature, 475(7356):368-372, 2011. |
| Quan et al.: Parallel on-chip gene synthesis and application to optimization of protein expression, Nature Biotechnology, 29(5):449-452, 2011. |
| Rafalski and Morgante. Corn and humans: recombination and linkage disequilibrium in two genomes of similar size. Trends in Genetics, 20(2):103-111, 2004. |
| Raje and Murma. A Review of electrohydrodynamic-inkjet printing technology. International Journal of Emerging Technology and Advanced Engineering, 4(5):174-183, 2014. |
| Rajpal et al.: A general method for greatly improving the affinity of antibodies by using combinatorial libraries. Proc. Natl. Acad. Sci. 102(24):8466-8471 (2005). |
| Rastegari et al.: XNOR-Net: ImageNet Classification Using Binary Convolutional Neural Networks, in ECCV 2016, Part IV, LNCS 9908, p. 525-542, 2016. |
| Reimagine SequenceSpace, Reimagine Research, Twist Bioscience, Product Brochure, Published Apr. 6, 2016 online at: www2.twistbioscience.com/TB_Product_Brochure_04.2016, 8 pages. |
| RF Electric discharge type excimer lamp. Products Catalog. Excimer lamp light source flat excimer, 16 pages dated Jan. 2016. From: http://www.hamamatsu.com/jp/en/product/category/1001/3026/index.html. |
| Richmond et al.: Amplification and assembly of chip-eluted Dna (AACED): a method for high-throughput gene synthesis. Nucleic Acids Res. Sep. 24, 2004;32(17):5011-8. Print 2004. |
| Roche. Restriction Enzymes from Roche Applied Science-A Tradition of Premium Quality and Scientific Support. FAQS and Ordering Guide. Roche Applied Science. Accessed Jan. 12, 2015, 37 pages. |
| Rogozin et al.: Origin and evolution of spliceosomal introns. Biology Direct, 7:11, 2012. |
| Ruminy et al.: Long-range identification of hepatocyte nuclear factor-3 (FoxA) high and low-affinity binding Sites with a chimeric nuclease, J. Mol. Bioi., vol. 310, 523-535 (2001). |
| Saaem et al.: In situ synthesis of DNA microarray on functionalized cyclic olefin copolymer substrate ACS Applied Materials & Interfaces, 2(2):491-497, 2010. |
| Saboulard et al.: High-throughput site-directed mutagenesis using oligonucleotides synthesized on DNA chips. Biotechniques. Sep. 2005;39(3):363-8. |
| Sacconi et al.: Three-dimensional magneto-optic trap for micro-object manipulation, Optics Letters, vol. 26, No. 17, 1359-1361 (Sep. 1, 2001). |
| Saiki et al.: Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature 324:163-166 (1986). |
| Sandhu et al.: Dual asymmetric PCR: one-step construction of synthetic genes. Biotechniques. Jan. 1992;12(1):14-6. |
| Sargolzaei et al.: Extent of linkage disequilibrium in Holstein cattle in North America. J.Dairy Science, 91:2106-2117, 2007. |
| Schaller et al.: Studies on Polynucleotides. XXV.1 The Stepwise Synthesis of Specific Deoxyribopolynucleotides (5). Further Studies on the Synthesis of Internucleotide Bond by the Carbodiimide Method. The Synthesis of Suitably Protected Dinucleotides as Intermediates in the Synthesis of Higher Oligonucleotides. J. Am. Chem. Soc. 1963; 85(23):3828-3835. |
| Shipman et al.: Molecular recordings by directed CRISPR spacer acquisition. Science. 353(6298):1-16 (2016). |
| Schmalzing et al.: Microchip electrophoresis: a method for high-speed SNP detection. Nucleic Acids Res 28(9):E43 (2000). |
