Methods and devices for de novo oligonucleic acid assembly

Information

  • Patent Grant
  • 10669304
  • Patent Number
    10,669,304
  • Date Filed
    Wednesday, February 3, 2016
    8 years ago
  • Date Issued
    Tuesday, June 2, 2020
    4 years ago
Abstract
Methods and devices are provided herein for surfaces for de novo nucleic acid synthesis which provide for low error rates. In addition, methods and devices are provided herein for increased nucleic acid mass yield resulting from de novo nucleic acid synthesis.
Description
SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on Jan. 25, 2016, is named 44854_708_201_SL.txt and is 5,878 bytes in size.


BACKGROUND

Highly efficient chemical gene synthesis with high fidelity and low cost has a central role in biotechnology and medicine, and in basic biomedical research. De novo gene synthesis is a powerful tool for basic biological research and biotechnology applications. While various methods are known for the synthesis of relatively short fragments in a small scale, these techniques suffer from scalability, automation, speed, accuracy, and cost. There is a need for devices for simple, reproducible, scalable, less error-prone and cost-effective methods that guarantee successful synthesis of desired genes and are amenable to automation.


BRIEF SUMMARY

Provided herein are methods for preparing a surface for oligonucleic acid synthesis, comprising: providing a structure comprising a surface, wherein the structure comprises silicon dioxide; depositing a first molecule on the surface at a first region, wherein the first molecule binds to the surface and lacks a reactive group that binds to a nucleoside phosphoramidite; depositing a second molecule on the surface at a second region, wherein the second region comprises a plurality of loci surrounded by the first region, wherein the second molecule binds to the surface and lacks a reactive group that binds to the nucleoside phosphoramidite; and depositing a mixture on the surface at the second region, wherein the mixture comprises the second molecule and a third molecule, wherein the third molecule binds to the surface and nucleoside phosphoramidite, and wherein the mixture comprises a greater amount of the second molecule than the third molecule. Methods are further provided wherein the second molecule and the third molecule both have a higher surface energy than a surface energy of the first molecule, and wherein surface energy is a measurement of water contact angle on a smooth planar surface. Methods are further provided wherein the difference in water contact angle between the first region and the second region is at least 10, 20, 50, or 75 degrees. Methods are further provided wherein the third molecule is a silane. Methods are further provided wherein the third molecule is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane. Methods are further provided wherein the third molecule is 3-glycidoxypropyltrimethoxysilane. Methods are further provided wherein the silane is an aminosilane. Methods are further provided wherein the second molecule is propyltrimethoxysilane. Methods are further provided wherein the first molecule is a fluorosilane. Methods are further provided wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane. Methods are further provided wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 100:1 to about 2500:1. Methods are further provided wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 2000:1. Methods are further provided wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of 2000:1. Methods are further provided wherein the first molecule lacks a free hydroxyl, amino, or carboxyl group. Methods are further provided wherein the second molecule lacks a free hydroxyl, amino, or carboxyl group. Methods are further provided wherein the mixture is in a gaseous state when deposited on the surface. Methods are further provided wherein the first molecule is in a gaseous state when deposited on the surface. Methods are further provided wherein the surface comprises a layer of silicon oxide. Provided herein is a device for oligonucleic acid synthesis prepared by any one of the methods described herein.


Provide herein are methods for preparing a surface for oligonucleic acid synthesis, comprising: providing a structure comprising a surface, wherein the structure comprises silicon dioxide, and wherein the surface comprises a layer of silicon oxide; coating the surface with a light-sensitive material that binds silicon oxide; exposing predetermined regions of the surface to a light source to remove a portion of the light-sensitive material coated on the surface; depositing a first molecule on the surface, wherein the first molecule binds the surface at the predetermined regions and lacks a reactive group that binds to a nucleoside phosphoramidite; removing a remaining portion of the light-sensitive material coated on the surface to expose loci, wherein each of the loci are surrounded by the predetermined regions comprising the first molecule; depositing a second molecule on the surface at the loci, wherein the second molecule binds to the loci and lacks a reactive group that binds to the nucleoside phosphoramidite; and depositing a mixture on the surface at the loci, the mixture comprises the second molecule and a third molecule, and wherein the third molecule binds to the surface and nucleoside phosphoramidite. Methods are further provided wherein the second molecule and the third molecule both have a higher surface energy than a surface energy of the first molecule, wherein the second molecule and the third molecule both have a higher surface energy than a surface energy of the first molecule, and wherein surface energy is a measurement of water contact angle on a smooth planar surface. Methods are further provided wherein the difference in water contact angle between the first region and the second region is at least 10, 20, 50, or 75 degrees. Methods are further provided wherein the difference in water contact angle between the first region and the second region is at least 50 degrees. Methods are further provided wherein the third molecule is a silane. Methods are further provided wherein the third molecule is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane. Methods are further provided wherein the third molecule is 3-glycidoxypropyltrimethoxysilane. Methods are further provided wherein the silane is an aminosilane. Methods are further provided wherein the second molecule is propyltrimethoxysilane. Methods are further provided wherein the first molecule is a fluorosilane. Methods are further provided wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane. Methods are further provided wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 100:1 to about 2500:1. Methods are further provided wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 2000:1. Methods are further provided wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of 2000:1. Methods are further provided wherein the first molecule lacks a free hydroxyl, amino, or carboxyl group. Methods are further provided wherein the second molecule lacks a free hydroxyl, amino, or carboxyl group. Methods are further provided wherein the mixture is in a gaseous state when deposited on the surface. Methods are further provided wherein the first molecule is in a gaseous state when deposited on the surface. Methods are further provided wherein the method further comprises applying oxygen plasma to the surface prior to coating the surface with the light-sensitive material that binds silicon oxide. Methods are further provided wherein the method further comprises applying oxygen plasma to the surface after exposing predetermined regions of the surface to light. Provided herein is a device for oligonucleic acid synthesis prepared by any one of the methods described herein.


Provided herein are methods for oligonucleic acid synthesis, comprising: providing predetermined sequences for at least 30,000 non-identical oligonucleic acids; providing a structure comprising a patterned surface, wherein the structure comprises silicon dioxide; wherein the patterned surface is generated by: depositing a first molecule on the surface at a first region, wherein the first molecule binds to the surface and lacks a reactive group that binds to a nucleoside phosphoramidite; and depositing a second molecule on the surface at a second region, wherein the second region comprises a plurality of loci surrounded by the first region, wherein the second molecule binds to the surface and lacks a reactive group that binds to the nucleoside phosphoramidite; and depositing a mixture on the surface at the second region, wherein the mixture comprises the second molecule and a third molecule, wherein the third molecule binds to the surface and nucleoside phosphoramidite, wherein the mixture comprises a greater amount of the second molecule than the third molecule; and synthesizing the at least 30,000 non-identical oligonucleic acids each at least 10 bases in length, wherein the at least 30,000 non-identical oligonucleic acids encode sequences with an aggregate deletion error rate of less than 1 in 1500 bases compared to the predetermined sequences, and wherein each of the at least 30,000 non-identical oligonucleic acids extends from a different locus. Methods are further provided wherein the second molecule and the third molecule both have a higher surface energy than a surface energy of the first molecule, and surface energy is a measurement of water contact angle on a smooth planar surface. Methods are further provided wherein a difference in water contact angle between the first region and the second region is at least 10, 20, 50, or 75 degrees. Methods are further provided wherein the difference in water contact angle between the first region and the second region is at least 50 degrees. Methods are further provided wherein the third molecule is a silane. Methods are further provided wherein the third molecule is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane. Methods are further provided wherein the silane is an aminosilane. Methods are further provided wherein the third molecule is 3-glycidoxypropyltrimethoxysilane. Methods are further provided wherein the second molecule is propyltrimethoxysilane. Methods are further provided wherein the first molecule is a fluorosilane. Methods are further provided wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane. Methods are further provided wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 100:1 to about 2500:1. Methods are further provided wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 2000:1. Methods are further provided wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of 2000:1. Methods are further provided wherein the first molecule lacks a free hydroxyl, amino, or carboxyl group. Methods are further provided wherein the second molecule lacks a free hydroxyl, amino, or carboxyl group. Methods are further provided wherein the mixture is in a gaseous state when deposited on the surface. Methods are further provided wherein the first molecule is in a gaseous state when deposited on the surface. Methods are further provided wherein each of the at least 30,000 non-identical oligonucleic acids is at least 30 bases in length. Methods are further provided wherein each of the at least 30,000 non-identical oligonucleic acids is 10 bases to 1 kb in length. Methods are further provided wherein each of the at least 30,000 non-identical oligonucleic acids is about 50 to about 120 bases in length. Methods are further provided wherein the aggregate deletion error rate is less than about 1 in 1700 bases compared to the predetermined sequences. Methods are further provided wherein the aggregate deletion error rate is achieved without correcting errors. Methods are further provided wherein the at least 30,000 non-identical oligonucleic acids synthesized encode sequences with an aggregate error rate of less than 1 in 1500 bases compared to the predetermined sequences without correcting errors. Methods are further provided wherein the aggregate error rate is less than 1 in 2000 bases compared to the predetermined sequences. Methods are further provided wherein the aggregate error rate is less than 1 in 3000 bases compared to the predetermined sequences. Methods are further provided wherein the surface comprises a layer of silicon oxide.


Provided herein are methods for nucleic acid synthesis, comprising: providing predetermined sequences for at least 200 preselected nucleic acids; providing a structure comprising a patterned surface, wherein the structure comprises silicon dioxide; wherein the patterned surface is generated by: depositing a first molecule on the surface at a first region, wherein the first molecule binds to the surface and lacks a reactive group that binds to a nucleoside phosphoramidite; and depositing a second molecule on the surface at a second region, wherein the second region comprises a plurality of loci surrounded by the first region, wherein the second molecule binds to the surface and lacks a reactive group that binds to the nucleoside phosphoramidite; and depositing a mixture on the surface at the second region, wherein the mixture comprises the second molecule and a third molecule, wherein the third molecule binds to the surface and nucleoside phosphoramidite, wherein the mixture comprises a greater amount of the second molecule than the third molecule; and synthesizing at least 20,000 non-identical oligonucleic acids each at least 50 bases in length, wherein each of the at least 20,000 non-identical oligonucleic acids extends from a different locus of the patterned surface; releasing the at least 20,000 non-identical oligonucleic acids from the patterned surface; suspending the at least 20,000 non-identical oligonucleic acids in a solution; and subjecting the solution comprising at least 20,000 non-identical oligonucleic acids to a polymerase chain assembly reaction to assemble at least 200 genes, wherein the assembled at least 200 preselected nucleic acids encode sequences with an aggregate deletion error rate of less than 1 in 1500 bases compared to the predetermined sequences. Methods are further provided wherein the second molecule and the third molecule both have a higher surface energy than a surface energy of the first molecule, and wherein surface energy is a measurement of water contact angle on a smooth planar surface. Methods are further provided wherein the difference in water contact angle between the first region and the second region is at least 10, 20, 50, or 75 degrees. Methods are further provided wherein the difference in water contact angle between the first region and the second region is at least 50 degrees. Methods are further provided wherein the third molecule is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane. Methods are further provided wherein the third molecule is a silane. Methods are further provided wherein the third molecule is 3-glycidoxypropyltrimethoxysilane. Methods are further provided wherein the silane is an aminosilane. Methods are further provided wherein the second molecule is propyltrimethoxysilane. Methods are further provided wherein the first molecule is a fluorosilane. Methods are further provided wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane. Methods are further provided wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 100:1 to about 2500:1. Methods are further provided wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of about 2000:1. Methods are further provided wherein the mixture comprises the second molecule and the third molecule present in a molar ratio of 2000:1. Methods are further provided wherein the first molecule lacks a free hydroxyl, amino, or carboxyl group. Methods are further provided wherein the second molecule lacks a free hydroxyl, amino, or carboxyl group. Methods are further provided wherein each of the at least 20,000 non-identical oligonucleic acids is about 50 to about 120 bases in length. Methods are further provided wherein the aggregate deletion error rate is less than about 1 in 1700 bases compared to the predetermined sequences. Methods are further provided wherein the aggregate deletion error rate is achieved without correcting errors. Methods are further provided wherein the assembled at least 200 preselected nucleic acids encode sequences with an aggregate error rate of less than 1 in 1500 bases compared to the predetermined sequences without correcting errors. Methods are further provided wherein the aggregate error rate is less than 1 in 2000 bases compared to the predetermined sequences. Methods are further provided wherein the surface comprises a layer of silicon oxide.


Provided here are devices for oligonucleic acid synthesis, comprising: a structure having a surface, wherein the structure comprises silicon dioxide; a plurality of recesses or posts on the surface, wherein each recess or post comprises: a width length that is 6.8 nm to 500 nm, a pitch length that is about twice the width length, and a depth length that is about 60% to about 125% of the pitch length; a plurality of loci on the surface, wherein each locus has a diameter of 0.5 to 100 μm, wherein each locus comprises at least two of the plurality of recesses or posts; and a plurality of clusters on the surface, wherein each of the clusters comprise 50 to 500 loci and has a cross-section of 0.5 to 2 mm. Devices are further provided wherein each of the clusters comprise 100 to 150 loci. Devices are further provided wherein the structure comprises at least 30,000 loci. Devices are further provided wherein the pitch length is 1 μm or less. Devices are further provided wherein the depth length is 1 μm or less. Devices are further provided wherein each of the loci has a diameter of 0.5 μm. Devices are further provided wherein each of the loci has a diameter of 10 μm. Devices are further provided wherein each of the loci has a diameter of 50 μm. Devices are further provided wherein the cross-section of each of the clusters is about 1.125 mm. Devices are further provided wherein each of the clusters has a pitch of about 1.125 mm. Devices are further provided wherein each locus comprises a molecule that binds to the surface and a nucleoside phosphoramidite. Devices are further provided wherein the molecule that binds to the surface and the nucleoside phosphoramidite is a silane. Devices are further provided wherein the molecule that binds to the surface and the nucleoside phosphoramidite is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane. Devices are further provided wherein the silane is 3-glycidoxypropyltrimethoxysilane. Devices are further provided wherein the silane is an aminosilane. Devices are further provided wherein a region surrounding the plurality of loci comprises a molecule that binds to the surface and lacks a nucleoside phosphoramidite. Devices are further provided wherein the molecule that binds to the surface and lacks the nucleoside phosphoramidite is a fluorosilane. Devices are further provided wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane or perfluorooctyltrichlorosilane. Devices are further provided wherein the surface comprises a layer of silicon oxide.


Provided herein are methods for oligonucleic acid synthesis, comprising: providing predetermined sequences; providing a device for oligonucleic acid synthesis prepared by any one of methods described herein; synthesizing a plurality of non-identical oligonucleic acids at least 10 bases in length, wherein each of the non-identical oligonucleic acids extends from a different locus. Methods are further provided wherein the aggregate deletion error rate is less than about 1 in 1700 bases compared to the predetermined sequences. Methods are further provided wherein the aggregate deletion error rate is achieved without correcting errors. Methods are further provided wherein the plurality of non-identical oligonucleic acids synthesized encode sequences with an aggregate error rate of less than 1 in 1500 bases compared to the predetermined sequences without correcting errors. Methods are further provided wherein the aggregate error rate is less than 1 in 2000 bases compared to the predetermined sequences. Methods are further provided wherein the aggregate error rate is less than 1 in 3000 bases compared to the predetermined sequences.


Provided herein are methods for nucleic acid synthesis, comprising: providing predetermined sequences for at least 200 preselected nucleic acids; providing the device described herein; synthesizing at least 20,000 non-identical oligonucleic acids each at least 50 bases in length, wherein each of the at least 20,000 non-identical oligonucleic acids extends from a different locus; releasing the at least 20,000 non-identical oligonucleic acids from the surface; suspending the at least 20,000 non-identical oligonucleic acids in a solution; and subjecting the solution comprising at least 20,000 non-identical oligonucleic acids to a polymerase chain assembly reaction to assemble at least 200 genes, wherein the assembled at least 200 preselected nucleic acids encode sequences with an aggregate deletion error rate of less than 1 in 1500 bases compared to the predetermined sequences. Methods are further provided wherein the aggregate deletion error rate is less than about 1 in 1700 bases compared to the predetermined sequences. Methods are further provided wherein the aggregate deletion error rate is achieved without correcting errors. Methods are further provided wherein the assembled at least 200 preselected nucleic acids encode sequences with an aggregate error rate of less than 1 in 1500 bases compared to the predetermined sequences without correcting errors. Methods are further provided wherein the aggregate error rate is less than 1 in 2000 bases compared to the predetermined sequences.


Provide herein are devices for oligonucleic acid synthesis, comprising: a structure having a surface, wherein the structure comprises silicon dioxide; a plurality of recesses or posts on the surface, wherein each recess or post comprises (i) a width length that is 6.8 nm to 500 nm, (ii) a pitch length that is about twice the width length, and (iii) a depth length that is about 60% to about 125% of the pitch length; a plurality of loci on the surface, wherein each locus has a diameter of 0.5 to 100 um, wherein each locus comprises at least two of the plurality of recesses or posts; a plurality of clusters on the surface, wherein each of the clusters comprise 50 to 500 loci and has a cross-section of 0.5 to 2 mm, wherein the plurality of loci comprise a less than saturating amount of a molecule that binds the surface and couples to nucleoside phosphoramidite; and a plurality of regions surrounding each loci comprise a molecule that binds the surface and does not couple to nucleoside phosphoramidite, wherein the plurality of loci have a higher surface energy than the plurality of regions surrounding each loci. Devices are further provided wherein the molecule that binds the surface and couples to nucleoside phosphoramidite is a silane disclosed herein. Devices are further provided wherein the molecule that binds the surface and does not couple to nucleoside phosphoramidite is a fluorosilane disclosed herein. Devices are further provided wherein the plurality of loci are coated with a molecule that binds the surface, does not couple to nucleoside phosphoramidite, and has a higher surface energy than the molecule on plurality of regions surrounding each loci. Devices are further provided wherein the surface comprises a layer of silicon oxide.