| Schmitt et al.: New strategies in engineering T-cell receptor gene-modified T cells to more effectively target malignancies. Clinical Cancer Research, 21(23):5191-5197, 2015. |
| Seelig et al.: Enzyme-Free Nucleic Acid Logic Circuits, Science 314(5805):1585-1588, 2006. |
| Sharan et al.: Recombineering: a homologous recombination-based method of genetic engineering. Nat Profile 4(2):1-37 (originally pp. 206-223) (2009). |
| Sharpe and Mount. Genetically modified T cells in cancer therapy: opportunities and challenges. Disease Models and Mechanisms, 8:337-350, 2015. |
| Sierzchala et al.: Solid-phase oligodeoxynucleotide synthesis : a two-step cycle using peroxy anion deprotection, J. Am. Chem. Soc., vol. 125, No. 44, 13427-13441 (2003). |
| Simonyan and Zisserman. Very Deep Convolutional Networks for Large-Scale Image Recognition, Published as a conference paper at Int. Conf. Learn. Represent., pp. 1-14, 2015. |
| Singh-Gasson et al.: Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array, Nature Biotechnology, vol. 17, 974-978 (Oct. 1999). |
| Skerra. Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity. Nucleic Acids Res. Jul. 25, 1992; 20(14):3551-4. |
| Smith et al.: Direct mechanical measurements of the elasticity of single DNA molecules using magnetic beads, Science, vol. 258, 1122-1126 (Nov. 13, 1992). |
| Smith et al.: Generating a synthetic genome by whole genome assembly: phix174 bacteriophage from synthetic oligonucleotides. Proc Natl Acad Sci U S A. Dec. 23, 2003;100(26):15440-5. Epub Dec. 2, 2003. |
| Smith et al.: Generation of cohesive ends on PCR products by UDG-mediated excision of dU, and application for cloning into restriction digest-linearized vectors. PCR Methods Appl. May 1993;2(4):328-32. |
| Smith et al.: Mutation detection with MutH, MutL, and MutS mismatch repair proteins, Proc. Natl. Acad. Sci. USA, vol. 93, 4374-4379 (Apr. 1996). |
| Smith et al.: Removal of Polymerase-Produced mutant sequences from PCR products, Proc. Natl. Acad. Sci. USA, vol. 94, 6847-6850 (Jun. 1997). |
| Solomon et al.: Genomics at Agilent: Driving Value in DNA Sequencing.https://www.agilent.com/labs/features/2010_genomics.html, 8 pages (Aug. 5, 2010). |
| Soni et al.: Progress toward ultrafast DNA sequencing using solid-state nanopores. Clin Chem. Nov. 2007;53(11):1996-2001. Epub Sep. 21, 2007. |
| Southern et al.: Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models. Genomics. Aug. 1992;13(4):1008-17. |
| Sproat et al.: An efficient method for the isolation and purification of oligoribonucleotides. Nucleosides & Nucleotides. 1995; 14(1&2):255-273. |
| Srivannavit et al.: Design and fabrication of microwell array chips for a solution-based, photogenerated acid-catalyzed parallel oligonucleotide DNA synthesis. Sensors and Actuators A, 116:150-160, 2004. |
| Srivastava et al.: RNA synthesis: phosphoramidites for RNA synthesis in the reverse direction. Highly efficient synthesis and application to convenient introduction of ligands, chromophores and modifications of synthetic RNA at the 3′-end, Nucleic Acids Symposium Series, 52(1):103-104, 2008. |
| Steel. The Flow-Thru Chip A Three-dimensional biochip platform. In: Schena, Microarray Biochip Technology, Chapter 5, Natick, MA: Eaton Publishing, 2000, 33 pages. |
| Stemmer et al.: Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene. Oct. 16, 1995;164(1):49-53. |
| Stryer. DNA Probes and genes can be synthesized by automated solid-phase methods. Biochemistry, 3rd edition, New York: W.H. Freeman and Company, 1988; 123-125. |
| Stutz et al.: Novel fluoride-labile nucleobase-protecting groups for the synthesis of 3′(2′)-O-amino-acylated RNA sequences. Helv. Chim. Acta. 2000; 83(9):2477-2503. |
| Sullivan et al.: Library construction and evaluation for site saturation mutagenesis. Enzyme Microb. Technol. 53:70-77 (2013). |
| Sun et al.: Structure-Guided Triple-Code Saturation Mutagenesis: Efficient Tuning of the Stereoselectivity of an Epoxide Hydrolase. ACS Catal. 6:1590-1597 (2016). |
| Takahashi. Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers. Biotechniques. Jul. 2009;47(1):609-15. doi: 10.2144/000113155. |
| Tanase et al.: Magnetic trapping of multicomponent nanowires, The Johns Hopkins University, Baltimore, Maryland, p. 1-3 (Jun. 25, 2001). |
| Taylor et al.: Impact of surface chemistry and blocking strategies on DNA microarrays. Nucleic Acids Research, 31(16):e87, 19 pages, 2003. |
| The SLIC. Gibson, CPEC and SLiCE assembly methods (and GeneArt Seamless, In-Fusion Cloning). 5 pages, available online Sep. 2, 2010. |
| Tian et al.: Accurate multiplex gene synthesis from programmable DNA microchips. Nature. Dec. 23, 2004;432(7020):1050-4. |
| Tsai et al.: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing Nat. Biotechnol., 32(6):569-576, 2014. |
| Twist Bioscience | White Paper. DNA-Based Digital Storage. Retrieved from the internet, Twistbioscience.com, Mar. 27, 2018, 5 pages. |
| U.S. Appl. No. 14/241,874 Final Office Action dated Jan. 28, 2019. |
| U.S. Appl. No. 14/241,874 Office Action dated Feb. 27, 2017. |
| U.S. Appl. No. 14/241,874 Office Action dated Jul. 14, 2016. |
| U.S. Appl. No. 14/241,874 Office Action dated May 4, 2018. |
| U.S. Appl. No. 14/452,429 Notice of Allowance dated Jun. 7, 2016. |
| U.S. Appl. No. 14/452,429 Office Action mailed Apr. 9, 2015. |
| U.S. Appl. No. 14/452,429 Office Action mailed Oct. 21, 2015. |
| U.S. Appl. No. 14/452,429 Restriction Requirement mailed Dec. 12, 2014. |
| U.S. Appl. No. 14/885,962 Notice of Allowance dated Nov. 8, 2017 and Sep. 29, 2017. |
| U.S. Appl. No. 14/885,962 Office Action dated Dec. 16, 2016. |
| U.S. Appl. No. 14/885,962 Office Action dated Sep. 8, 2016. |
| U.S. Appl. No. 14/885,962 Restriction Requirement dated Mar. 1, 2016. |
| U.S. Appl. No. 14/885,963 Notice of Allowance dated May 24, 2016. |
| U.S. Appl. No. 14/885,963 Office Action dated Feb. 5, 2016. |
| U.S. Appl. No. 14/885,965 Office Action dated Aug. 28, 2018. |
| U.S. Appl. No. 14/885,965 Office Action dated Aug. 30, 2017. |
| U.S. Appl. No. 14/885,965 Office Action dated Feb. 10, 2017. |
| U.S. Appl. No. 14/885,965 Office Action dated Feb. 18, 2016. |
| U.S. Appl. No. 14/885,965 Office Action dated Jan. 4, 2018. |
| U.S. Appl. No. 14/885,965 Office Action dated Jul. 7, 2016. |
| U.S. Appl. No. 15/015,059 Final Office Action dated Jul. 17, 2019. |
| U.S. Appl. No. 15/015,059 Office Action dated Aug. 19, 2019. |
| U.S. Appl. No. 15/015,059 Office Action dated Feb. 7, 2019. |