INCORPORATION BY REFERENCE

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.





BRIEF DESCRIPTION OF THE DRAWINGS


FIGS. 1A-1F depict a process flow for patterning multiple chemical layers. FIG. 1A depicts optional cleaning of the surface of a structure. FIG. 1B depicts deposition of a photosensitive lack. FIG. 1C depicts an optical photolithography step. FIG. 1D depicts deposition of chemical layer to be patterned. FIG. 1E depicts removal of the photosensitive lack. FIG. 1F depicts deposition of a second chemical layer.



FIG. 2A illustrates a region of a surface for oligonucleic acid synthesis coated with a silane that binds the surface and couples nucleoside.



FIG. 2B illustrates a region of a surface for oligonucleic acid synthesis coated with a mixture of silanes, one silane that binds the surface and couples oligonucleic acid, and another silane that binds the surface and does not couple to nucleoside.



FIG. 3A illustrates an arrangement of an array of posts.



FIG. 3B illustrates an arrangement of an array of wells.



FIG. 4 illustrates loci within a cluster, a section of a cluster, a locus and a textured surface.



FIGS. 5A-5C and 5D-5F illustrate the process to manufacture a textured microfluidic device. FIG. 5A illustrates a silicon chip with one side polished. A textured layer is formed via pass printing scheme lithography (FIG. 5B), followed by silicon reactive ion etching and addition of a resist strip (FIG. 5C). Oxidation of the chip (FIG. 5D), printing of a fiducial layer via lithography (FIG. 5E), followed by a final oxide etching results in a textured silicon chip, depicted in FIG. 5F.



FIG. 6 illustrates a textured surface having small wells.



FIGS. 7A-7G illustrate a method for patterning a surface with a chemical layer. FIG. 7A depicts the optional cleaning of the surface of a structure. FIG. 7B depicts deposition of a first chemical layer, an active functional agent to be patterned. FIG. 7C depicts deposition of a photosensitive lack. FIG. 7D depicts an optical photolithography step. FIG. 7E depicts patterning of the chemical layer using the photosensitive lack as mask. FIG. 7F depicts removal of the photosensitive lack. FIG. 7G depicts deposition of a second chemical layer, a passive functionalization agent.



FIG. 8 illustrates a structure having fiducial markings.



FIG. 9 illustrates another structure having fiducial markings.



FIG. 10 is a diagram demonstrating a process workflow from oligonucleic synthesis to gene shipment.



FIG. 11 illustrates an outline of a system for nucleic acid synthesis, including an oligonucleic acid synthesizer, a structure (wafer), schematics outlining the alignment of the system elements in multiple directions, and exemplary setups for reagent flow.



FIG. 12 illustrates phosphoramidite chemistry for oligonucleotide synthesis.



FIG. 13 illustrates a pass-printing scheme.



FIG. 14 illustrates a computer system.



FIG. 15 is a block diagram illustrating a first architecture of a computer system.



FIG. 16 is a diagram demonstrating a network configured to incorporate a plurality of computer systems, a plurality of cell phones and personal data assistants, and Network Attached Storage (NAS).



FIG. 17 is a block diagram of a multiprocessor computer system using a shared virtual address memory space.



FIG. 18A depicts results from a photolithographic process performed using a chemical layer patterned as an adhesion promoter.



FIG. 18B depicts a close up view of one cluster depicted in FIG. 18A, the cluster having 80 μm discs.



FIG. 19, part A illustrates a functionalized surface. FIG. 19, part B illustrates corresponding BioAnalyzer data for each of the five spots in FIG. 19, part A.



FIG. 20 indicates BioAnalyzer data of surface extracted 100-mer oligonucleotides synthesized on a silicon oligonucleotide synthesis device.



FIG. 21 represents a sequence alignment, where “x” denotes a single base deletion, “star” denotes single base mutation, and “+” denotes low quality spots in Sanger sequencing.



FIG. 22 represents a sequence alignment, where “x” denotes a single base deletion, “star” denotes single base mutation, and “+” denotes low quality spots in Sanger sequencing.



FIG. 23 is a histogram for oligonucleotides encoding for 240 genes, with the length of oligonucleotide as the x-axis and number of oligonucleotide as the y-axis.



FIG. 24 is a histogram for oligonucleic acids collectively encoding for a gene, with the length of oligonucleotide as the x-axis and number of oligonucleotide as the y-axis.



FIG. 25 illustrates plots for DNA thickness per device (part A) and DNA mass per device (part B) for oligonucleic acids of 30, 50, and 80-mers when synthesized a surface.



FIG. 26 illustrates the deletion rate at a given index of synthesized oligonucleotides for various silane solutions.





DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides devices and methods for the rapid parallel synthesis of oligonucleic acids with low error rates. The oligonucleotide synthesis steps described here are “de novo,” meaning that oligonucleotides are built one monomer at a time to form a polymer. During de novo synthesis of oligonucleic acids, the crowding of single stranded oligonucleic acids extending from a surface results in an increase in error rates. To reduce the frequency of crowding-related errors, methods are provided herein to reduce the density of nucleoside-coupling agent bound to specific regions of the surface.


An exemplary method for generating a surface having a reduction in nucleoside-coupling agent density is illustrated in FIGS. 1A-1F. As a first step, a structure 101 is optionally cleaned with oxygen plasma, FIG. 1A. Exemplary structures include those made of silicon dioxide or silicon oxide. Directly after cleaning, the structure is coated with a photosensitive lack 103 (e.g., photoresist), FIG. 1B. Optical lithography is then performed, FIG. 1C, where electromagnetic wavelength 105 is projected through a shadow mask 107, resulting in removal of the photosensitive lack at predetermined locations and remaining photosensitive lack 109 at other locations. Next, a first molecule (e.g., a fluorosilane) is deposited on the surface and coats the surface at regions exposed as a result of photolithography 111, FIG. 1D. The first molecule is one that does not couple to nucleoside. The photosensitive lack is then stripped away (FIG. 1E), revealing exposed regions 113. The next step involves a two-part deposition process. First, a second molecule that binds the surface and does not bind nucleoside is deposited on the surface. Next, a mixture is deposited on the surface comprising the second molecule, and a third molecule, where the third molecule binds the surface and is also able to couple nucleoside, FIG. 1F. This two-step deposition process results in predetermined sites 115 on the surface having a low concentration of activating agent. To assist with efficiency of the reactions during the oligonucleic acid synthesis process, the region for oligonucleic acid extension (a locus) has a higher surface energy than the region of the surface surrounding the locus. When oligonucleic acids 201 are extended from a surface having a saturating amount of nucleoside-coupling molecule, they are relatively crowded, FIG. 2A. In contrast, when oligonucleic acids are extended on a surface having a mixture of a nucleoside-coupling molecule 203 and a non-nucleoside-coupling molecule 204, less crowding results, FIG. 2B, and a lower error rate is observed.


As a consequence of reducing the amount of nucleoside coupling molecules on the surface of a structure, the oligonucleic acid yields are also reduced. If the oligonucleic acid yields are too low, insufficient amounts of material will be produced for subsequent downstream molecular biology processes utilizing oligonucleic acids synthesized, e.g., as gene assembly. In order to increase the oligonucleic acid yields, methods are also provided for increasing the surface area at the site of oligonucleic acid extension by creating textured surface. To increase the surface area, the surface of a device may have a field of protrusions (see, for example, FIG. 3A) or a field of recesses (FIG. 3B), e.g., posts or rivets. To optimize surface area for oligonucleic acid synthesis methods, the height 300, width 305, and pitch 310 of the protrusions or height 315, width 320, and pitch 325 of the recesses are designed such that there is sufficient space for two times the length of the expected oligonucleic acid and sufficient depth for efficient washing. In one example, the width length is at least twice the desired oligonucleic acid length to be synthesized on a surface disclosed herein, the depth length is about 60% to about 125% of the pitch length, and the pitch length is about twice the width length.


A number of steps are performed to make a textured surface. A exemplary structure 101 (e.g., silicon-based) disclosed herein is polished (FIG. 5A), a textured layer pattern 501 is formed via printing lithography (FIG. 5B), a silicon reactive ion etching and resist strip is performed to leave indents 503 in the surface (FIG. 5C), the surface is subject to oxidation 507 (FIG. 5D), a fiducial layer is optionally printed on via lithography using a photosensitive lack 509 (FIG. 5E), after which a final oxide etching results in a textured silicon surface having a fiducial structure 513 (FIG. 5F). The structure may then be additionally exposed to functionalization agents as described above and elsewhere herein, to result in a structure having a patterned surface with loci coated with a molecule for coupling nucleoside 515 (alone or as a mixture depicted in FIG. 2B) surrounding by regions coated with an agent that does not couple nucleoside 517, FIG. 6.


DEFINITIONS

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 oligonucleotide or polynucleotide being known and chosen before the synthesis or assembly of the nucleic acid molecules.


Surface Materials


Structures for oligonucleic acid synthesis provided herein are fabricated from a variety of materials capable of modification to support a de novo oligonucleic acid synthesis reaction. In some cases, the structures are sufficiently conductive, e.g., are able to form uniform electric fields across all or a portion of the structure. A structure described herein can comprise a flexible material. Exemplary flexible materials include, without limitation, modified nylon, unmodified nylon, nitrocellulose, and polypropylene. A structure described herein can 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). Structure disclosed herein can be fabricated from a material comprising silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), glass, or any combination thereof. In some cases, a structure disclosed herein is manufactured with a combination of materials listed herein or any other suitable material known in the art. In some cases, a structure disclosed herein comprises a silicon dioxide base and a surface layer of silicon oxide. Alternatively, the structure can have a base of silicon oxide. Surface of the structure provided here can be textured, resulting in an increase overall surface area for oligonucleic acid synthesis. Structure disclosed herein may comprise at least 5%, 10%, 25%, 50%, 80%, 90%, 95%, or 99% silicon. A device disclosed herein may be fabricated from a silicon on insulator (SOI) wafer.


Patterned Surfaces


Disclosed herein are surfaces for the synthesis of oligonucleic acids at a low error rate. Surfaces disclosed herein comprise predetermined regions having varying wettability characteristics and varying ability to couple nucleoside. For example, a surface disclosed herein comprises high energy regions comprising a molecule that binds to the surface and also couples to nucleoside, and the high energy regions are surrounded by low energy regions comprising a molecule that binds to the surface and does not couple to nucleoside. In some instances, the high energy regions comprise a mixture of molecules having varying ability to couple nucleoside, e.g., a nucleoside phosphoramidite.


To set stage for coupling monomers extending from a structure, surfaces of a structure disclosed herein can be coated with a layer of material comprising an active functionalization agent, such as a nucleoside-coupling agent. An nucleoside-coupling agent is one that binds to the surface and also binds to nucleic acid monomer, thereby supporting a coupling reaction to the surface. Nucleoside-coupling agents are molecules having a reactive group, for example, a hydroxyl, amino or carboxyl group, available for binding to a nucleoside in a coupling reaction. A surface can be additionally coated with a layer of material comprising a passive functionalization agent. A passive functionalization agent or material binds to the surface but does not efficiently bind to nucleic acid, thereby preventing nucleic acid attachment at sites where passive functionalization agent is bound. Passive functionalization agents are molecules lacking an available reactive group (e.g., a hydroxyl, amino or carboxyl group) for binding a nucleoside in a coupling reaction. Surfaces can be configured for both active and passive functionalization agents bound to the surface at within predetermined regions of the surface, generating distinct regions for oligonucleic acid synthesis, wherein the region comprises a less than saturating amount of active functionalization agent, compare FIGS. 2A and 2B.


A first exemplary method of functionalizing a surface is discussed above with reference to FIGS. 1A-1F. In FIG. 1, the active functionalizing agent is deposited as a last step, FIG. 1F. Alternatively, the active functionalization agent may be deposited earlier in the process and function as an adhesion promoter for a photosensitive lack. FIG. 7 provides an illustrative representation of this alternative method. As a first step, a structure 101 is optionally cleaned with oxygen plasma, FIG. 7A. After cleaning, the structure is coated with a first chemical layer, a molecule that binds the surface and binds nucleoside is deposited on the surface 703 (e.g., an aminosilane), FIG. 7B. The surface of the device is then coated with a photosensitive lack 103, FIG. 7C. Optical lithography is then performed, FIG. 7D, where electromagnetic wavelength 105 is projected through a shadow mask 107, resulting in removal of the photosensitive lack 103 at predetermined locations and remaining photosensitive lack 109 (e.g., photoresist) at other locations. The use of a photoresist mask results in patterning of the first chemical layer 705, FIG. 7E. A second chemical layer, a molecule that binds the surface and does not bind nucleoside 707, is deposited on the surface, FIG. 7F. The photosensitive lack is then stripped away (FIG. 7G), revealing patterned regions 709. The resulting surface is patterned with loci comprising nucleoside-coupling molecules for oligonucleic acid extension reactions.


The surface energy of a chemical layer coated on a surface can facilitate localization of droplets on the surface. Depending on the patterning arrangement selected, the proximity of loci and/or area of fluid contact at the loci can be altered. In the context of silicon surfaces, certain aminosilane molecules can bind their silicon atom to the oxygen atom of the surface and also have additional chemical interactions for both binding photoresist or biomolecules. For example, for both (3-aminopropyl)trimethoxysilane (APTMS), or (3-aminopropyl)triethoxysilane (APTES), the silicon atom binds to the oxygen atom of the surface and the amine groups bind to the organic molecules. Exemplary activate functionalizing chemical coating agents include (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane or N-(3-triethoxysilylpropyl)-4-hydroxybutyramide.


Surfaces, or more specifically resolved loci, onto which nucleic acids or other molecules are deposited, e.g., for oligonucleic acid synthesis, can be smooth or substantially planar or are textured, having raised or lowered features (e.g., three-dimensional features). Surfaces disclosed herein can be layered with one or more different layers of compounds. Such modification layers of interest include, without limitation, inorganic and organic layers such as metals, metal oxides, polymers, small organic molecules and the like. Non-limiting polymeric layers include peptides, proteins, nucleic acids or mimetics thereof (e.g., peptide nucleic acids and the like), polysaccharides, phospholipids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyetheyleneamines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and any other suitable compounds described herein or otherwise known in the art. In some cases, polymers are heteropolymeric. In some cases, polymers are homopolymeric. In some cases, polymers comprise functional moieties or are conjugated.


Loci disclosed herein can be functionalized with one or more molecules that increase and/or decrease surface energy. The molecule can be chemically inert. The surface energy, or hydrophobicity, of a surface is a factor for determining the affinity of a nucleotide to attach onto the surface. Provided herein is a method for functionalization of a surface disclosed herein comprises: (a) providing a structure having a surface that comprises silicon dioxide; and (b) silanizing the surface using, a suitable silanizing agent described herein or otherwise known in the art, for example, an organofunctional alkoxysilane molecule. In some cases, the organofunctional alkoxysilane molecule comprises dimethylchloro-octodecyl-silane, methyldichloro-octodecyl-silane, trichloro-octodecyl-silane, trimethyl-octodecyl-silane, triethyl-octodecyl-silane, or any combination thereof. In some cases, a surface comprises functionalization with polyethylene/polypropylene (functionalized by gamma irradiation or chromic acid oxidation, and reduction to hydroxyalkyl surface), highly crosslinked polystyrene-divinylbenzene (derivatized by chloromethylation, and aminated to benzylamine functional surface), nylon (the terminal aminohexyl groups are directly reactive), or etched with reduced polytetrafluoroethylene.


A surface disclosed herein can be functionalized by contact with a derivatizing composition that contains a mixture of silanes, under reaction conditions effective to couple the silanes to the surface. Silanization generally can be used to cover a surface through self-assembly with organofunctional alkoxysilane molecules. A variety of siloxane functionalizing reagents can further be used as currently known in the art, e.g., for lowering or increasing surface energy. The organofunctional alkoxysilanes are classified according to their organic functions. Non-limiting examples of siloxane functionalizing reagents include hydroxyalkyl siloxanes (silylate surface, functionalizing with diborane and oxidizing the alcohol by hydrogen peroxide), diol (dihydroxyalkyl) siloxanes (silylate surface, and hydrolyzing to diol), aminoalkyl siloxanes (amines require no intermediate functionalizing step), glycidoxysilanes (3-glycidoxypropyl-dimethyl-ethoxysilane, glycidoxy-trimethoxysilane), mercaptosilanes (3-mercaptopropyl-trimethoxysilane, 3-4 epoxycyclohexyl-ethyltrimethoxysilane or 3-mercaptopropyl-methyl-dimethoxysilane), bicyclohepthenyl-trichlorosilane, butyl-aldehyde-trimethoxysilane, or dimeric secondary aminoalkyl siloxanes. The hydroxyalkyl siloxanes can include 3-(chloro)allyltrichlorosilane turning into 3-hydroxypropyl, or 7-octenyltrichlorosilane turning into 8-hydroxyoctyl. The diol (dihydroxyalkyl) siloxanes include glycidyl trimethoxysilane-derived (3-glycidyloxypropyl)trimethoxysilane (GOPS). The aminoalkyl siloxanes include 3-aminopropyl trimethoxysilane turning into 3-aminopropyl (3-aminopropyl-triethoxysilane, 3-aminopropyl-diethoxy-methylsilane, 3-aminopropyl-dimethyl-ethoxysilane, or 3-aminopropyl-trimethoxysilane). The dimeric secondary aminoalkyl siloxanes can be bis (3-trimethoxysilylpropyl) amine turning into bis(silyloxylpropyl)amine. In some cases, the functionalizing agent comprises 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane and N-(3-triethoxysilylpropyl)-4-hydroxybutyramide.