| U.S. Appl. No. 15/135,434 Notice of Allowance dated Feb. 9, 2018. |
| U.S. Appl. No. 15/135,434 Office Action dated Nov. 30, 2017. |
| U.S. Appl. No. 15/135,434 Restriction Requirement dated Jul. 12, 2017. |
| U.S. Appl. No. 15/151,316 Final Office Action dated Jul. 9, 2020. |
| U.S. Appl. No. 15/151,316 Final Office Action dated Feb. 21, 2019. |
| U.S. Appl. No. 15/151,316 Office Action dated Jun. 7, 2018. |
| U.S. Appl. No. 15/151,316 Office Action dated Oct. 4, 2019. |
| U.S. Appl. No. 15/154,879 Notice of Allowance dated Feb. 1, 2017. |
| U.S. Appl. No. 15/156,134 Final Office Action dated Jan. 3, 2020. |
| U.S. Appl. No. 15/156,134 Office Action dated Apr. 4, 2019. |
| U.S. Appl. No. 15/156,134 Office Action dated Nov. 25, 2020. |
| U.S. Appl. No. 15/187,714 Final Office Action dated Sep. 17, 2019. |
| U.S. Appl. No. 15/187,714 Office Action dated Apr. 4, 2019. |
| U.S. Appl. No. 15/187,714 Restriction Requirement dated Sep. 17, 2018. |
| U.S. Appl. No. 15/187,721 Notice of Allowance dated Dec. 7, 2016. |
| U.S. Appl. No. 15/187,721 Office Action dated Oct. 14, 2016. |
| U.S. Appl. No. 15/233,835 Notice of Allowance dated Oct. 4, 2017. |
| U.S. Appl. No. 15/233,835 Office Action dated Feb. 8, 2017. |
| U.S. Appl. No. 15/233,835 Office Action dated Jul. 26, 2017. |
| U.S. Appl. No. 15/233,835 Restriction Requirement dated Nov. 4, 2016. |
| U.S. Appl. No. 15/245,054 Office Action dated Mar. 21, 2017. |
| U.S. Appl. No. 15/245,054 Office Action dated Oct. 19, 2016. |
| U.S. Appl. No. 15/268,422 Final Office Action dated Oct. 3, 2019. |
| U.S. Appl. No. 15/268,422 Office Action dated Mar. 1, 2019. |
| U.S. Appl. No. 15/268,422 Restriction Requirement dated Oct. 4, 2018. |
| U.S. Appl. No. 15/272,004 Office Action dated Jun. 12, 2020. |
| U.S. Appl. No. 15/377,547 Final Office Action dated Feb. 8, 2019. |
| U.S. Appl. No. 15/377,547 Office Action dated Jul. 27, 2018. |
| U.S. Appl. No. 15/377,547 Office Action dated Mar. 24, 2017. |
| U.S. Appl. No. 15/377,547 Office Action dated Nov. 30, 2017. |
| U.S. Appl. No. 15/433,909 Non-Final Office Action dated Feb. 8, 2019. |
| U.S. Appl. No. 15/433,909 Restriction Requirement dated Sep. 17, 2018. |
| U.S. Appl. No. 15/602,991 Final Office Action dated Dec. 13, 2018. |
| U.S. Appl. No. 15/602,991 Notice of Allowance dated Oct. 25, 2017. |
| U.S. Appl. No. 15/602,991 Office Action dated May 31, 2018. |
| U.S. Appl. No. 15/602,991 Office Action dated May 31, 2019. |
| U.S. Appl. No. 15/602,991 Office Action dated Sep. 21, 2017. |
| U.S. Appl. No. 15/603,013 Final Office Action dated Nov. 6, 2019. |
| U.S. Appl. No. 15/603,013 Office Action dated Jun. 26, 2019. |
| U.S. Appl. No. 15/603,013 Office Action dated Jan. 30, 2018. |
| U.S. Appl. No. 15/603,013 Office Action dated Jul. 10, 2018. |
| U.S. Appl. No. 15/603,013 Office Action dated Oct. 20, 2017. |
| U.S. Appl. No. 15/619,322 Final Office Action dated Mar. 30, 2020. |
| U.S. Appl. No. 15/619,322 Office Action dated Aug. 14, 2019. |
| U.S. Appl. No. 15/619,322 Office Action dated Nov. 4, 2020. |
| U.S. Appl. No. 15/682,100 Office Action dated Jan. 2, 2018. |
| U.S. Appl. No. 15/682,100 Restriction Requirement dated Nov. 8, 2017. |
| U.S. Appl. No. 15/709,274 Notice of Allowance dated Apr. 3, 2019. |
| U.S. Appl. No. 15/729,564 Final Office Action dated Dec. 13, 2018. |
| U.S. Appl. No. 15/729,564 Office Action dated Jan. 8, 2018. |
| U.S. Appl. No. 15/729,564 Office Action dated Jun. 6, 2018. |
| U.S. Appl. No. 15/729,564 Office Action dated May 30, 2019. |
| U.S. Appl. No. 15/816,995 Office Action dated May 19, 2020. |
| U.S. Appl. No. 15/816,995 Office Action dated Sep. 20, 2019. |
| U.S. Appl. No. 15/816,995 Restriction Requirement dated Apr. 4, 2019. |
| U.S. Appl. No. 15/835,342 Final Office Action dated Sep. 8, 2020. |
| U.S. Appl. No. 15/835,342 Office Action dated Dec. 2, 2019. |
| U.S. Appl. No. 15/835,342 Restriction Requirement dated Sep. 10, 2019. |
| U.S. Appl. No. 15/844,395 Office Action dated Jan. 24, 2020. |
| U.S. Appl. No. 15/844,395 Restriction Requirement dated May 17, 2019. |
| U.S. Appl. No. 15/860,445 Final Office Action dated Dec. 13, 2018. |
| U.S. Appl. No. 15/860,445 Office Action dated May 30, 2018. |
| U.S. Appl. No. 15/921,479 Final Office Action dated Jun. 15, 2020. |
| U.S. Appl. No. 15/921,479 Office Action dated Nov. 12, 2019. |
| U.S. Appl. No. 15/921,479 Restriction Requirement dated May 24, 2019. |
| U.S. Appl. No. 15/921,537 Office Action dated Apr. 1, 2020. |
| U.S. Appl. No. 15/960,319 Office Action dated Aug. 16, 2019. |
| U.S. Appl. No. 15/991,992 Office Action dated May 21, 2020. |
| U.S. Appl. No. 15/991,992 Restriction Requirement dated Mar. 10, 2020. |
| U.S. Appl. No. 16/006,581 Office Action dated Sep. 25, 2019. |
| U.S. Appl. No. 16/031,784 Office Action dated May 12, 2020. |
| U.S. Appl. No. 16/039,256 Office Action dated Aug. 20, 2020. |
| U.S. Appl. No. 16/039,256 Restriction Requirement dated May 18, 2020. |
| U.S. Appl. No. 16/128,372 Office Action dated Oct. 8, 2020. |
| U.S. Appl. No. 16/128,372 Restriction Requirement dated May 18, 2020. |
| U.S. Appl. No. 16/165,952 Office Action dated Mar. 12, 2020. |
| U.S. Appl. No. 16/239,453 Office Action dated May 11, 2020. |
| U.S. Appl. No. 16/239,453 Office Action dated Nov. 7, 2019. |
| U.S. Appl. No. 16/384,678 Final Office Action dated Oct. 15, 2020. |
| U.S. Appl. No. 16/384,678 Office Action dated Jan. 21, 2020. |
| U.S. Appl. No. 16/409,608 Office Action dated Sep. 9, 2019. |
| U.S. Appl. No. 16/530,717 Final Office Action dated Apr. 15, 2020. |
| U.S. Appl. No. 16/530,717 Office Action dated Sep. 6, 2019. |
| U.S. Appl. No. 16/535,777 Final Office Action dated Oct. 20, 2020. |
| U.S. Appl. No. 16/535,777 Office Action dated Feb. 8, 2021. |
| U.S. Appl. No. 16/535,777 Office Action dated Jan. 23, 2020. |
| U.S. Appl. No. 16/535,779 First Action Interview dated Feb. 10, 2020. |
| U.S. Appl. No. 16/798,275 Office Action dated Feb. 10, 2021. |
| Unger et al.: Monolithic microfabricated valves and pumps by multilayer soft lithography. Science. Apr. 7, 2000;288(5463):113-6. |
| Vaijayanthi et al.: Recent advances in oligonucleotide synthesis and their applications. Indian J Biochem Biophys. Dec. 2003;40(6):377-91. |
| Van Den Brulle et al.: A novel solid phase technology for high-throughput gene synthesis. Biotechniques. 2008; 45(3):340-343. |
| Van der Velde: Thesis. Finding the Strength of Glass. Delft University of Technology. 1-16 (2015). |