Desired surface tensions, wettabilities, water contact angles, and/or contact angles for other suitable solvents are achieved by providing a surface with a suitable ratio of functionalization agents. A surface disclosed herein can be functionalized to enable covalent binding of molecular moieties that can lower the surface energy so that wettability can be reduced. A portion of a surface disclosed herein may be prepared to have a high surface energy and increased wettability. Surfaces disclosed herein can also be modified to comprise reactive hydrophilic moieties such as hydroxyl groups, carboxyl groups, thiol groups, and/or substituted or unsubstituted amino groups. Suitable materials that can be used for solid phase chemical synthesis, e.g., cross-linked polymeric materials (e.g., divinylbenzene styrene-based polymers), agarose (e.g., Sepharose®), dextran (e.g., Sephadex®), cellulosic polymers, polyacrylamides, silica, glass (particularly controlled pore glass, or “CPG”), ceramics, and the like. The supports may be obtained commercially and used as is, or they may be treated or coated prior to functionalization.


Dilution of Active Functionalization Agent to Form Regions of High Surface Energy


To achieve surfaces with low density of nucleoside-coupling agents, a mixture of both active and passive functionalization agents is mixed and deposited at a predetermined region of a surface disclosed herein. Such a mixture provides for a region having a less than saturating amount of active functionalization agent bound to the surface and therefore lowers the density of functionalization agent in a particular region. A mixture of agents that bind to a surface disclosed herein can be deposited on predetermined region of the surface, wherein coated surface provides for a density of synthesized oligonucleic acids that is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% less than the density of synthesized oligonucleic acids extended from a region of the surface comprising only the active functionalization agent. The mixture can comprise (i) a silane that binds the surface and couples nucleoside and (ii) a silane that binds the surface and does not couple nucleoside is deposited on predetermined region of a surface disclosed herein, wherein coated surface provides for a density of synthesized oligonucleic acids that is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% less than the density of synthesized oligonucleic acids extended from a region of the surface comprising (i) the silane that binds the surface and couples nucleoside and not (ii) the silane that binds the surface and does not couple nucleoside. A predetermined region of a surface can be treated with a diluted amount of an active functionalization agent disclosed herein provides for a reduction in density of synthesized oligonucleic acids that is about 50% compared to an identical surface coated with a non-diluted amount of the active functionalization agents. One exemplary active functionalization agent is 3-glycidoxypropyltrimethoxysilane, which can optionally be included in a mixture disclosed herein. For example, the mixture can include 3-glycidoxypropyltrimethoxysilane or propyltrimethoxysilane; or 3-glycidoxypropyltrimethoxysilane and propyltrimethoxysilane. Regions surrounding those regions deposited with the mixture can be coated with a passive functionalization agent having a lower surface energy. In some cases, the passive functionalization agent having a lower surface energy is a fluorosilane. Exemplary fluorosilanes include (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane and perfluorooctyltrichlorosilane.


More broadly, active functionalization agent can comprise a silane, such as an aminosilane. In some cases, the active functionalization agent comprises a silane that, once activated, couples nucleoside, e.g., a nucleoside phosphoramidite. The active functionalization agent can be a silane that has a higher surface energy than the passive functionalization agent deposited on areas of the surface located outside of predetermined regions where the silane is deposited. In some cases, both molecules types in the mixture comprise silanes, and the mixture is deposited on the surface. For example, one of the molecules in the mixture is a silane that binds the surface and couples to nucleoside, and another one of the molecules in the surface is a silane that binds the surface and does not couple to nucleoside. In such cases, both molecules in the mixture, when deposited on the surface, provide for a region having a higher surface energy than regions surrounding where the mixture is deposited.


Agents in a mixture disclosed here are chosen from suitable reactive and inert moieties, thus diluting the surface density of reactive groups to a desired level for downstream reactions, where both molecules have similar surface energy. In some cases, the density of the fraction of a surface functional group that reacts to form a growing oligonucleotide in an oligonucleotide synthesis reaction is about 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 7.0, 10.0, 15.0, 20.0, 50.0, 75.0, 100.0 μmol/m2.


Mixtures disclosed herein can comprise at least 2, 3, 4, 5 or more different types of functionalization agents. A mixture can comprises 1, 2, 3 or more silanes. The ratio of the at least two types of surface functionalization agents in a mixture deposited on a surface disclosed herein may range from about 1:1 to 1:100 with the active functionalization agent being diluted to a greater amount compared to a functionalization agent that does not couple nucleoside. In some cases, the ratio of the at least two types of surface functionalization agents in a mixture deposited on a surface disclosed herein is about 1:100 to about 1:2500, with the active functionalization agent being diluted to a greater amount compared to a functionalization agent that does not couple nucleoside. Exemplary ratios of the at least two types of surface functionalization agents in a mixture deposited on a surface disclosed herein include at least 1:10, 1:50, 1:100, 1:200, 1:500, 1:1000, 1:2000, 1:2500, 1:3000, or 1:5000, with the active functionalization agent being diluted to a greater amount compared to a functionalization agent that does not couple nucleoside. An exemplary specific ratio of the at least two types of surface functionalization agents in a mixture is about 1:2000, with the active functionalization agent being diluted to a greater amount compared to a functionalization agent that does not couple nucleoside. Another exemplary specific ratio of the at least two types of surface functionalization agents in a mixture is 1:2000, with the active functionalization agent being diluted to a greater amount compared to a functionalization agent that does not couple nucleoside. The passive functionalization agent deposited on the surface can be a fluorosilane molecule. Exemplary fluorosilane molecules are (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane and perfluorooctyltrichlorosilane. The mixture deposited on a surfaced disclosed herein may comprise a silane that binds the surface and nucleoside phosphoramidite and is diluted about 1:100 to about 1:2500 with a silane that bind the surface and does not bind nucleoside phosphoramidite. In some cases, the silane molecule deposited on a surfaced disclosed herein is diluted about 1:2000.


To attain a reduction in active agent density at particular locations on a surface disclosed herein, deposition at regions for nucleic acid extension with the non-nucleoside molecule of the mixture occurs prior to deposition with the mixture itself. In some case, the mixture deposited on a surfaced disclosed herein comprises 3-glycidoxypropyltrimethoxysilane diluted at a ratio of about 1:2000. The mixture deposited on a surfaced disclosed herein may comprise 3-glycidoxypropyltrimethoxysilane diluted at a ratio of about 1:2000 in propyltrimethoxysilane. In one example, a surface disclosed herein is first deposited at regions for nucleic acid extension with propyltrimethoxysilane prior to deposition of a mixture of propyltrimethoxysilane and 3-glycidoxypropyltrimethoxysilane. In some cases, a silane deposited at sites of oligonucleic acid synthesis are selected from the group consisting of 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidyloxypropyl/trimethoxysilane and N-(3-triethoxysilylpropyl)-4-hydroxybutyramide.


Hydrophilic and Hydrophobic Surfaces


The surface energy, or hydrophobicity of a surface, can be evaluated or measured by measuring a water contact angle. Water contact angle is the angle between the drop surface and a solid surface where a water droplet meets the solid surface. A surface with a water contact angle of smaller than 90°, the solid surface can be considered hydrophilic or polar. A surface with a water contact angle of greater than 90°, the solid surface can be considered hydrophobic or apolar. Highly hydrophobic surfaces with low surface energy can have water contact angle that is greater than 120°.


Surface characteristics of coated surfaces can be adjusted in various ways suitable for oligonucleotide synthesis. A surface described herein is selected to be inert to the conditions of ordinary oligonucleotide synthesis; e.g. the solid surface may be devoid of free hydroxyl, amino, or carboxyl groups to the bulk solvent interface during monomer addition, depending on the selected chemistry. In some cases, the surface may comprise reactive moieties prior to the start of a first cycle, or first few cycles of an oligonucleotide synthesis process, wherein the reactive moieties can be quickly depleted to unmeasurable densities after one, two, three, four, five, or more cycles of the oligonucleotide synthesis reaction. The surface can further be optimized for well or pore wetting, e.g., by common organic solvents such as acetonitrile and the glycol ethers or aqueous solvents, relative to surrounding surfaces.


Without being bound by theory, the wetting phenomenon is understood to be a measure of the surface tension or attractive forces between molecules at a solid-liquid interface, and is expressed in dynes/cm2. For example, fluorocarbons have very low surface tension, which is typically attributed to the unique polarity (electronegativity) of the carbon-fluorine bond. In tightly structured Langmuir-Blodgett type films, surface tension of a layer can be primarily determined by the percent of fluorine in the terminus of the alkyl chains. For tightly ordered films, a single terminal trifluoromethyl group can render a surface nearly as lipophobic as a perfluoroalkyl layer. When fluorocarbons are covalently attached to an underlying derivatized solid (e.g. a highly crosslinked polymeric) support, the density of reactive sites can be lower than Langmuir-Blodgett and group density. For example, surface tension of a methyltrimethoxysilane surface can be about 22.5 mN/m and aminopropyltriethoxysilane surface can be about 35 mN/m. Briefly, hydrophilic behavior of surfaces is generally considered to occur when critical surface tensions are greater than 45 mN/m. As the critical surface tension increases, the expected decrease in contact angle is accompanied with stronger adsorptive behavior. Hydrophobic behavior of surfaces is generally considered to occur when critical surface tensions are less than 35 mN/m. At first, the decrease in critical surface tension is associated with oleophilic behavior, i.e. the wetting of the surfaces by hydrocarbon oils. As the critical surface tensions decrease below 20 mN/m, the surfaces resist wetting by hydrocarbon oils and are considered both oleophobic as well as hydrophobic. For example, silane surface modification can be used to generate a broad range of critical surface tensions. Devices and methods disclosed herein include surface coatings, e.g. those involving silanes, to achieve surface tensions of less than 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 115, 120 mN/m, or higher. Further, in some cases, the methods and devices disclosed herein use surface coatings, e.g. those involving silanes, to achieve surface tensions of more than 115, 110, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6 mN/m or less. The water contact angle and the surface tension of non-limiting examples of surface coatings, e.g., those involving silanes, are described in Table 1 and Table 2 of Arkles et al. (Silanes and Other Coupling Agents, Vol. 5v: The Role of Polarity in the Structure of Silanes Employed in Surface Modification. 2009), which is incorporated herein by reference in its entirety. The tables are replicated below.









TABLE 1





Contact angles of water (degrees) on smooth surfaces


















Heptadecafluorodecyltrimethoxysilane
113-115



Poly(tetrafluoroethylene)
108-112



Polypropylene
108 



Octadecyldimethylchlorosilane
110 



Octadecyltrichlorosilane
102-109



Tris(trimethylsiloxy)silylethyldimethylchlorosilane
103-104



Octyldimethylchlorosilane
104 



Butyldimethylchlorosilane
100 



Trimethylchlorosilane
 90-100



Polyethylene
 88-103



Polystyrene
94



Poly(chlorotrifluoroethylene)
90



Human skin
75-90



Diamond
87



Graphite
86



Silicon (etched)
86-88



Talc
82-90



Chitosan
80-81



Steel
70-75



Methoxyethoxyundecyltrichlorosilane
73-74



Methacryloxypropyltrimethoxysilane
70



Gold, typical (see gold, clean)
66



Intestinal mucosa
50-60



Kaolin
42-46



Platinum
40



Silicon nitride
28-30



Silver iodide
17



[Methoxy(polyethyleneoxy)propyl]trimethoxysilane
15-16



Sodalime glass
<15 



Gold, clean
<10 



Trimethoxysilylpropyl substituted poly(ethyleneimine),
<10 



hydrochloride

















TABLE 2





Critical surface tensions (mN/m)


















Heptadecafluorodecyltrichlorosilane
12



Poly(tetrafluoroethylene)
18.5



Octadecyltrichlorosilane
20-24



Methyltrimethoxysilane
22.5



Nonafluorohexyltrimethoxysilane
23



Vinyltriethoxysilane
25



Paraffin wax
25.5



Ethyltrimethoxysilane
27.0



Propyltrimethoxysilane
28.5



Glass, sodalime (wet)
30.0



Poly(chlorotrifluoroethylene)
31.0



Polypropylene
31.0



Poly(propylene oxide)
32



Polyethylene
33.0



Trifluoropropyltrimethoxysilane
33.5



3-(2-Aminoethyl)aminopropyltrimethoxysilane
33.5



Polystyrene
34



p-Tolyltrimethoxysilane
34



Cyanoethyltrimethoxysilane
34



Aminopropyltriethoxysilane
35



Acetoxypropyltrimethoxysilane
37.5



Poly(methyl methacrylate)
39



Poly(vinyl chloride)
39



Phenyltrimethoxysilane
40.0



Chloropropyltrimethoxysilane
40.5



Mercaptopropyltrimethoxysilane
41



Glycidoxypropyltrimethoxysilane
42.5



Poly(ethylene terephthalate)
43



Copper (dry)
44



Poly(ethylene oxide)
43-45



Aluminum (dry)
45



Nylon 6/6
45-46



Iron (dry)
46



Glass, sodalime (dry)
47



Titanium oxide (anatase)
91



Ferric oxide
107



Tin oxide
111










Provided herein are surfaces, or a portion of the surface, functionalized or modified to be more hydrophilic or hydrophobic as compared to the surface or the portion of the surface prior to the functionalization or modification. One or more surfaces may be modified to have a difference in water contact angle of greater than 90°, 85°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15° or 10° as measured on one or more uncurved, smooth or planar equivalent surfaces. In some cases, the surface of microstructures, channels, wells, resolved loci, resolved reactor caps or other parts of structure is modified to have a differential hydrophobicity corresponding to a difference in water contact angle that is greater than 90°, 85°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15° or 10° as measured on uncurved, smooth or planar equivalent surfaces of such structures.


Provided herein are surfaces have a predetermined first region comprising two or more molecules having different ability to couple nucleoside and have a similarity in water contact angle. In some cases, the similarity is less than 40°, 35°, 30°, 25°, 20°, 15° or 10° as measured on one or more smooth or planar equivalent surfaces. In some cases, the first region is surrounded by a second region, where the first and second region have a difference in water contact angle of greater than 90°, 85°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15° or 10° as measured on one or more smooth or planar equivalent surfaces. In some cases, the first region is surrounded by a second region, where the first and second region have a difference in water contact angle of at least 90°, 85°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15° or 10° as measured on one or more smooth or planar equivalent surfaces. Unless otherwise stated, water contact angles mentioned herein correspond to measurements that would be taken on uncurved, smooth or planar equivalents of the surfaces in question.


Surface Preparation


Surfaces provided herein comprise or are modified to support oligonucleic acid synthesis at predetermined locations and with a resulting low error rate. A common method for functionalization comprises selective deposition of an organosilane molecule onto a surface of a structure disclosed herein. Selective deposition refers to a process that produces two or more distinct areas on a structure, wherein at least one area has a different surface or chemical property that another area of the same structure. Such properties include, without limitation, surface energy, chemical termination, surface concentration of a chemical molecule, and the like. Any suitable process that changes the chemical properties of the surface described herein or known in the art may be used to functionalize the surface, for example chemical vapor deposition of an organosilane. Typically, this results in the deposition of a self-assembled monolayer (SAM) of the functionalization species.


Provided herein are methods for functionalizing a surface of a structure disclosed herein for oligonucleic acid synthesis which include photolithography. An exemplary photolithographic method comprises 1) applying a photoresist to a surface, 2) exposing the resist to light, e.g., using a binary mask opaque in some areas and clear in others, and 3) developing the resist; wherein the areas that were exposed are patterned. The patterned resist can then serve as a mask for subsequent processing steps, for example, etching, ion implantation, and deposition. After processing, the resist is typically removed, for example, by plasma stripping or wet chemical removal. Oxygen plasma cleaning may optionally be used to facilitate the removal of residual organic contaminants in resist cleared areas, for example, by using a typically short plasma cleaning step (e.g., oxygen plasma). Resist can be stripped by dissolving it in a suitable organic solvent, plasma etching, exposure and development, etc., thereby exposing the areas of the surface that had been covered by the resist. Resist can be removed in a process that does not remove functionalization groups or otherwise damage the functionalized surface.


Provided herein is a method for functionalizing a surface of a structure disclosed herein for oligonucleic acid synthesis comprises a resist or photoresist coat. Photoresist, in many cases, refers to a light-sensitive material useful in photolithography to form patterned coatings. It is applied as a liquid to solidify on a surface as volatile solvents in the mixture evaporate. In some cases, the resist is applied in a spin coating process as a thin film, e.g., 1 μm to 100 μm. The coated resist can be patterned by exposing it to light through a mask or reticle, changing its dissolution rate in a developer. In some cases, the resist coat is used as a sacrificial layer that serves as a blocking layer for subsequent steps that modify the underlying surface, e.g., etching, and then is removed by resist stripping. Surface of a structure can be functionalized while areas covered in resist are protected from active or passive functionalization.


Provide herein are methods where a chemical cleaning is a preliminary step in surface preparation. In some exemplary methods, active functionalization is performed prior to lithography. A structure may be first cleaned, for example, using a piranha solution. An example of a cleaning process includes soaking a structure 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 structure (e.g., nitrogen gas). The process optionally includes a post piranha treatment comprising soaking the piranha treated structure in a basic solution (e.g., NH4OH) followed by an aqueous wash (e.g., water). Alternatively, a structure can be plasma cleaned, optionally following the piranha soak and optional post piranha treatment. An example of a plasma cleaning process comprises an oxygen plasma etch.