| Van Der Werf et al.: Limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14 belongs to a novel class of epoxide hydrolases. J. Bacteriol. 180:5052-5057 (1998). |
| Van Tassell et al.: SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries. Nature Methods, 5:247-252, 2008. |
| Vargeese et al.: Efficient activation of nucleoside phosphoramidites with 4,5-dicyanoimidazole during oligonucleotide synthesis. Nucleic Acids Res. Feb. 15, 1998;26(4):1046-50. |
| Verma et al.: Modified oligonucleotides: synthesis and strategy for users. Annu Rev Biochem 67:99-134 (1998). |
| Vincent et al.: Helicase-dependent isothermal DNA amplification. EMBO Rep. Aug. 2004;5(8):795-800. |
| Visscher et al.: Construction of multiple-beam optical traps with nanometer-resolution position sensing, IEEE Journal of Selected Topics in Quantum Electronics, vol. 2, No. 4, 1066-1076 (Dec. 1996). |
| Voldmans et al.: Holding forces of single-particle dielectrophoretic traps. Biophysical Journal, vol. 80, No. 1, 531-541 (Jan. 2001). |
| Vos et al.: AFLP:A new technique for DNA fingerprinting. Nucleic Acids Res. Nov. 11, 1995;23(21):4407-14. |
| Wagner et al.: Nucleotides, Part LXV, Synthesis of 2′-Deoxyribonucleoside 5′-Phosphoramidites: New Building Blocks for the Inverse (5′-3′)-Oligonucleotide Approach. Helvetica Chimica Acta, 83(8):2023-2035, 2000. |
| Wah et al.: Structure of Fok I has implications for DNA cleavage, Proc. Natl. Acad. Sci. USA, vol. 95, 10564-10569 (Sep. 1998). |
| Wah et al.: Structure of the multimodular endonuclease Fok I bound to DNA, Nature, vol. 388, 97-100 ( Jul. 1997). |
| Walker et al.: Strand displacement amplification—an isothermal, in vitro DNA amplification technique. Nucleic Acids Res. Apr. 11, 1992;20(7):1691-6. |
| Wan et al.: Deep Learning for Content-Based Image Retrieval: A comprehensive study. in Proceedings of the 22nd ACM International Conference on Multimedia—Nov. 3-7, 2014, Orlando, FL, p. 157-166, 2014. |
| Warr et al.: Exome Sequencing: current and future perspectives. G3: (Bethesda) 5(8):1543-1550 (2015). |
| Weber et al.: A modular cloning system for standardized assembly of multigene constructs. PLoS One. Feb. 18, 2011;6(2):e16765. doi: 10.1371/journal.pone.0016765. |
| Welz et al.: 5-(Benzylmercapto)-1H-tetrazole as activator for 2′-O-TBDMS phosphoramidite building blocks in RNA synthesis. Tetrahedron Lett. 2002; 43(5):795-797. |
| Westin et al.: Anchored multiplex amplification on a microelectronic chip array Nature Biotechnology, 18:199-202 (2000) (abstract only). |
| Whitehouse et al.: Analysis of the mismatch and insertion/deletion binding properties of Thermus thermophilus, HB8, MutS, Biochemical and Biophysical Research Communications, vol. 233, 834-837 (1997). |
| Wiedenheft et al.: RNA-guided genetic silencing systems in bacteria and archaea. Nature 482:331-338 (2012). |
| Wijshoff. Structure and fluid-dynamics in Piezo inkjet printheads. Thesis. Venio, The Netherlands, published 2008, p. 1-185. |
| Wirtz. Direct measurement of the transport properties of a single DNA molecule, Physical Review Letters, vol. 75, No. 12, 2436-2439 (Sep. 18, 1995). |
| Withers-Martinez et al.: PCR-based gene synthesis as an efficient approach for expression of the A+ T-rich malaria genome, Protein Engineering, vol. 12, No. 12, 1113-1120 (1999). |