Provided herein are methods for surface preparation where, an active chemical vapor (CVD) deposition step is done after photolithography. An exemplary first step includes optionally cleaning the surface cleaning the surface of a structure using cleaning methods disclosed herein. Cleaning can include oxygen plasma treatment. In some cases, the CVD step is for deposition of a mixture, the mixture having at least two molecules resulting in a high surface energy region and the region coated with the first chemical layer is a lower surface energy region. The mixture may comprise a molecule that binds the surface and couple nucleoside phosphoramidite mixed with a greater amount of a molecule that binds the surface and does not couple nucleoside phosphoramidite. For the two step dilution protocol, prior to depositing the mixture on the surface, a step includes deposition of 100% of the mixture ingredient molecule that binds the surface and does not couple nucleoside phosphoramidite. The first chemical layer can comprise a fluorosilane disclosed herein, for example, tridecafluoro-1,1,2,2-tetrahydrooctyl)-trichlorosilane. The second chemical layer can comprise at least two silanes disclosed herein. In some cases, the two silanes are GOPS and propyltrimethoxysilane. In an exemplary method, the surface is treated with propyltrimethoxysilane prior to treatment with the mixture. The above workflow is an example process and any step or component may be omitted or changed in accordance with properties desired of the final functionalized surface.


A surface of a structure disclosed herein can be coated with a resist, subject to functionalization and/or after lithography, and then treated to remove the resist. 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. After stripping resist, the surface can be further subjected to deposition of an active functionalization agent binding to exposed areas to create a desired differential functionalization pattern. In some cases, the active functionalization areas comprise one or more different species of silanes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more silanes. One of the one or more silanes may b present in the functionalization composition in an amount greater than another silane. The composition and density of functionalization agent can contribute to a low error rate of oligonucleic acid synthesis, e.g., an error rate of less than 1 in 1000, less than 1 in 1500, less than 1 in 2000, less than 1 in 3000, less than 1 in 4000, less than 1 in 5000 bases).


Provided herein are methods which include applying an adhesion promoter to the surface. The adhesion promoter is applied in addition to applying the light sensitive lack. In some cases, applying both the adhesion promoter and light sensitive lack is done to surfaces including, without limitation, glass, silicon, silicon dioxide and silicon nitride. Exemplary adhesion promoters include silanes, e.g., aminosilanes. Exemplary aminosilanes include, without limitation, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane and N-(3-triethoxysilylpropyl)-4-hydroxybutyramide. In addition, a passive layer can be deposited on the surface, which may or may not have reactive oxide groups. The passive layer can comprise silicon nitride (Si3N4) or polyamide. The photolithographic step can be used to define regions where loci form on the passivation layer.


Provided here are methods for producing a substrate having a plurality of loci starts with a structure. The structure (e.g., silicon) can have any number of layers disposed upon it, including but not limited to a conducting layer such as a metal (e.g., silicon dioxide, silicon oxide, or aluminum). The structure can have a protective layer (e.g., titanium nitride). The layers can be deposited with the aid of various deposition techniques, such as, for example, 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) and physical vapor deposition (e.g., sputter deposition, evaporative deposition).


An oxide layer can be deposited on the structure. The oxide layer can comprise silicon dioxide. The silicon dioxide can be deposited using tetraethyl orthosilicate (TEOS), high density plasma (HDP), or any combination thereof. The silicon dioxide can be deposited to a thickness suitable for the manufacturing of suitable microstructures described in further detail elsewhere herein. In some cases, silicon dioxide is grown in a conformal way on a silicon substrate. Growth on a silicon substrate can performed in a wet or dry atmosphere. An exemplary wet growth method is provided where wet growth is conducted at high temperatures, e.g., about 1000 degrees Celsius and in water vapor. The dry growth method can be conducted in the presence of oxygen.


Loci can be created using photolithographic techniques such as those used in the semiconductor industry. For example, a photo-resist (e.g., a material that changes properties when exposed to electromagnetic radiation) can be coated onto the silicon dioxide (e.g., by spin coating of a wafer) to any suitable thickness. Exemplary coating thicknesses include about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 μm. An exemplary photoresist material is MEGAPOSIT SPR 3612 photoresist (Dow Electronic Material) or a similar product. The substrate including the photo-resist can be exposed to an electromagnetic radiation source. A mask can be used to shield radiation from portions of the photo-resist in order to define the area of the loci. The photo-resist can be a negative resist or a positive resist (e.g., the area of the loci can be exposed to electromagnetic radiation or the areas other than the loci can be exposed to electromagnetic radiation as defined by the mask). The area overlying the location in which the resolved loci are to be created is exposed to electromagnetic radiation to define a pattern that corresponds to the location and distribution of the resolved loci in the silicon dioxide layer. The photoresist can be exposed to electromagnetic radiation through a mask defining a pattern that corresponds to the resolved loci. Next, the exposed portion of the photoresist can be removed, such as, e.g., with the aid of wet chemical etching and a washing operation. The removed portion of the mask can then be exposed to a chemical etchant to etch the substrate and transfer the pattern of resolved loci into the silicon dioxide layer. The etchant can include an acid, such as, for example, buffered HF in the case of silicon dioxide.


Various etching procedures can be used to etch the silicon in the area where the resolved loci are to be formed. The etch can be an isotropic etch (i.e., the etch rate alone one direction substantially equal or equal to the etch rate along an orthogonal direction), or an anisotropic etch (i.e., the etch rate along one direction is less than the etch rate alone an orthogonal direction), or variants thereof. The etching techniques can be both wet silicon etches such as KOH, TMAH, EDP and the like, and dry plasma etches (for example DRIE). Both may be used to etch micro structures wafer through interconnections.


The dry etch can be an anisotropic etch that etches substantially vertically (e.g., toward the substrate) but not laterally or substantially laterally (e.g., parallel to the substrate). In some cases, the dry etch comprises etching with a fluorine based etchant such as CF4, CHF3, C2F6, C3 F6, or any combination thereof. In some cases, the etching is performed for 400 seconds with an Applied Materials eMax-CT machine having settings of 100 mT, 1000 W, 20 G, and 50 CF4. The substrates described herein can be etched by deep reactive-ion etching (DRIE). DRIE is a highly anisotropic etch process used to create deep penetration, steep-sided holes and trenches in wafers/substrates, typically with high aspect ratios. The substrates can be etched using two main technologies for high-rate DRIE: cryogenic and Bosch. Methods of applying DRIE are described in the U.S. Pat. No. 5,501,893, which is herein incorporated by reference in its entirety.


The wet etch can be an isotropic etch that removes material in all directions. In some cases, the wet oxide etches are performed at room temperature with a hydrofluoric acid base that can be buffered (e.g., with ammonium fluoride) to slow down the etch rate. In some cases, a chemical treatment can be used to etch a thin surface material, e.g., silicon dioxide or silicon nitride. Exemplary chemical treatments include buffered oxide etch (BOE), buffered HF and/or NH4F. The etch time needed to completely remove an oxide layer is typically determined empirically. In one example, the etch is performed at 22° C. with 15:1 BOE (buffered oxide etch).


The silicon dioxide layer can be etched up to an underlying material layer. For example, the silicon dioxide layer can be etched until a titanium nitride layer. In some cases, the silicon dioxide is grown at a temperature of 1000 degrees Celsius and the underlying layer is typically silicon.


An additional surface layer can be added on top of an etched silicon layer subsequent to etching. In an exemplary arrangement, the additional surface layer is one that effectively binds to an adhesion promoter. Exemplary additional surface layers include, without limitation, silicon dioxide and silicon nitride. In the case of silicon dioxide, the additional layer can be added by conformal growth of a thin layer of this material on the silicon.


Clusters and Loci


Provided herein are devices having surfaces which comprises 50 to 10000 clusters 400, each cluster located in a predetermined position. The surface of a device disclosed herein can comprise more than 10000 clusters, each cluster located in a predetermined position. The term “locus” as used herein refers to a discrete region on the surface of a structure which provides support for synthesis of oligonucleotides encoding for a single sequence to extend from the surface. In an exemplary arrangement, each cluster comprises 121 loci 425, each loci being located in a predetermined position. The loci can comprise a molecule that binds the surface and also couples to nucleoside. Moreover, loci can comprise a mixture of (i) a molecule that binds the surface and couples to a nucleoside; and (ii) a molecule that binds the surface and does not couple to nucleoside. In some cases, the regions surrounding the loci comprise a molecule that binds the surface and does not couple to nucleoside, wherein the surface of regions surrounding the loci results a lower surface energy than the surface energy at the loci.


The loci can be located on a substantially planar surface. In some arrangements, a locus is located on a well, microwell, channel, post, or other raised or lowered feature of a surface disclosed herein. A region of a locus can span a plurality of wells, microwells, channels, posts, or other raised or lowered features of a surface disclosed herein.


Provided herein are structures comprising a surface that supports the synthesis of a plurality of oligonucleic acids having different predetermined sequences at addressable locations on a common support. The surface of a structure disclosed herein can 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 non-identical oligonucleic acids. In some case, at least a portion of the oligonucleic acids have an identical sequence or are configured to be synthesized with an identical sequence. Structures disclosed herein provides for a surface environment for the growth of oligonucleic acids having at least about 10, 20, 30, 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, 1000, 2000 bases or more in length.


Provided herein are surfaces in which oligonucleic acids are synthesized on distinct loci of a surface disclosed herein, wherein each locus supports the synthesis of a population of oligonucleic acids. In some cases, each locus supports the synthesis of a population of oligonucleic acids having a different sequence than a population of oligonucleic acids grown on another locus. Loci of a surface disclosed herein are each located within a cluster of a plurality of clusters. Provided herein are surfaces which comprise at least 10, 100, 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; 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, 200, 500 or more loci. In some cases, each cluster includes 50 to 500, 50 to 200, 50 to 150, or 100 to 150 loci. In some cases, each cluster includes 100 to 150 loci. In exemplary arrangements, each cluster includes 109, 121, 130 or 137 loci.


The number of distinct oligonucleic acids synthesized on a surface disclosed herein can be dependent on the number of distinct loci available on the surface. Provided herein are structures wherein the density of loci within a cluster of a structure disclosed herein 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 or more. In some cases, a the surface of a structure disclosed herein comprises from about 10 loci per mm2 to about 500 mm2, from about 25 loci per mm2 to about 400 mm2, or from about 50 loci per mm2 to about 200 mm2.


Provided herein are structures wherein the distance between the centers of two adjacent loci within a cluster is from about 10 μm to about 500 μm, from about 10 μm to about 200 μm, or from about 10 μm to about 100 μm. Provided herein are structures wherein the distance between two centers of adjacent loci is greater than about 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm or 100 μm. In some cases, the distance between the centers of two adjacent loci is less than about 200 μm, 150 μm, 100 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm or 10 μm.


Provided herein are structures wherein the number of distinct nucleic acids or genes assembled from a plurality of oligonucleic acids synthesized on the structure is dependent on the number of clusters available in the surface of the structure. In some case, the density of clusters within a region of the surface is at least or about 1 cluster per 100 mm2, 1 cluster per 10 mm2, 1 cluster per 5 mm2, 1 cluster per 4 mm2, 1 cluster per 3 mm2, 1 cluster per 2 mm2, 1 cluster per 1 mm2, 2 clusters per 1 mm2, 3 clusters per 1 mm2, 4 clusters per 1 mm2, 5 clusters per 1 mm2, 10 clusters per 1 mm2, 50 clusters per 1 mm2 or more. In some cases, a region of a surface disclosed herein comprises from about 1 cluster per 10 mm2 to about 10 clusters per 1 mm2. Provided herein are structures wherein the distance between the centers of two adjacent clusters is less than about 50 μm, 100 μm, 200 μm, 500 μm, 1000 μm, or 2000 μm or 5000 μm. Provided herein are structures wherein the distance between the centers of two adjacent clusters is 1.125 mm. In some cases, the distance between the centers of two adjacent clusters is between about 50 μm and about 100 μm, between about 50 μm and about 200 μm, between about 50 μm and about 300 μm, between about 50 μm and about 500 μm, or between about 100 μm to about 2000 μm. In some cases, the distance between the centers of two adjacent clusters is between about 0.05 mm to about 50 mm, between about 0.05 mm to about 10 mm, between about 0.05 mm and about 5 mm, between about 0.05 mm and about 4 mm, between about 0.05 mm and about 3 mm, between about 0.05 mm and about 2 mm, between about 0.1 mm and 10 mm, between about 0.2 mm and 10 mm, between about 0.3 mm and about 10 mm, between about 0.4 mm and about 10 mm, between about 0.5 mm and 10 mm, between about 0.5 mm and about 5 mm, or between about 0.5 mm and about 2 mm. In some cases, the distance between two clusters is about or at least about 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm, 3 mm, 3.2 mm, 3.4 mm, 3.6 mm, 3.8 mm, 4 mm, 4.2 mm, 4.4 mm, 4.6 mm, 4.8 mm, 5 mm, 5.2 mm, 5.4 mm, 5.6 mm, 5.8 mm, 6 mm, 6.2 mm, 6.4 mm, 6.6 mm, 6.8 mm, 7 mm, 7.2 mm, 7.4 mm, 7.6 mm, 7.8 mm, 8 mm, 8.2 mm, 8.4 mm, 8.6 mm, 8.8 mm, or 9 mm. The distance between two clusters may range between 0.3-9 mm, 0.4-8 mm, 0.5-7 mm, 0.6-6 mm, 0.7-5 mm, 0.7-4 mm, 0.8-3 mm, or 0.9-2 mm. Those of skill in the art appreciate that the distance may fall within any range bound by any of these values, for example 0.8 mm-2 mm.


Provided herein are structures having a surface wherein one or more loci on the surface comprise a channel or a well. In some cases, the channels or wells are accessible to reagent deposition via a deposition device such as an oligonucleic acid synthesizer. In some cases, reagents and/or fluids may collect in a larger well in fluid communication one or more channels or wells. In some case, a structure disclosed herein comprises a plurality of channels corresponding to a plurality of loci with a cluster, and the plurality of channels or wells are in fluid communication with one well of the cluster. In some cases, a library of oligonucleic acids is synthesized in a plurality of loci of a cluster, followed by the assembly of the oligonucleic acids into a large nucleic acid such as gene, wherein the assembly of the large nucleic acid optionally occurs within a well of the cluster, e.g., by using PCA.


Provided herein are structures wherein a cluster located on a surface disclosed herein has the same or different width, height, and/or volume as another cluster of the surface. A well located on a surface disclosed herein may have the same or different width, height, and/or volume as another well of the surface. Provided herein are structures wherein a channel located on a surface disclosed herein has the same or different width, height, and/or volume as another channel of the surface. Provided herein are structures wherein the diameter of a cluster is between about 0.05 mm to about 50 mm, between about 0.05 mm to about 10 mm, between about 0.05 mm and about 5 mm, between about 0.05 mm and about 4 mm, between about 0.05 mm and about 3 mm, between about 0.05 mm and about 2 mm, between about 0.05 mm and about 1 mm, between about 0.05 mm and about 0.5 mm, between about 0.05 mm and about 0.1 mm, between about 0.1 mm and 10 mm, between about 0.2 mm and 10 mm, between about 0.3 mm and about 10 mm, between about 0.4 mm and about 10 mm, between about 0.5 mm and 10 mm, between about 0.5 mm and about 5 mm, or between about 0.5 mm and about 2 mm. Provided herein are structures wherein the diameter of a cluster is less than or about 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 0.5 mm, 0.1 mm, 0.09 mm, 0.08 mm, 0.07 mm, 0.06 mm or 0.05 mm. In some cases, the diameter of a cluster is between about 1.0 and about 1.3 mm. In some cases, the diameter of a cluster is between about 0.5 to 2.0 mm. In some cases, the diameter of a cluster is about 1.150 mm. In some cases, the diameter of a cluster or well, or both is about 0.08 mm.


Provided herein are structures wherein the height of a well is from about 20 μm to about 1000 μm, from about 50 μm to about 1000 μm, from about 100 μm to about 1000 μm, from about 200 μm to about 1000 μm, from about 300 μm to about 1000 μm, from about 400 μm to about 1000 μm, or from about 500 μm to about 1000 μm. In some cases, the height of a well is less than about 1000 μm, less than about 900 μm, less than about 800 μm, less than about 700 μm, or less than about 600 μm.


Provided herein are structures wherein the structure disclosed herein comprises a plurality of channels corresponding to a plurality of loci within a cluster, wherein the height or depth of a channel is from about 5 μm to about 500 μm, from about 5 μm to about 400 μm, from about 5 μm to about 300 μm, from about 5 μm to about 200 μm, from about 5 μm to about 100 μm, from about 5 μm to about 50 μm, or from about 10 μm to about 50 μm. In some cases, the height of a channel is less than 100 μm, less than 80 μm, less than 60 μm, less than 40 μm or less than 20 μm.


Provided herein are structures wherein the diameter of a channel, well, or substantially planar locus is from about 0.5 μm to about 1000 μm, from about 0.5 μm to about 500 μm, from about 0.5 μm to about 200 μm, from about 0.5 μm to about 100 μm, from about 1 μm to about 100 μm, from about 5 μm to about 100 μm, or from about 10 μm to about 100 μm. In some cases, the diameter of a locus is about 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 1 μm, or 0.5 μm. In some cases, the diameter of a channel, well, or substantially planar locus is less than about 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 1 μm, or 0.5 μm. In some cases, the distance between the center of two adjacent loci is from about 1 μm to about 500 μm, from about 1 μm to about 200 μm, from about 1 μm to about 100 μm, from about 5 μm to about 200 μm, from about 5 μm to about 100 μm, from about 5 μm to about 50 μm, or from about 5 μm to about 30 μm, for example, about 20 μm.


Each of the resolved loci on the substrate can have any shape that is known in the art, or the shapes that can be made by methods known in the art. For example, each of the resolved loci can have an area that is in a circular shape, a rectangular shape, elliptical shape, or irregular shape. In some instances, the resolved loci can be in a shape that allows liquid to easily flow through without creating air bubbles. Resolved loci can have a circular shape, with a diameter that can be about, at least about, or less than about 0.5, 1 micrometers (μm), 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm or 750 μm. In some cases, the resolved loci have a diameter of 80 μm. The resolved loci may have a monodisperse size distribution, i.e. all of the microstructures may have approximately the same width, height, and/or length. A resolved loci of may have a limited number of shapes and/or sizes, for example the resolved loci may be represented in 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or more distinct shapes, each having a monodisperse size. The same shape can be repeated in multiple monodisperse size distributions, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or more monodisperse size distributions. A monodisperse distribution may be reflected in a unimodular distribution with a standard deviation of less than 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1%, 0.1%, 0.05%, 0.01%, 0.001% of the mode or smaller.


Textured Surfaces


Structures disclosed herein can be manufactured to increase the surface area such that, at particular regions for oligonucleic acid growth, the yield is increased. For example a textured surface is provided which comprises raised or recessed textured features, such as posts, wells, or other shapes. Texture features of the surfaces (e.g., posts or wells) are measured by the following parameters: S=surface area per unit cell; S0=surface area without texture; d=depth length; w=width length; and p=pitch length. In exemplary arrangements, the ratio of pitch length to width length is about 2. If the ratio of pitch length to width length is 2, then the following equation may be used to calculate the surface area of a chip with texture:









S
=



p
2



(

1
+


π





d


2





p



)


.





Equation





1







If the ratio of pitch length to width length is 2, then the following equation may be used to calculate the surface area of a chip with texture:









S
=



S
0



(

1
+


π





d


2





p



)


.





Equation





2







Provided herein are structures wherein the width length is at least twice the desired oligonucleic acid length to be synthesized on a surface disclosed herein. In some instances, the width length of a textured feature disclosed herein is greater than about 0.68 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 15 nm, 20 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm. In some instances, the width length of a textured feature disclosed herein is about 0.1 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 15 nm, 20 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm. Those of skill in the art appreciate that the width length may fall within any range bound by any of these values, for example 10 nm to 400 nm, 100 nm to 800 nm, or 200 nm to 1 μm. In some instances, the width length is 7 nm to 500 nm. In some case, the width length is 6.8 nm to 500 nm.


Provided herein are structures wherein the pitch length of a textured feature disclosed herein is greater than about 0.1 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 15 nm, 20 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm. In some instances, the pitch length of a textured feature disclosed herein is about 0.1 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 15 nm, 20 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm. Those of skill in the art appreciate that the pitch length may fall within any range bound by any of these values, for example 10 nm to 400 nm, 100 nm to 800 nm, or 200 nm to 1 μm. In some cases, the pitch length is 14 nm to 1 μm. In some cases, the pitch length is 13.6 nm to 1 μm. In some instances, the pitch length is about twice the width length.


Provided herein are structures wherein the depth length of a textured feature disclosed herein is greater than about 0.1 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 15 nm, 20 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm. In some cases, the depth length of a textured feature disclosed herein is about 0.1 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 15 nm, 20 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm. Those of skill in the art appreciate that the depth length may fall within any range bound by any of these values, for example 10 nm to 400 nm, 100 nm to 800 nm, or 200 nm to 1 μm. In some instances, the depth length is 8 nm to 1 μm. In some cases, the depth length is 13.6 nm to 2 μm. In some instances, the depth length is about 60% to about 125% of the pitch length.


Provided herein are structures wherein the width length and the pitch length are of a predetermined ratio. The ratio of pitch length to width length can be greater than about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10. The ratio of pitch length to width length can be about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10. In some instances, the ratio of pitch length to width length is about 1. In some instances, the ratio of pitch length to width length is about 2.


Provided herein are structures wherein the depth length and the pitch length are of a predetermined ratio. In some instances, the ratio of pitch length to width length is greater than about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10. In some instances, the ratio of pitch length to width length is about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10. In some instances, the ratio of depth length to pitch length is about 0.5. In some instances, the ratio of depth length to pitch length is about 0.6 to about 1.2. In some instances, the ratio of pitch length to width length is about 1. In some instances, the ratio of depth length to pitch length is about 0.6 to 1.25, or about 0.6 to about 2.5.


Provided herein are structures wherein a surface disclosed herein comprises a plurality of clusters, each cluster comprising a plurality of loci 400, wherein in each loci has a distance apart from each other 415 of about 25.0 μm, a diameter 410 of about 50 μm, a distance from the center of one loci to the center of another loci 405, 420 of 75 μm in X and Y axis directions, FIG. 4, and the surface is optionally textured 430. As exemplary arrangement is illustrated in FIG. 4, where one locus 425 from a cluster of loci 1000 located on a textured surface 403. In some cases, a structure disclosed herein comprises loci having depth to width ratios that greater than about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1. The loci can have a depth to width ratio that is greater than about 5:1. In some cases, the loci have a depth to width ratio that is about 10:1 or greater than 10:1.


Provided herein are structures wherein the surface of a structure described herein is substantially planar or comprises recesses/lowered or protruding/raised features. In some cases, the protrusions comprise wells and/or channels. The raised or lowered features may have sharp or rounded edges and may have cross-sections (widths) of any desired geometric shape, such as rectangular, circular, etc. The lowered features may form channels along the entire substrate surface or a portion of it.


The raised or lowered features may have an aspect ratio of at least or about at least 1:20, 2:20, 3:20, 4:20, 5:20, 6:20, 10:20, 15:20, 20:20, 20:10, 20:5, 20:1, or more. The raised or lowered features may have an aspect ratio of at most or about at most 20:1, 20:5, 20:10, 20:20, 20:15, 20:10, 20:10, 6:20, 5:20, 4:20, 3:20, 2:20, 1:20, or less. The raised or lowered features may have an aspect ratio that falls between 1:20-20:1, 2:20-20:5, 3:20-20:10, 4-20:20:15, 5:20-20:20, 6:20-20:20. Those of skill in the art appreciate that the raised or lowered features may have an aspect ratio that may fall within any range bound by any of these values, for example 3:20-4:20. In some cases, the raised or lowered features have an aspect ratio that falls within any range defined by any of the values serving as endpoints of the range.


Provided herein are structures wherein raised or lowered features have cross-sections of at least or about at least 10 nanometers (nm), 11 nm, 12 nm, 20 nm, 30 nm, 100 nm, 500 nm, 1000 nm, 10000 nm, 100000 nm, 1000000 nm, or more. The raised or lowered features may have cross-sections of at least or most or about at most 1000000 nm, 100000 nm, 10000 nm, 1000 nm, 500 nm, 100 nm, 30 nm, 20 nm, 12 nm, 11 nm, 10 nm, or less. The raised or lowered features may have cross-sections that fall between 10 nm-1000000 nm, 11 nm-100000 nm, 12 nm-10000 nm, 20 nm-1000 nm, 30 nm-500 nm. Those of skill in the art appreciate that the raised or lowered features may have cross-sections that may fall within any range bound by any of these values, for example 10 nm-100 nm. The raised or lowered features may have cross-sections that fall within any range defined by any of the values serving as endpoints of the range.


Provided herein are structures wherein, raised or lowered features have heights of at least or about at least 10 nanometers (nm), 11 nm, 12 nm, 20 nm, 30 nm, 100 nm, 500 nm, 1000 nm, 10000 nm, 100000 nm, 1000000 nm, or more. The raised or lowered features may have heights of at most or about at most 1000000 nanometers (nm), 100000 nm, 10000 nm, 1000 nm, 500 nm, 100 nm, 30 nm, 20 nm, 12 nm, 11 nm, 10 nm, or less. The raised or lowered features may have heights that fall between 10 nm-1000000 nm, 11 nm-100000 nm, 12 nm-10000 nm, 20 nm-1000 nm, 30 nm-500 nm. Those of skill in the art appreciate that the raised or lowered features may have heights that may fall within any range bound by any of these values, for example 100 nm-1000 nm. The raised or lowered features may have heights that fall within any range defined by any of the values serving as endpoints of the range. The individual raised or lowered features may be separated from a neighboring raised or lowered feature by a distance of at least or at least about 5 nanometers (nm), 10 nm, 11 nm, 12 nm, 20 nm, 30 nm, 100 nm, 500 nm, 1000 nm, 10000 nm, 100000 nm, 1000000 nm, or more. The individual raised or lowered features may be separated from a neighboring raised or lowered feature by a distance of at most or about at most 1000000 nanometers (nm), 100000 nm, 10000 nm, 1000 nm, 500 nm, 100 nm, 30 nm, 20 nm, 12 nm, 11 nm, 10 nm, 5 nm, or less. The raised or lowered features may have heights that fall between 5-1000000 nm, 10-100000 nm, 11-10000 nm, 12-1000 nm, 20-500 nm, 30-100 nm. Those of skill in the art appreciate that the individual raised or lowered features may be separated from a neighboring raised or lowered feature by a distance that may fall within any range bound by any of these values, for example 100-1000 nm. The individual raised or lowered features may be separated from a neighboring raised or lowered feature by a distance that falls within any range defined by any of the values serving as endpoints of the range. In some cases, the distance between two raised or lowered features is at least or about at least 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 5.0, 10.0 times, or more, the cross-section (width) or average cross-section of the raised or lowered features. The distance between the two raised or lowered features is at most or about at most 10.0, 5.0, 3.0, 2.0, 1.0, 0.5, 0.2, 0.1 times, or less, the cross-section (width) or average cross-section of the raised or lowered features. The distance between the two raised or lowered features may be between 0.1-10, 0.2-5.0, 1.0-3.0 times, the cross-section (width) or average cross-section of the raised or lowered features. Those of skill in the art appreciate that the distance between the two raised or lowered features may be between any times the cross-section (width) or average cross-section of the raised or lower features within any range bound by any of these values, for example 5-10 times. The distance between the two raised or lowered features may be within any range defined by any of the values serving as endpoints of the range.


Provided herein are structures wherein groups of raised or lowered features are separated from each other. Perimeters of groups of raised or lowered features may be marked by a different type of structural feature or by differential functionalization. A group of raised or lowered features may be dedicated to the synthesis of a single oligonucleotide. A group of raised or lowered features may span an area that is at least or about at least 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 20 μm, 50 μm, 70 μm, 90 μm, 100 μm, 150 μm, 200 μm, or wider in cross section. A group of raised or lowered features may span an area that is at most or about at most 200 μm, 150 μm, 100 μm, 90 μm, 70 μm, 50 μm, 20 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, or narrower in cross section. A group of raised or lowered features may span an area that is between 10-200 μm, 11-150 μm, 12-100 μm, 13-90 μm, 14-70 μm, 15-50 μm, 13-20 μm, wide in cross-section. Those of skill in art appreciate that a group of raised or lowered features may span an area that falls within any range bound by any of these values, for example 12-200 μm. A group of raised or lowered features may span an area that fall within any range defined by any of the values serving as endpoints of the range.


Provided herein are structures wherein a structure comprising a surface is about the size of a standard 96 well plate, for example between about 100 and 200 mm by between about 50 and 150 mm. In some cases, a structure comprising a surface disclosed 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 cases, the diameter of a structure comprising a surface disclosed herein 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 structure size include about 300 mm, 200 mm, 150 mm, 130 mm, 100 mm, 76 mm, 51 mm and 25 mm. In some cases, a structure comprising a surface disclosed herein has a planar surface area of at least about 100 mm2; 200 mm2; 500 mm2; 1,000 mm2; 2,000 mm2; 5,000 mm2; 10,000 mm2; 12,000 mm2; 15,000 mm2; 20,000 mm2; 30,000 mm2; 40,000 mm2; 50,000 mm2 or more. In some cases, the thickness of a structure comprising a surface disclosed herein is between about 50 um and about 2000 um, between about 50 um and about 1000 um, between about 100 um and about 1000 um, between about 200 um and about 1000 um, or between about 250 um and about 1000 um. Non-limiting examples of structure thickness include 275 um, 375 um, 525 um, 625 um, 675 um, 725 um, 775 um and 925 um. In some cases, the thickness of a structure varies with diameter and depends on the composition of the structure. For example, a structure comprising materials other than silicon has 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. Provided herein is a wafer comprises multiple structures (e.g., 1 to 30 or more) disclosed herein.


Provided herein are structures wherein a surface is configured to allow for controlled flow and mass transfer paths for oligonucleic acid synthesis on a surface. The configuration allows for the controlled and even distribution of mass transfer paths, chemical exposure times, and/or wash efficacy during oligonucleic acid synthesis. In some case, the configuration allows for increased sweep efficiency, for example by providing sufficient volume for a growing an oligonucleic acid such that the excluded volume by the growing oligonucleic acid does not take up more than 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or less of the initially available volume that is available or suitable for growing the oligonucleic acid.


Provided herein are structures wherein a surface of the structure comprises features with a density of about or greater than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400 or 500 features per mm2. Standard silicon wafer processes can be employed to create a structure surface that will have a high surface area as described above and a managed flow, allowing rapid exchange of chemical exposure. In some cases, the oligonucleotide synthesis surface comprises series of structural features with sufficient separation to allow oligomer chains greater than at least or about at least 20 mer, 25 mer, 30 mer, 50 mer, 100 mer, 200 mer, 250 mer, 300 mer, 400 mer, 500 mer, or more to be synthesized without substantial influence on the overall channel or pore dimension, for example due to excluded volume effects, as the oligonucleotide grows. In some cases, the oligonucleotide synthesis surface comprises a series of structures with sufficient separation to allow oligomer chains greater than at most or about at most 500 mer, 200 mer, 100 mer, 50 mer, 30 mer, 25 mer, 20 mer, or less to be synthesized without substantial influence on the overall channel or pore dimension, for example due to excluded volume effects, as the oligonucleotide grows.


Provided herein are structures wherein the distance between the features is greater than at least or about at least 5 nm, 10 nm, 20 nm, 100 nm, 1000 nm, 10000 nm, 100000 nm, 1000000 nm, or more. In some case, the distance between the features is greater than at most or about at most 1000000 nm, 100000 nm, 10000 nm, 1000 nm, 100 nm, 20 nm, 10 nm, 5 nm, or less. In some case, the distance between the features falls between 5-1000000 nm, 10-100000 nm, 20-10000 nm, or 100-1000 nm. In some case, the distance between the features is greater than 200 nm. The features may be created by any suitable MEMS processes described elsewhere herein or otherwise known in the art, such as a process employing a timed reactive ion etch process. Such semiconductor manufacturing processes can typically create feature sizes smaller than 200 nm, 100 nm, 50 nm, 40 nm, 30 nm, 25 nm, 20 nm, 10 nm, 5 nm, or less. Those of skill in the art appreciate that the feature size smaller than 200 nm can be between any of these values, for example, 20-100 nm. The feature size can fall within any range defined by any of these values serving as endpoints of the range. In some cases, an array of 40 μm wide posts are etched with 30 μm depth, which about doubles the surface area available for synthesis.


Provided herein are devices for oligonucleic acid synthesis, the device comprising a structure having a surface disclosed herein; a plurality of recesses or posts on the surface, wherein each recess or post comprises (i) a width length that is 6.8 nm to 500 nm, (ii) a pitch length that is about twice the width length, and (iii) a depth length that is about 60% to about 125% of the pitch length; a plurality of loci on the surface, wherein each locus has a diameter of 0.5 to 100 um, wherein each locus comprises at least two of the plurality of recesses or posts; a plurality of clusters on the surface, wherein each of the clusters comprise 50 to 500 loci and has a cross-section of 0.5 to 2 mm, wherein the plurality of loci comprise a less than saturating amount of a molecule that binds the surface and couples to nucleoside phosphoramidite; and a plurality of regions surrounding each loci comprise a molecule that binds the surface and does not couple to nucleoside phosphoramidite, wherein the plurality of loci have a higher surface energy than the plurality of regions surrounding each loci. In some cases, the molecule that binds the surface and couples to nucleoside phosphoramidite is a silane disclosed herein. In some cases, the molecule that binds the surface and does not couple to nucleoside phosphoramidite is a fluorosilane disclosed herein. In some cases, the plurality of loci are coated with a molecule that binds the surface, does not couple to nucleoside phosphoramidite, and has a higher surface energy than the molecule on plurality of regions surrounding each loci.


Preparation of Textured Surfaces


As discussed above, FIGS. 5A-5F and FIG. 6 describe a method for generating a textured surface. In some cases, photolithography is applied structure to create a mask of photoresist. In a subsequent step, a deep reactive-ion etching (DRIE) step is used to etch vertical side-walls (e.g., until an insulator layer in a structure comprising an insulator layer) at locations devoid of the photoresist. In a following step, the photoresist is stripped. Photolithography, DRIE and photoresist strip steps may be repeated on the structure handle side. In cases wherein the structure comprises an insulator layer such as silicon dioxide, buried oxide (BOX) is removed using an etching process. Thermal oxidation can then be applied to remove contaminating polymers that may have been deposited on the side walls during the method. In a subsequent step, the thermal oxidation is stripped using a wet etching process.


To resist coat only a small region of the surface (e.g., lowered features such as a well and/or channel), a droplet of resist may be deposited into the lowered feature where it optionally spreads. In some cases, a portion of the resist is removed, for example, by etching (e.g., oxygen plasma etch) to leave a smooth surface covering only a select area.


The surface can be wet cleaned, for example, using a piranha solution. Alternatively, the surface can be plasma cleaned, for example, by dry oxygen plasma exposure. The photoresist may be coated by a process governed by wicking into the device layer channels. The photoresist may be patterned using photolithography to expose areas that are desired to be passive (i.e., areas where oligonucleic acid synthesis is not designed to take place). Patterning by photolithography may occur by exposing the resist to light through a binary mask that has a pattern of interest. After exposure, the resist in the exposed regions may be removed in developer solution.


Alignment Marks


During the deposition process, references points on a surface are used by a machine for calibration purposes. Surfaces described herein may comprise fiducial marks, global alignment marks, lithography alignment marks or a combination thereof. Fiducial marks are generally placed on the surface of a structure, such as an array of clusters 800 to facilitate alignment of such devices with other components of a system, FIG. 8 illustrates an exemplary arrangement. The surface of a structure disclosed herein may have one or more fiducial marks, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10. In some cases, fiducial marks may are used for global alignment of the microfluidic device.