| Wood et al.: Human DNA repair genes, Science, vol. 291, 1284-1289 (Feb. 16, 2001). |
| Wosnick et al.: Rapid construction of large synthetic genes: total chemical synthesis of two different versions of the bovine prochymosin gene. Gene. 1987;60(1):115-27. |
| Wright and Church. An open-source oligomicroarray standard for human and mouse. Nature Biotechnology, 20:1082-1083, 2002. |
| Wu et al.: An improvement of the on-line electrophoretic concentration method for capillary electrophoresis of proteins an experimental factors affecting he concentration effect, Analytical Sciences, vol. 16, 329-331 (Mar. 2000). |
| Wu et al.: RNA-mediated gene assembly from DNA arrays. Angew Chem Int Ed Engl. May 7, 2012;51(19):4628-32. doi: 10.1002/anie.201109058. |
| Wu et al.: Sequence-Specific Capture of Protein-DNA Complexes for Mass Spectrometric Protein Identification PloS ONE. Oct. 20, 2011, vol. 6, No. 10. |
| Wu et al.: Specificity of the nick-closing activity of bacteriophage T4 DNA ligase. Gene. 1989;76(2):245-54. |
| Xiong et al.: A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Res. 2004, 32(12):e98. |
| Xiong et al.: Chemical gene synthesis: Strategies, softwares, error corrections, and applications. FEMS Microbiol. Rev., 32:522-540, 2008. |
| Xiong et al.: Non-polymerase-cycling-assembly-based chemical gene synthesis: Strategies, methods, and progress. Biotechnology Advances. 26(2):121-134, 2008. |
| Xu et al.: Design of 240,000 orthogonal 25mer DNA barcode probes. PNAS, 106(7):2289-2294, 2009. |
| Xu et al.: Coordination between the Polymerase and 5 ′-Nuclease Components of DNA Polymerase I of Escherichia coli. The Journal of Biological Chemistry. 275(27):20949-20955 (2000). |
| Yang et al.: Purification, cloning, and characterization of the CEL I nuclease, Biochemistry, 39(13):3533-35, 2000. |
| Yazdi et al.: A Rewritable, Random-Access DNA-Based Storage System, Scientific Reports, 5, Article No. 14138, 27 pages, 2015. |
| Yazdi et al.: DNA-Based Storage: Trends and Methods. IEEE Transactions on Molecular, Biological and Multi-Scale Communications. IEEE. 1(3):230-248 (2016). |
| Yehezkel et al.: De novo DNA synthesis using single molecule PCR Nucleic Acids Research, 36(17):e107, 2008. |
| Yes HMDS vapor prime process application note Prepared by UC Berkeley and University of Texas at Dallas and re-printed by Yield Engineering Systems, Inc., 6 pages (http://www.yieldengineering.com/Portals/0/HMDS%20Application%20Note.pdf (Published online Aug. 23, 2013). |
| Youil et al.: Detection of 81 of 81 known mouse Beta-Globin promoter mutations with T4 Endonuclease VII. The EMC Method. Genomics, 32:431-435, 1996. |
| Young et al.: Two-step total gene synthesis method. Nucleic Acids Res. 32(7):e59, 2004. |
| Zhang and Seelig. Dynamic DNA nanotechnology using strand-displacement reactions, Nat. Chem., 3(2):103-113, 2011. |
| Zheleznaya et al.: Nicking endonucleases. Biochemistry (Mosc). 74(13):1457-66, 2009. |
| Zheng et al.: Manipulating the Stereoselectivity of Limonene Epoxide Hydrolase by Directed Evolution Based on Iterative Saturation Mutagenesis. J. Am. Chem. Soc. 132:15744-15751 (2010). |
| Zhirnov et al.: Nucleic acid memory. Nature Materials, 15:366, 2016. |