Fiducial marks may have various shapes and sizes. In some cases, a fiducial mark has the shape of a square, circle, triangle, cross, “X”, addition or plus sign, subtraction or minus sign, or any combination thereof. In some one example, a fiducial mark is in the shape of an addition or a plus sign 805. In some cases, a fiducial mark comprises a plurality of symbols. Exemplary fiducial mark may comprises one or more plus signs 810, e.g., 2, 3, 4, or more plus signs. In one example, a fiducial mark comprises 4 plus signs.


Fiducial marks may be located on the surface of structures disclosed herein. A fiducial mark may be about 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 100 μm, 1000 μm, 2000 μm, 5000 μm, 7000 μm, 8000 μm, 9000 μm, or 10,000 μm, from the center of the surface. In some cases, the fiducial mark is located from about 0.1 mm to about 10 mm from the edge of the surface portion, e.g., about 0.5 mm from the edge. In some case, the fiducial is located from about 1 mm to about 10 mm form a cluster, e.g., 1.69 mm. In some instances, a distance from the center of a fiducial mark and a nearest corner of a surface in one dimension is from about 0.5 mm to about 10 mm, e.g., about 1 mm. In some instances, a length of a fiducial mark in one dimension is from about 0.5 mm to about 5 mm, e.g., about 1 mm. In some instances, the width of a fiducial mark is from about 0.01 mm to about 2 mm, e.g., 0.05 mm.


Global alignment marks may have various shapes and sizes. Global alignment marks are placed on the surface of a structure described herein to facilitate alignment of such devices with other components of a system, FIG. 9 illustrates an exemplary arrangement. Exemplary global alignment marks have the shape of a square, circle, triangle, cross, “X”, addition or plus sign, subtraction or minus sign, or any combination thereof. Exemplary global alignment marks include the shape of a circle 925 or a plus mark 945. In some cases, a global alignment mark is located near an edge of the substrate portion, as shown by the location of marks 905, 910, 915, 920, 930, 935, and 940. A global alignment mark can comprise a plurality of symbols. In some case, a global alignment mark comprises one or more circles, e.g., 2, 3, 4, or more plus signs.


A global alignment mark may be located on the surface of a structure disclosed herein. In some cases, the global alignment mark is about 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 100 μm, 1000 μm, 2000 μm, 5000 μm, 7000 μm, 8000 μm, 9000 μm, or 10,000 μm, from the center of the surface. In some case, the global alignment mark is about 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 100 μm, 1000 μm, 2000 μm, 5000 μm, 7000 μm, 8000 μm, 9000 μm, or 10,000 μm, from the edge of the surface. In some cases, the global alignment mark is about 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 750 μm, or 1000 μm in size. In an example arrangement, the global alignment mark is about 125 μm in diameter and is located about 1000 μm from the edge of the surface of the structure. Surface disclosed herein may comprise one or more global alignment marks, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, or more marks. The distance between the global alignment marks may be about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 100 μm, 500 μm, 1000 μm, 2000 μm, 5000 μm, 7000 μm, 8000 μm, 9000 μm, or 10,000 μm.


Oligonucleic Acid Synthesis


De Novo Synthesis Workflow


Structures having modified surfaces described herein may be used for de novo synthesis processes. An exemplary workflow for one such process is divided generally into phases: (1) de novo synthesis of a single stranded oligonucleic acid library, (2) joining oligonucleic acids to form larger fragments, (3) error correction, (4) quality control, and (5) shipment, FIG. 10. Prior to de novo synthesis, an intended nucleic acid sequence or group of nucleic acid sequences is preselected. For example, a group of genes is preselected for generation.


Once preselected nucleic acids for generation are selected, a predetermined library of oligonucleic acids is designed for de novo synthesis. Various suitable methods are known for generating high density oligonucleic acid arrays. In the workflow example, a surface layer 1001 is provided. In the example, chemistry of the surface is altered in order to improve the oligonucleic acid synthesis process. Areas of low surface energy are generated to repel liquid while areas of high surface energy are generated to attract liquids. The surface itself may be in the form of a planar surface or contain variations in shape, such as protrusions or microwells which increase surface area. In the workflow example, high surface energy molecules selected serve a dual function of supporting DNA chemistry, as disclosed in International Patent Application Publication WO/2015/021080, which is herein incorporated by reference in its entirety.


In situ preparation of oligonucleic acid arrays is generated on a solid support and utilizes single nucleotide extension process to extend multiple oligomers in parallel. A device, such as an oligonucleic acid synthesizer, is designed to release reagents in a step wise fashion such that multiple oligonucleic acids extend, in parallel, one residue at a time to generate oligomers with a predetermined nucleic acid sequence 1002. In some cases, oligonucleic acids are cleaved from the surface at this stage. Cleavage may include gas cleavage, e.g., with ammonia or methylamine.


The generated oligonucleic acid libraries are placed in a reaction chamber. In this exemplary workflow, the reaction chamber (also referred to as “nanoreactor”) is a silicon coated well, containing PCR reagents and lowered onto the oligonucleic acid library 1003. Prior to or after the sealing 1004 of the oligonucleic acids, a reagent is added to release the oligonucleic acids from the surface. In the exemplary workflow, the oligonucleic acids are released subsequent to sealing of the nanoreactor 1005. Once released, fragments of single stranded oligonucleic acids hybridize in order to span an entire long range sequence of DNA. Partial hybridization 1005 is possible because each synthesized oligonucleic acid is designed to have a small portion overlapping with at least one other oligonucleic acid in the pool.


After hybridization, a PCA reaction is commenced. During the polymerase cycles, the oligonucleic acids anneal to complementary fragments and gaps are filled in by a polymerase. Each cycle increases the length of various fragments randomly depending on which oligonucleic acids find each other. Complementarity amongst the fragments allows for forming a complete large span of double stranded DNA 1006.


After PCA is complete, the nanoreactor is separated from the surface 1007 and positioned for interaction with a polymerase 1008. After sealing, the nanoreactor is subject to PCR 1009 and the larger nucleic acids are formed. After PCR 1010, the nanochamber is opened 1011, error correction reagents are added 1012, the chamber is sealed 1013 and an error correction reaction occurs to remove mismatched base pairs and/or strands with poor complementarity from the double stranded PCR amplification products 1014. The nanoreactor is opened and separated 1015. Error corrected product is next subject to additional processing steps, such as PCR and molecular bar coding, and then packaged 1022 for shipment 1023.


In some cases, quality control measures are taken. After error correction, quality control steps include for example interaction with a wafer having sequencing primers for amplification of the error corrected product 1016, sealing the wafer to a chamber containing error corrected amplification product 1017, and performing an additional round of amplification 1018. The nanoreactor is opened 1019 and the products are pooled 1020 and sequenced 1021. After an acceptable quality control determination is made, the packaged product 1022 is approved for shipment 1023.


The structures described herein comprise actively functionalized surfaces configured to support the attachment and synthesis of oligonucleic acids. Synthesized oligonucleic acids include oligonucleic acids comprising modified and/or non-canonical bases and/or modified backbones. In various methods, a library of oligonucleic acids having pre-selected sequences is synthesized on a structure disclosed herein. In some cases, one or more of the oligonucleic acids has a different sequence and/or length than another oligonucleic acid in the library. The stoichiometry of each oligonucleic acid synthesized on a surface is controlled and tunable by varying one or more features of the surface (e.g., functionalized surface) and/or oligonucleic acid sequence to be synthesized; one or more methods for surface functionalization and/or oligonucleic acid synthesis; or a combination thereof. In many instances, controlling the density of a growing oligonucleic acid on a resolved locus of a structure disclosed herein allows for oligonucleic acids to be synthesized with a low error rate.


Oligonucleic acids synthesized using the methods and/or devices described herein include at least about 50, 60, 70, 75, 80, 90, 100, 120, 150, 200, 300, 400, 500, 600, 700, 800 or more bases. A library of oligonucleic acids may be synthesized, wherein a population of distinct oligonucleic acids are assembled to generate a larger nucleic acid comprising at least about 500; 1,000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000; 10,000; 11,000; 12,000; 13,000; 14,000; 15,000; 16,000; 17,000; 18,000; 19,000; 20,000; 25,000; 30,000; 40,000; or 50,000 bases. Oligonucleic acid synthesis methods described herein are useful for the generation of an oligonucleic acid library comprising at least 500; 1,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000; 1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000; 2,200,000; 2,400,000; 2,600,000; 2,800,000; 3,000,000; 3,500,000; 4,00,000; or 5,000,000 distinct oligonucleic acids. In some case, at least about 1 pmol, 10 pmol, 20 pmol, 30 pmol, 40 pmol, 50 pmol, 60 pmol, 70 pmol, 80 pmol, 90 pmol, 100 pmol, 150 pmol, 200 pmol, 300 pmol, 400 pmol, 500 pmol, 600 pmol, 700 pmol, 800 pmol, 900 pmol, 1 nmol, 5 nmol, 10 nmol, 100 nmol or more of an oligonucleic acid is synthesized within a locus.


Oligonucleic acids are synthesized on a surface described herein using a system comprising an oligonucleic acid synthesizer that deposits reagents necessary for synthesis, FIG. 11. Reagents for oligonucleic acid synthesis include, for example, reagents for oligonucleic acid extension and wash buffers. As non-limiting examples, the oligonucleic acid synthesizer deposits coupling reagents, capping reagents, oxidizers, de-blocking agents, acetonitrile and gases such as nitrogen gas. In addition, the oligonucleic acid synthesizer optionally deposits reagents for the preparation and/or maintenance of structure integrity. The oligonucleic acid synthesizer comprises material deposition devices that can move in the X-Y direction to align with the location of the surface of the structure. The oligonucleic acid synthesizer can also move in the Z direction to seal with the surface of the structure, forming a resolved reactor.


Methods are provided herein where at least or about at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 10000, 50000, 100000 or more nucleic acids can be synthesized in parallel. Total molar mass of nucleic acids synthesized within the device or the molar mass of each of the nucleic acids may be at least or at least about 10, 20, 30, 40, 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 25000, 50000, 75000, 100000 picomoles, or more.


Oligonucleic acid synthesis methods disclosed herein include those which are enzyme independent. An example of a synthesis method that is useful with the devices provided herein is one that incorporates phosphoramidite chemistry, FIG. 12. Typically, after the deposition of a monomer, e.g., a mononucleotide, a dinucleotide, or a longer oligonucleotide with suitable modifications for phosphoramidite chemistry one or more of the following steps may be performed at least once to achieve the step-wise synthesis of high-quality polymers in situ: 1) Coupling, 2) Capping, 3) Oxidation, 4) Sulfurization, and 5) Deblocking (detritylation). Washing steps typically intervene steps 1 to 5.


Provided herein are methods wherein an oligonucleic acid error rate is dependent on the efficiency of one or more chemical steps of oligonucleic acid synthesis. In some cases, oligonucleic acid synthesis comprises a phosphoramidite method, wherein a base of a growing oligonucleic acid chain is coupled to phosphoramidite. Coupling efficiency of the base is related to the error rate. For example, higher coupling efficiency correlates to lower error rates. In some cases, the devices and/or synthesis methods described herein allow for a coupling efficiency greater than 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99%. In some cases, an oligonucleic acid synthesis method comprises a double coupling process, wherein a base of a growing oligonucleic acid chain is coupled with a phosphoramidite, the oligonucleic acid is washed and dried, and then treated a second time with a phosphoramidite. Efficiency of deblocking in a phosphoramidite oligonucleic acid synthesis method also contributes to error rate. In some cases, the devices and/or synthesis methods described herein allow for removal of 5′-hydroxyl protecting groups at efficiencies greater than 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99%. Error rate may be reduced by minimization of depurination side reactions.


Methods for the synthesis of oligonucleic acids typically involve an iterating sequence of the following steps: application of a protected monomer to an actively functionalized surface (e.g., locus) to link with either the activated surface, a linker or with a previously deprotected monomer; deprotection of the applied monomer so that it can react with a subsequently applied protected monomer; and application of another protected monomer for linking. One or more intermediate steps include oxidation or sulfurization. In some cases, one or more wash steps precede or follow one or all of the steps.


In an exemplary method, at least 20,000 or more non-identical oligonucleic acids each at least 10, 50, 100 or more bases in length are synthesized, wherein each of the at least 20,000 non-identical oligonucleic acids extends from a different locus of the patterned surface. Methods disclosed herein provides for at least 20,000 non-identical oligonucleic acids collectively encoding for at least 200 preselected nucleic acids, and have an aggregate error rate of less than 1 in 1500 bases compared to predetermined sequences without correcting errors. In some cases, the aggregate error rate is less than 1 in 2000, less than 1 in 3000 bases or less compared to the predetermined sequences. Surfaces provided herein provide for the low error rates.


Oligonucleotide Libraries with Low Error Rates


The term “error rate” may also be referred to herein as a comparison of the collective sequence encoded by oligonucleic acids generated compared to the sequence of one or more predetermined longer nucleic acid, e.g., a gene. An aggregate “error rate” refers to the collective error rate of synthesized nucleic acids compared to the predetermined sequences for which the nucleic acids are intended to encode. Error rates include mismatch error rate, deletion error rate, insertion error rate, insertion/deletion error rate, any combination thereof. Methods and devices herein provide for low error rates are for synthesized oligonucleic acid libraries having at least 20,000; 40,000; 60,000; 80,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 1,000,000; or 2,000,000 or more oligonucleic acids. Loci may be configured to comprise a population of oligonucleic acids, wherein the population may be configured to comprise oligonucleic acids having the same or different sequences.


Devices and methods described herein provide for a low overall error rate for the individual types of errors are achieved. Individual types of error rates include deletions, insertions, or substitutions for an oligonucleic acid library synthesized. In some cases, oligonucleic acids synthesized have an average error rate of about 1:500, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000 or less. These error rates may be for at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or more of the oligonucleic acids synthesized.


Methods described herein provide synthesis of oligonucleic acids having an average deletion error rate of about 1:500, 1:1000, 1:1700, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000 or less. Methods described herein provide synthesis of oligonucleic acids having an deletion error rate of about 1:500, 1:1000, 1:1700, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000 or less. Methods described herein provide synthesis of oligonucleic acids having an average insertion error rate of about 1:500, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000 or less. Methods described herein provide synthesis of oligonucleic acids having an insertion error rate of about 1:500, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000 or less. Methods described herein provide synthesis of oligonucleic acids having an average substitution error rate of about 1:500, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000 or less. Methods described herein provide synthesis of oligonucleic acids having a substitution error rate of about 1:500, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000 or less. The overall error rate or error rates for individual types of errors such as deletions, insertions, or substitutions for each oligonucleotide synthesized, may be for at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or more of the oligonucleotides synthesized.


Oligonucleic Acid Release and Assembly


Oligonucleic acids synthesized using the methods and devices described herein, are optionally released from the surface from which they were synthesized. In some cases, oligonucleic acids are cleaved from the surface at this stage. Cleavage may include gaseous cleavage, e.g., with gaseous ammonia or gaseous methylamine. Loci in a single cluster collectively correspond to sequence encoding for a single gene, and, when cleaved, may remain on the surface of the loci within a cluster. The application of ammonia gas is used to simultaneously deprotect phosphates groups protected during the synthesis steps, i.e. removal of electron-withdrawing cyano group. Once released from the surface, oligonucleic acids may be assembled into larger nucleic acids. Synthesized oligonucleic acids are useful, for example, as components for gene assembly/synthesis, site-directed mutagenesis, nucleic acid amplification, microarrays, and sequencing libraries.


Provided herein are methods where oligonucleic acids of predetermined sequence are designed to collectively span a large region of a target sequence, such as a gene. In some cases, larger oligonucleic acids are generated through ligation reactions to join the synthesized oligonucleic acids. One example of a ligation reaction is polymerase chain assembly (PCA). In some cases, at least of a portion of the oligonucleic acids are designed to include an appended region that is a substrate for universal primer binding. For PCA reactions, the presynthesized oligonucleic acids include overlaps with each other (e.g., 4, 20, 40 or more bases with overlapping sequence). During the polymerase cycles, the oligonucleic acids anneal to complementary fragments and then are filled in by polymerase. Each cycle thus increases the length of various fragments randomly depending on which oligonucleic acids find each other. Complementarity amongst the fragments allows for forming a complete large span of double stranded DNA. In some cases, after the PCA reaction is complete, an error correction step is conducted using mismatch repair detecting enzymes to remove mismatches in the sequence. Once larger fragments of a target sequence are generated, they can be amplified. For example, in some cases, a target sequence comprising 5′ and 3′ terminal adaptor sequences is amplified in a polymerase chain reaction (PCR) which includes modified primers, e.g., uracil containing primers the hybridize to the adaptor sequences.


Provided herein are methods wherein following oligonucleic acid synthesis, oligonucleic acids within one cluster are released from their respective surfaces and pooled into the common area, such as a ell. In some cases, the pooled oligonucleic acids are assembled into a larger nucleic acid, such as a gene, within the well. In some cases, at least about 1, 10, 50, 100, 200, 240, 500, 1000, 10000, 20000, 50000, 100000, 1000000 or more nucleic acids are assembled from oligonucleic acids synthesized on a surface disclosed herein. A pass-printing scheme may be used to deliver reagents to loci in a cluster, as wells as to transfer synthesis reaction products to another location. At least 2, 3, 4, 5, 6, 7, 8, 9, or 10 passes may be used to deliver reagents. For example, four passes of the may be used to deliver reagents to a second structure for assembly or analysis, FIG. 13. In some cases, assembled nucleic acids generated by methods described herein have a low error rate compared to a predetermined sequence without correcting errors. In some cases, assembled nucleic acids generated by methods described herein have an error rate of less than 1:1000, 1:1500, 1:2000, 1:2500, 1:3000 bases compared to a predetermined sequence without correcting errors.


Computer Systems


Methods are provided herein for attachment of pre-synthesized oligonucleotide and/or polynucleotide sequences to a support and in situ synthesis of the same using light-directed methods, flow channel and spotting methods, inkjet methods, pin-based methods and/or bead-based methods are used. In some cases, pre-synthesized oligonucleotides are attached to a support or are synthesized using a spotting methodology wherein monomers solutions are deposited drop wise by a dispenser that moves from region to region. In one example, oligonucleotides are spotted on a support using a mechanical wave actuated dispenser.


The systems described herein can further include a member for providing a droplet to a first spot (or feature) having a plurality of support-bound oligonucleotides. The droplet can include one or more compositions comprising nucleotides or oligonucleotides (also referred herein as nucleotide addition constructs) having a specific or predetermined nucleotide to be added and/or reagents that allow one or more of hybridizing, denaturing, chain extension reaction, ligation, and digestion. In some cases, different compositions or different nucleotide addition constructs may be deposited at different addresses on the support during any one iteration so as to generate an array of predetermined oligonucleotide sequences (the different features of the support having different predetermined oligonucleotide sequences). One particularly useful way of depositing the compositions is by depositing one or more droplet, each droplet containing the desired reagent (e.g. nucleotide addition construct) from a pulse jet device spaced apart from the support surface, onto the support surface or features built into the support surface.


A substrate with resolved features is “addressable” when it has multiple regions of different moieties (e.g., different polynucleotide sequences) such that a region (i.e., a “feature” or “spot” of the substrate) at a particular predetermined location (i.e., an “address”) on the substrate will detect a particular target or class of targets (although a feature may incidentally detect non-targets of that location). Substrate features are typically, but need not be, separated by intervening spaces. In some cases, features may be built into a substrate and may create one-, two-, or three-dimensional microfluidic geometries. A “substrate layout” refers to one or more characteristics of the features, such as feature positioning on the substrate, one or more feature dimensions, and an indication of a molecule at a given location.


Any of the systems described herein, may be operably linked to a computer and may be automated through a computer either locally or remotely. Methods and disclosed herein may further comprise software programs on computer systems and use thereof. Accordingly, computerized control for the synchronization of the dispense/vacuum/refill functions such as orchestrating and synchronizing the material deposition device movement, dispense action and vacuum actuation are within the bounds of the invention. The computer systems may be programmed to interface between the user specified base sequence and the position of a material deposition device to deliver the correct reagents to specified regions of the substrate.


The computer system 1400 illustrated in FIG. 14 may be understood as a logical apparatus that can read instructions from media 1411 and/or a network port 1405, which can optionally be connected to server 1409 having fixed media 1412. The system can include a CPU 1401, disk drives 1403, optional input devices such as keyboard 1415 and/or mouse 1416 and optional monitor 1407. Data communication can be achieved through the indicated communication medium to a server at a local or a remote location. The communication medium can include any means of transmitting and/or receiving data. For example, the communication medium can be a network connection, a wireless connection or an internet connection. Such a connection can provide for communication over the World Wide Web. It is envisioned that data relating to the present disclosure can be transmitted over such networks or connections for reception and/or review by a party 1422.



FIG. 15 is a block diagram illustrating a first example architecture of a computer system 1500 that can be used in connection with example embodiments of the present invention. An example computer system can include a processor 1502 for processing instructions. Non-limiting examples of processors include: Intel Xeon™ processor, AMD Opteron™ processor, Samsung 32-bit RISC ARM 1176JZ(F)-S v1.0™ processor, ARM Cortex-A8 Samsung S5PC100™ processor, ARM Cortex-A8 Apple A4™ processor, Marvell PXA 930™ processor, or a functionally-equivalent processor. Multiple threads of execution can be used for parallel processing. In some embodiments, multiple processors or processors with multiple cores can also be used, whether in a single computer system, in a cluster, or distributed across systems over a network comprising a plurality of computers, cell phones, and/or personal data assistant devices.


A high speed cache 1504 can be connected to, or incorporated in, the processor 1502 to provide a high speed memory for instructions or data that have been recently, or are frequently, used by processor 1502. The processor 1502 is connected to a north bridge 1506 by a processor bus 1508. The north bridge 1506 is connected to random access memory (RAM) 1510 by a memory bus 1512 and manages access to the RAM 1510 by the processor 1502. The north bridge 1506 is also connected to a south bridge 1514 by a chipset bus 1516. The south bridge 1514 is, in turn, connected to a peripheral bus 1518. The peripheral bus can be, for example, PCI, PCI-X, PCI Express, or other peripheral bus. The north bridge and south bridge are often referred to as a processor chipset and manage data transfer between the processor, RAM, and peripheral components on the peripheral bus 1518. In some alternative architectures, the functionality of the north bridge can be incorporated into the processor instead of using a separate north bridge chip. In some embodiments, system 1500 can include an accelerator card 1522 attached to the peripheral bus 1518. 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 1524 and can be loaded into RAM 1510 and/or cache 1504 for use by the processor. The system 1500 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 1500 also includes network interface cards (NICs) 1520 and 1521 connected to the peripheral bus for providing network interfaces to external storage, such as Network Attached Storage (NAS) and other computer systems that can be used for distributed parallel processing.



FIG. 16 a diagram showing a network 1600 with a plurality of computer systems 1602a, and 1602b, a plurality of cell phones and personal data assistants 1602c, and Network Attached Storage (NAS) 1604a, and 1604b. In example embodiments, systems 1602a, 1602b, and 1602c can manage data storage and optimize data access for data stored in Network Attached Storage (NAS) 1604a and 1604b. A mathematical model can be used for the data and be evaluated using distributed parallel processing across computer systems 1602a, and 1602b, and cell phone and personal data assistant systems 1602c. Computer systems 1602a, and 1602b, and cell phone and personal data assistant systems 1602c can also provide parallel processing for adaptive data restructuring of the data stored in Network Attached Storage (NAS) 1604a and 1604b. FIG. 16 illustrates an example only, and a wide variety of other computer architectures and systems can be used in conjunction with the various embodiments of the present invention. For example, a blade server can be used to provide parallel processing. Processor blades can be connected through a back plane to provide parallel processing. Storage can also be connected to the back plane or as Network Attached Storage (NAS) through a separate network interface. In some cases, 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.



FIG. 17 is a block diagram of a multiprocessor computer system 1700 using a shared virtual address memory space in accordance with an example embodiment. The system includes a plurality of processors 1702a-f that can access a shared memory subsystem 1704. The system incorporates a plurality of programmable hardware memory algorithm processors (MAPs) 1706a-f in the memory subsystem 1704. Each MAP 1706a-f can comprise a memory 1708a-f and one or more field programmable gate arrays (FPGAs) 1710a-f. The MAP provides a configurable functional unit and particular algorithms or portions of algorithms can be provided to the FPGAs 1710a-f for processing in close coordination with a respective processor. For example, the MAPs can be used to evaluate algebraic expressions regarding the data model and to perform adaptive data restructuring in example embodiments. In this example, each MAP is globally accessible by all of the processors for these purposes. In one configuration, each MAP can use Direct Memory Access (DMA) to access an associated memory 1708a-f, allowing it to execute tasks independently of, and asynchronously from, the respective microprocessor 1702a-f. In this configuration, a MAP can feed results directly to another MAP for pipelining and parallel execution of algorithms.


The above computer architectures and systems are examples only, and a wide variety of other computer, cell phone, and personal data assistant architectures and systems can be used in connection with example 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 cases, 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.


The computer system can be implemented using software modules executing on any of the above or other computer architectures and systems. In other examples, 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, such as accelerator card 1522.


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.


EXAMPLES
Example 1
Patterning of an Wet Deposited Aminosilane on a Silicon Dioxide Surface

In this example, a silicon dioxide wafer was treated with a single organic layer deposited at different locations on the wafer to create loci with a high surface energy and coupling ability to nucleoside. A surface of 1000 Angstroms of silicon dioxide on top of polished silicon was selected. A controlled surface density of hydroxyl groups was achieved on the surface by a wet process using a 1% solution of N-(3-TRIETHOXYSILYLPROPYL-4HYDROXYBUTYRAMIDE in ethanol and acetic acid deposited on the surface and treated for 4 hours, followed by placing the wafers on a hot plate at 150 degrees C. for 14 hours.


A layer of MEGAPOSIT SPR 3612 photoresist was deposited on top of the aminosilane. In this case, the organic layer was an adhesion promoter for the photoresist. The photoresist layer was patterned by exposure to ultraviolet light through a shadow mask. The photoresist pattern was transferred into the organic layer by oxygen plasma. The photoresist was then stripped, revealing a pattern of regions for biomolecular coupling. Clusters of 80 discs with a diameter of about 80 μm were well resolved.


Oligonucleic acids were extended from the surface. The photolithographic process performed without adhesion promoter layer did not result in organized loci having oligonucleic acids extended (data not shown). Oligonucleic acids extension performed a surface treated with the photolithographic process performed using the aminosilane layer resulted in clarified small discs of oligonucleic acids 80 μm in diameter (FIG. 18B) located within a cluster of discs (FIG. 18A).


Example 2
Patterning of a Gaseous Deposited Aminosilane on a Silicon Dioxide Surface

A CVD process was performed by delivering silane to the surface in gaseous state and applying a controlled deposition pressure of about 200 mTor and a controlled temperature of about 150 degrees C. (Data not shown.)


Example 3
Synthesis of a 50-Mer Sequence on an Oligonucleotide Synthesis Device

A oligonucleic acid synthesis device was assembled into a flowcell, which was connected to an Applied Biosystems (ABI394 DNA Synthesizer). The oligonucleic acid synthesis device was uniformly functionalized with N-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest, CAS No. 156214-80-1) and was used to synthesize an exemplary oligonucleotide of 50 bp (“50-mer oligonucleotide”) using oligonucleotide synthesis methods described herein. The sequence of the 50-mer was as described in SEQ ID NO.: 1.


5′AGACAATCAACCATTTGGGGTGGACAGCCTTGACCTCTAGACTTCGGCA T##TTTTTTTTTT3′ (SEQ ID NO.: 1), where # denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 from ChemGenes), which is a cleavable linker enabling the release of oligos from the surface during deprotection. The synthesis was done using standard DNA synthesis chemistry (coupling, capping, oxidation, and deblocking) according to the protocol in Table 3 and an ABI synthesizer.










TABLE 3







General DNA Synthesis
Table 3









Process Name
Process Step
Time (sec)












WASH (Acetonitrile Wash
Acetonitrile System Flush
4


Flow)
Acetonitrile to Flowcell
23



N2 System Flush
4



Acetonitrile System Flush
4


DNA BASE ADDITION
Activator Manifold Flush
2


(Phosphoramidite +
Activator to Flowcell
6


Activator Flow)
Activator +
6



Phosphoramidite to



Flowcell



Activator to Flowcell
0.5



Activator +
5



Phosphoramidite to



Flowcell



Activator to Flowcell
0.5



Activator +
5



Phosphoramidite to



Flowcell



Activator to Flowcell
0.5



Activator +
5



Phosphoramidite to



Flowcell



Incubate for 25 sec
25


WASH (Acetonitrile Wash
Acetonitrile System Flush
4


Flow)
Acetonitrile to Flowcell
15



N2 System Flush
4



Acetonitrile System Flush
4


DNA BASE ADDITION
Activator Manifold Flush
2


(Phosphoramidite +
Activator to Flowcell
5


Activator Flow)
Activator +
18



Phosphoramidite to



Flowcell



Incubate for 25 sec
25


WASH (Acetonitrile Wash
Acetonitrile System Flush
4


Flow)
Acetonitrile to Flowcell
15



N2 System Flush
4



Acetonitrile System Flush
4


CAPPING (Cap A + B, 1:1,
CapA + B to Flowcell
15


Flow)


WASH (Acetonitrile Wash
Acetonitrile System Flush
4


Flow)
Acetonitrile to Flowcell
15



Acetonitrile System Flush
4


OXIDATION (Oxidizer
Oxidizer to Flowcell
18


Flow)


WASH (Acetonitrile Wash
Acetonitrile System Flush
4


Flow)
N2 System Flush
4



Acetonitrile System Flush
4



Acetonitrile to Flowcell
15



Acetonitrile System Flush
4



Acetonitrile to Flowcell
15



N2 System Flush
4



Acetonitrile System Flush
4



Acetonitrile to Flowcell
23



N2 System Flush
4



Acetonitrile System Flush
4


DEBLOCKING (Deblock
Deblock to Flowcell
36


Flow)


WASH (Acetonitrile Wash
Acetonitrile System Flush
4


Flow)
N2 System Flush
4



Acetonitrile System Flush
4



Acetonitrile to Flowcell
18



N2 System Flush
4.13



Acetonitrile System Flush
4.13



Acetonitrile to Flowcell
15









The phosphoramidite/activator combination was delivered similar to the delivery of bulk reagents through the flowcell. No drying steps were performed as the environment stays “wet” with reagent the entire time. The flow restrictor was removed from the ABI 394 synthesizer to enable faster flow. Without flow restrictor, flow rates for amidites (0.1M in ACN), Activator, (0.25M Benzoylthiotetrazole (“BTT”; 30-3070-xx from GlenResearch) in ACN), and Ox (0.02M 12 in 20% pyridine, 10% water, and 70% THF) were roughly ˜100 uL/sec, for acetonitrile (“ACN”) and capping reagents (1:1 mix of CapA and CapB, wherein CapA is acetic anhydride in THF/Pyridine and CapB is 16% 1-methylimidizole in THF), roughly ˜200 uL/sec, and for Deblock (3% dichloroacetic acid in toluene), roughly ˜300 uL/sec (compared to ˜50 uL/sec for all reagents with flow restrictor). The time to completely push out Oxidizer was observed, the timing for chemical flow times was adjusted accordingly and an extra ACN wash was introduced between different chemicals. After oligonucleotide synthesis, the chip was deprotected in gaseous ammonia overnight at 75 psi. Five drops of water were applied to the surface to recover oligonucleic acids (FIG. 19A). The recovered oligonucleic acids were then analyzed on a BioAnalyzer small RNA chip (FIG. 19B).


Example 4
Synthesis of a 100-Mer Sequence on an Oligonucleotide Synthesis Device

The same process as described in Example 3 for the synthesis of the 50-mer sequence was used for the synthesis of a 100-mer oligonucleotide (“100-mer oligonucleotide”; 5′ CGGGATCCTTATCGTCATCGTCGTACAGATCCCGACCCATTTGCTGTCCACCAGTCA TGCTAGCCATACCATGATGATGATGATGATGAGAACCCCGCAT##TTTTTTTTTT3′, where # denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 from ChemGenes); SEQ ID NO.: 2) on two different silicon chips, the first one uniformly functionalized with N-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE and the second one functionalized with 5/95 mix of 11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane, and the oligos extracted from the surface were analyzed on a BioAnalyzer instrument (FIG. 20).


All ten samples from the two chips were further PCR amplified using a forward (5′ATGCGGGGTTCTCATCATC3′; SEQ ID NO.: 3) and a reverse (5′CGGGATCCTTATCGTCATCG3′; SEQ ID NO.: 4) primer in a 50 uL PCR mix (25 uL NEB Q5 mastermix, 2.5 uL 10 uM Forward primer, 2.5 uL 10 uM Reverse primer, luL oligo extracted from the surface, and water up to 50 uL) using the following thermalcycling program:


98 C, 30 sec


98 C, 10 sec; 63 C, 10 sec; 72 C, 10 sec; repeat 12 cycles


72 C, 2 min


The PCR products were also run on a BioAnalyzer (data not shown), demonstrating sharp peaks at the 100-mer position. Next, the PCR amplified samples were cloned, and Sanger sequenced. Table 4 summarizes the results from the Sanger sequencing for samples taken from spots 1-5 from chip 1 and for samples taken from spots 6-10 from chip 2.














TABLE 4







Spot

Error rate
Cycle efficiency





















1
1/763
bp
99.87%



2
1/824
bp
99.88%



3
1/780
bp
99.87%



4
1/429
bp
99.77%



5
1/1525
bp
99.93%



6
1/1615
bp
99.94%



7
1/531
bp
99.81%



8
1/1769
bp
99.94%



9
1/854
bp
99.88%



10
1/1451
bp
99.93%










Thus, the high quality and uniformity of the synthesized oligonucleotides were repeated on two chips with different surface chemistries. Overall, 89%, corresponding to 233 out of 262 of the 100-mers that were sequenced were perfect sequences with no errors.



FIGS. 21 and 22 show alignment maps for samples taken from spots 8 and 7, respectively, where “x” denotes a single base deletion, “star” denotes single base mutation, and “+” denotes low quality spots in Sanger sequencing. The aligned sequences in FIG. 21 together represent an error rate of about 97%, where 28 out of 29 reads correspond to perfect sequences. The aligned sequences in FIG. 22 together represent an error rate of about 81%, where 22 out of 27 reads correspond to perfect sequences.


Finally, Table 5 summarizes key error characteristics for the sequences obtained from the oligonucleotides samples from spots 1-10.










TABLE 5








Sample ID/Spot no.


