| Zhou et al.: Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences Nucleic Acids Research, 32(18):5409-5417 (2004). |
| Zhou et al.: Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane Scientific Reports. Vol. 4, No. 4912 (May 9, 2014). |
| Bai. A Novel Human scFv Library with Non-Combinatorial Synthetic CDR Diversity. PLoS One. 10(10):1-18 (2015). |
| Berg: Biochemistry. 5th ED. New York (2002) 148-149. |
| Borda et al.: Secret writing by DNA hybridization. Acta Technica Napocensis Electronics and Telecommunications. 50(2):21-24 (2008). |
| Fernández-Quintero et al.: Characterizing the Diversity of the CDR-H3 Loop Conformational Ensembles in Relationship to Antibody Binding Properties. Front. Immunol. 9:1-11 (2019). |
| GE Healthcare. AKTA oligopilot plus. Data File 18-114-66 AD ©. 8 pages (2006). |
| GE Healthcare. Robust and cost-efficient oligonucleotide synthesis. Application Note 28-4058-08 AA. 4 pages (2005). |
| Hudson: Matrix Assisted Synthetic Transformations: A Mosaic of Diverse Contributions. Journal of Combinatorial Chemistry. 1(6):403-457 (1999). |
| Kalva et al.: Gibson Deletion: a novel application of isothermal in vitro recombination. Biological Procedures Online. 20(1):1-10 (2018). |
| Lebl et al.: Economical Parallel Oligonucleotide and Peptide Synthesizer—Pet Oligator. Int. J. Peptide Res. Ther. 13(1-2):367-376 (2007). |
| Momentiv. Technical Data Sheet. Silquest A-1100. Momentiv. 1-6 (2020). |
| Opposition to European Patent No. 3030682 filed Mar. 3, 2021. |
| PCT/US2020/052291 International Search Report and Written Opinion dated Mar. 10, 2021. |
| PCT/US2020/052306 International Search Report and Written Opinion dated Mar. 2, 2021. |
| PCT/US2020/064106 International Search Report and Written Opinion dated Jun. 3, 2021. |
| PCT/US2020/064106 Invitation to Pay Additional Fees dated Apr. 9, 2021. |
| Pigott et al.: The Use of a Novel Discovery Platform to Identify Peptide-Grafted Antibodies that Activate GLP-1 Receptor Signaling. Innovative Targeting Solutions Inc. (2013) XP055327428 retrieved from the internet: http://www.innovativetargeting.com/wo-content/uploads/2013/12/Pigott-et-al-Antibody-Engineering-2013.pdf. |
| Ponsel. High Affinity, Developability and Functional Size: The Holy Grail of Combinatorial Antibody Library Generation. Molecules. 16:3675-3700 (2011). |
| Regep et al.: The H3 loop of antibodies shows unique structural characteristics. Proteins. 85(7):1311-1318 (2017). |
| U.S. Appl. No. 15/245,054 Notice of Allowance dated Dec. 14, 2017. |
| U.S. Appl. No. 15/272,004 Final Office Action dated Mar. 18, 2021. |
| U.S. Appl. No. 15/835,342 Office Action dated Apr. 16, 2021. |
| U.S. Appl. No. 15/902,855 Restriction Requirement dated Apr. 6, 2021. |
| U.S. Appl. No. 15/921,479 Office Action dated Apr. 27, 2021. |
| U.S. Appl. No. 16/039,256 Final Office Action dated Mar. 30, 2021. |
| U.S. Appl. No. 16/128,372 Final Office Action dated Mar. 18, 2021. |
| Number | Date | Country | |
|---|---|---|---|
| 20210142182 A1 | May 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62650231 | Mar 2018 | US | |
| 62617067 | Jan 2018 | US | |
| 62613728 | Jan 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16239453 | Jan 2019 | US |
| Child | 17154879 | US |