OSA_
OSA_
OSA_
OSA_
OSA_
OSA_
OSA_
OSA_
OSA_
OSA_



0046/1
0047/2
0048/3
0049/4
0050/5
0051/6
0052/7
0053/8
0054/9
0055/10




















Total Sequences
32
32
32
32
32
32
32
32
32
32


Sequencing
25 of 28
27 of 27
26 of 30
21 of 23
25 of 26
29 of 30
27 of 31
29 of 31
28 of 29
25 of 28


Quality












Oligo Quality
23 of 25
25 of 27
22 of 26
18 of 21
24 of 25
25 of 29
22 of 27
28 of 29
26 of 28
20 of 25


ROI Match
2500
2698
2561
2122
2499
2666
2625
2899
2798
2348


Count












ROI Mutation
2
2
1
3
1
0
2
1
2
1


ROI Multi Base
0
0
0
0
0
0
0
0
0
0


Deletion












ROI Small
1
0
0
0
0
0
0
0
0
0


Insertion












ROI Single Base
0
0
0
0
0
0
0
0
0
0


Deletion












Large Deletion
0
0
1
0
0
1
1
0
0
0


Count












Mutation: G > A
2
2
1
2
1
0
2
1
2
1


Mutation: T > C
0
0
0
1
0
0
0
0
0
0


ROI Error Count
3
2
2
3
1
1
3
1
2
1


ROI Error Rate
Err: ~1 in
Err: ~1 in
Err: ~1 in
Err: ~1 in
Err: ~1 in
Err: ~1 in
Err: ~1 in
Err: ~1 in
Err: ~1 in
Err: ~1 in



834
1350
1282
708
2500
2667
876
2900
1400
2349


ROI Minus
MP Err: ~1
MP Err: ~1
MP Err: ~1
MP Err: ~1
MP Err: ~1
MP Err: ~1
MP Err: ~1
MP Err: ~1
MP Err: ~1
MP Err: ~1


Primer Error
in 763
in 824
in 780
in 429
in 1525
in 1615
in 531
in 1769
in 854
in 1451


Rate









Example 5
Nanoreactor

A nanoreactor was sealed to a silicon wafer. The wafer contained nucleic acids generated from the DNA synthesis reaction. Gene assembly reagents were added to the reaction chamber. Gene amplification occurred in the resolved enclosure. The reaction chamber included nucleic acids encoding for different predetermined sequences. A series of enzymatic reactions resulted in the linking of amplified nucleic acids into a 2 kilobase gene.


Example 6
Error Correction of Assembled Nucleic Acids










TABLE 6






Nucleic Acid
Sequence








Assembled Gene,
5′ ATGACCATGATTACGGATTCACT



SEQ ID NO.: 5
GGCCGTCGTTTTACAACGTCGTGACT




GGGAAAACCCTGGCGTTACCCAACTT




AATCGCCTTGCAGCACATCCCCCTTT




CGCCAGCTGGCGTAATAGCGAAGAGG




CCCGCACCGATCGCCCTTCCCAACAG




TTGCGCAGCCTGAATGGCGAATGGCG




CTTTGCCTGGTTTCCGGCACCAGAAG




CGGTGCCGGAAAGCTGGCTGGAGTGC




GATCTTCCTGAGGCCGATACTGTCGT




CGTCCCCTCAAACTGGCAGATGCACG




GTTACGATGCGCCCATCTACACCAAC




GTGACCTATCCCATTACGGTCAATCC




GCCGTTTGTTCCCACGGAGAATCCGA




CGGGTTGTTACTCGCTCACATTTAAT




GTTGATGAAAGCTGGCTACAGGAAGG




CCAGACGCGAATTATTTTTGATGGCG




TTAACTCGGCGTTTCATCTGTGGTGC




AACGGGCGCTGGGTCGGTTACGGCCA




GGACAGTCGTTTGCCGTCTGAATTTG




ACCTGAGCGCATTTTTACGCGCCGGA




GAAAACCGCCTCGCGGTGATGGTGCT




GCGCTGGAGTGACGGCAGTTATCTGG




AAGATCAGGATATGTGGCGGATGAGC




GGCATTTTCCGTGACGTCTCGTTGCT




GCATAAACCGACTACACAAATCAGCG




ATTTCCATGTTGCCACTCGCTTTAAT




GATGATTTCAGCCGCGCTGTACTGGA




GGCTGAAGTTCAGATGTGCGGCGAGT




TGCGTGACTACCTACGGGTAACAGTT




TCTTTATGGCAGGGTGAAACGCAGGT




CGCCAGCGGCACCGCGCCTTTCGGCG




GTGAAATTATCGATGAGCGTGGTGGT




TATGCCGATCGCGTCACACTACGTCT




GAACGTCGAAAACCCGAAACTGTGGA




GCGCCGAAATCCCGAATCTCTAT




C 3′






Assembly
5′ ATGACCATGATTACGGATTCACT



Oligonucleotide 1,
GGCCGTCGTTTTACAACGTCGTGACT



SEQ ID NO.: 6
GGGAAAACCCTGGCGTTACCCAACTT




AATCGCCTTGCAGCACATCCCCCTTT




CGCCAGCTGGCGTAATAGCGAAGAGG




CCCGCACCGATCGCCCTTCCCAACAG




TTGCGCAGCC 3′






Assembly
5′ GATAGGTCACGTTGGTGTAGATG



Oligonucleotide 2,
GGCGCATCGTAACCGTGCATCTGCCA



SEQ ID NO.: 7
GTTTGAGGGGACGACGACAGTATCGG




CCTCAGGAAGATCGCACTCCAGCCAG




CTTTCCGGCACCGCTTCTGGTGCCGG




AAACCAGGCAAAGCGCCATTCGCCAT




TCAGGCTGCGCAACTGTTGGGA 3′






Assembly
5′ CCCATCTACACCAACGTGACCTA



Oligonucleotide 3,
TCCCATTACGGTCAATCCGCCGTTTG



SEQ ID NO.: 8
TTCCCACGGAGAATCCGACGGGTTGT




TACTCGCTCACATTTAATGTTGATGA




AAGCTGGCTACAGGAAGGCCAGACGC




GAATTATTTTTGATGGCGTTAACTCG




GCGTTTCATCTGTGGTGCAACGG 3′






Assembly
5′ GCCGCTCATCCGCCACATATCCT



Oligonucleotide 4,
GATCTTCCAGATAACTGCCGTCACTC



SEQ ID NO.: 9
CAGCGCAGCACCATCACCGCGAGGCG




GTTTTCTCCGGCGCGTAAAAATGCGC




TCAGGTCAAATTCAGACGGCAAACGA




CTGTCCTGGCCGTAACCGACCCAGCG




CCCGTTGCACCACAGATGAAACG 3′






Assembly
5′ AGGATATGTGGCGGATGAGCGGC



Oligonucleotide 5,
ATTTTCCGTGACGTCTCGTTGCTGCA



SEQ ID NO.: 10
TAAACCGACTACACAAATCAGCGATT




TCCATGTTGCCACTCGCTTTAATGAT




GATTTCAGCCGCGCTGTACTGGAGGC




TGAAGTTCAGATGTGCGGCGAGTTGC




GTGACTACCTACGGGTAACAGTT




T 3′






Assembly
5′ GATAGAGATTCGGGATTTCGGCG



Oligonucleotide 6,
CTCCACAGTTTCGGGTTTTCGACGTT



SEQ ID NO.: 11
CAGACGTAGTGTGACGCGATCGGCAT




AACCACCACGCTCATCGATAATTTCA




CCGCCGAAAGGCGCGGTGCCGCTGGC




GACCTGCGTTTCACCCTGCCATAAAG




AAACTGTTACCCGTAGGTAGTCAC




G 3′









A gene of about 1 kb (SEQ ID NO.: 5; Table 6) was assembled using 6 purchased oligonucleotides (5 nM each during PCA) (Ultramer; SEQ ID NO.: 6-11; Table 6) and assembled in a PCA reaction using a 1×NEB Q5 buffer with 0.02 U/uL Q5 hot-start high-fidelity polymerase and 100 uM dNTP as follows:


1 cycle: 98 C, 30 sec


15 cycles: 98 C, 7 sec; 62 C 30 sec; 72 C, 30 sec


1 cycle: 72 C, 5 min


Ultramer oligonucleotides are expected to have error rates of at least 1 in 500 nucleotides, more likely at least 1 in 200 nucleotides or more.


The assembled gene was amplified in a PCR reaction using a forward primer (5′ ATGACCATGATTACGGATTCACTGGCC3′ SEQ ID NO.: 12) and a reverse primer (5′GATAGAGATTCGGGATTTCGGCGCTCC3′ SEQ ID NO.: 13), using 1×NEB Q5 buffer with 0.02 U/uL Q5 hot-start high-fidelity polymerase, 200 uM dNTP, and 0.5 uM primers as follows:


1 cycle: 98 C, 30 sec


30 cycles: 98 C, 7 sec; 65 C 30 sec; 72 C, 45 sec


1 cycle: 72 C, 5 min


The amplified assembled gene was analyzed in a BioAnalyzer and cloned. Mini-preps from ˜24 colonies were Sanger sequenced. The BioAnalyzer analysis provided a broad peak and a tail for the uncorrected gene, indicated a high error rate. The sequencing indicated an error rate of 1/789 (data not shown). Two rounds of error correction were followed using CorrectASE (Life Technologies, www.lifetechnologies.com/order/catalog/product/A14972) according to the manufacturer's instructions. The resulting gene samples were similarly analyzed in the BioAnalyzer after round one and round two and cloned. (Data not shown.) 24 colonies were picked for sequencing. The sequencing results indicated an error rate of 1/5190 bp and 1/6315 bp after the first and second rounds of error correction, respectively.


Example 7
Oligonucleic Acid Distribution for Synthesizing Genes Under 1.8 Kb in Length

240 genes were selected for de novo synthesis wherein the genes ranged from 701 to 1796 base pairs in length. The gene sequence for each of the genes was divided into smaller fragments encoding for oligonucleic acids ranging between 50 to 90 nucleotides in length, with each nucleotide having 20 to 25 nucleotides overlapping sequences A distribution chart is depicted in FIG. 23, where the X axis depicts the oligonucleic acid length, and the Y axis depicted the number of oligonucleic acids synthesized. A total of roughly 5,500 oligonucleic acids were synthesized on an aminosilane coated surface using a protocol similar to that of Example 3, and assembled using a polymerase chain assembly reaction to anneal overlapping sequence of each oligonucleotide to a different oligonucleotide to form a gene.


Example 8
Oligonucleic Acid Distribution for Synthesizing a Long Gene Sequence

Gene sequence for a single gene that was larger than 1.8 kb in length was divided into smaller fragments encoding for oligonucleic acids ranging between 50 to 120 nucleotides in length, with each nucleotide having 20 to 25 nucleotides overlapping sequences. A total of 90 different design arrangement were synthesized. A distribution chart is depicted in FIG. 24, where the X axis depicts the oligonucleic acid length, and the Y axis depicted the number of oligonucleic acids synthesized. The oligonucleic acids were synthesized on an aminosilane coated surface using a protocol similar to that of Example 3, and assembled using a polymerase chain assembly reaction to anneal overlapping sequence of each oligonucleotide to a different oligonucleotide to form a gene.


Example 9
Two-Step Deposition Process for Dilution of Nucleoside Coupling Agent

Various methods for surface preparation were preformed, which include: (i) performing an active chemical vapor (CVD) deposition step before photolithography; (ii) performing an active chemical vapor (CVD) deposition step after photolithography; and (i) performing a dilution active chemical vapor (CVD) deposition step after photolithograph.


A first silicon dioxide structure having a silicon oxide layer was individually cleaned in an oxygen plasma (referred to as the “HAPS” chip. An aminosilane, N-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE, HAPS), was deposited on the silicon oxide at predetermined locations, referred to as loci. The surface was coated with AZ resist and then baked. The surface was cleaned again in an oxygen plasma, fluorinated (depositing (tridecafluoro-1,1,2,2-tetrahydrooctyl)-trichlorosilane), and then stripped.


A second silicon dioxide structure having a silicon oxide layer was individually cleaned in an oxygen plasma (referred to as the “100% GOPS-1” chip). A silane, 3-glycidoxypropyltrimethoxysilane (GOPS), was deposited on the silicon oxide at predetermined locations, referred to as loci. The surface was coated with AZ resist and then baked. The surface was cleaned again in an oxygen plasma, fluorinated (depositing (tridecafluoro-1,1,2,2-tetrahydrooctyl)-trichlorosilane), and then stripped.


A third silicon dioxide structure having a silicon oxide layer was individually cleaned in an oxygen plasma (referred to as the “100% GOPS-2” chip). Directly after cleaning, the surface was coated with AZ resist and then baked at 90 degrees Celsius for 7 min. The surface was cleaned again in an oxygen plasma, fluorinated in the YES CVD system (depositing (tridecafluoro-1,1,2,2-tetrahydrooctyl)-trichlorosilane), and then stripped. The surface was treated with 100% GOPS at 1 Torr for 1 hour at chamber temperature of 100 degrees Celsius. Lastly, the surface was activated in water for 30 minutes at room temperature.


For the diluted active agent deposition protocol, a fourth silicon dioxide structure having a silicon oxide layer was individually cleaned in an oxygen plasma (referred to as the “GOPS-diluted” chip). Directly after cleaning, the surface was coated with AZ resist and then baked at 90 degrees Celsius for 7 min. The surface was cleaned again in an oxygen plasma, fluorinated in the YES CVD system (depositing (tridecafluoro-1,1,2,2-tetrahydrooctyl)-trichlorosilane), and then stripped. The surface was treated with 100% propyltrimethoxysilane at 3.5 Torr for 15 hours at chamber temperature of 100 C degrees Celsius. The surface was treated with water for 30 minutes, followed by a second deposition step. In the second deposition step, a mixture of 0.05% GOPS and 99.95% propyltrimethoxysilane was deposited on the surface and treated at 3.5 Torr for 1.5 hours at chamber temperature of 100 C. Lastly, the surface was activated in water for 30 minute at room temperature. For each structure prepared, the molecules able to couple nucleoside (HAPS and GOPS) were deposited in described locations on the surfaces, loci, and the loci were arranged in clusters.


Example 10
Characterization of Low Density Oligonucleic Acid Surfaces

Oligonucleic acids of 30, 50 and 80 nucleotides in length were synthesized on the surfaces prepared in Example 9. A FRT tool (MicroProf 100, Fries Research and Technology, GmbH, Germany) was used to measure the thickness of DNA post-synthesis. The FRT tool scans across a cluster and measure reflectivity of light, which corresponds to the amount of material on the surface. A summary of the results is shown in FIG. 25A for growth of 30, 50 and 80-mers. In the case of synthesizing 80-mers, the GOPS-diluted surfaces produced a DNA thickness 52% lower than the 100% GOPS-2 and the HAPS chips.


Qubit analysis from fluorometric measurement of the clusters was also performed. A summary of the analysis from growth of 30, 50 and 80-mers is in the chart in FIG. 25B. For 30, 50 and 80-mers, the GOPS-diluted chip resulted in 42% less DNA density than the 100% GOPS-2 or the HAPS chips.


Example 11
Deletion Error Rate Analysis for Low Density Oligonucleic Acid Surfaces

Oligonucleic acids of 30, 50 and 80 nucleotides in length were synthesized on the surfaces prepared in Example 9, gas cleaved from the surface, and subject to sequence analysis using an Illumina MiSeq. Deletion error rates were determined for oligonucleic acids synthesized on the GOPS-diluted surfaces. The total deletion error rate was 0.060%, or 1 in 1674 bases. Sequencing of control oligonucleic acids from the GOPS-diluted chips resulted in a deletion rate of 0.070%. Analysis of the deletion error rate frequency at particular bases in terms of distance from the surface was performed, and results are shown in the plot in FIG. 26. Notably, the GOPS-diluted surfaces resulted in a deletion error rate frequency for bases closer to the surface which is less than twice the error rate frequency for bases further from the surface. In other words, compared to other surfaces analyzed, the GOPS-diluted surfaces reduce the increase in deletion error rate observed at bases closer to the surface. As a whole the GOPS-diluted surfaces resulted in lower average deletion error rate above background error rate levels, Table 7.











TABLE 7






Active agent added
Average deletion



before or after
error rate above


Surface
photoresist
background levels







GOPS-diluted (no. 2063)
After
0.03%


GOPS-100%-2 (no. 2059)
After
0.09%


GOPS-100%-1 (no. 2413)
Before
0.07%


GOPS-100%-1 (no. 2763)
Before
0.06%


GOPS-100%-1 (no. 2770)
Before
0.09%


GOPS-100%-1 (no. 2809)
Before
0.15%


GOPS-100%-1 (no. 2810)
Before
0.08%


HAPS (no. 1994)
Before
0.14%


HAPS (no. 2541)
Before
0.07%









Example 12
Textured Surface

A microfluidic device is manufactured to have increased surface area. An array of recesses or posts is etched into a silicon dioxide wafer to increase surface area by a factor of 2 to 3. A number of steps are performed to make a textured surface. To the starting silicon dioxide wafer, with one side polished, is added a textured layer via a pass printing scheme lithography. A silicon reactive ion etching and resist strip is added to the chip, followed by oxidation of the surface. The fiducial layer is printed on via lithography, after which a final oxide etching results in a textured silicon ship. The surface has a recess or post width that is about 2 times the length of the desired oligonucleic acids to be extended. A chart of exemplary widths is provided in Table 8 based on an approximate length of 0.34 nm/base.











TABLE 8





No. of bases
Oligo length (nm)
Width of post or recess (nm)

















1
0.34
0.68


10
3.4
6.8


100
34
68


200
68
136


300
102
204









A silicon dioxide structure is prepared having a 16×16 array of clusters. Each cluster includes multiple groups of 4 loci, wherein each loci resides on top of a different design feature, as outlined in Table 9.














TABLE 9







Locus
Width w (um)
Pitch p (um)
Depth d (um)









1
0.2
0.4
0.25-0.5 



2
0.3
0.6
0.4-0.8



3
0.4
0.8
0.5-1.0



4
No texture
No texture
No texture










Each loci is coated with an silane that binds the surface and couples to nucleoside phosphoramidite. Oligonucleic acid synthesis is perform on the structure and DNA thickness, DNA mass and error rates of the synthesized oligonucleic acids are measured.


Example 13
Array of 256 Clusters

A microfluidic device is manufactured. Each device is 200 mm, double-side polished. A SOI wafer has 21 chips arranged in a 200 mm wafer. Each chip is 32 mm×32 mm in size, and comprised a 16×16 array of clusters. A total of 256 clusters are present in the array. 121 reaction sites are located in a single cluster, providing 30,976 individually addressable oligo sites per chip. Each cluster pitch is 1.125 mm. Each of the reaction sites are about 50 μm.


Example 14
Fiducial Marks

A silicon dioxide structure is prepared having a 16×16 array of clusters. Each cluster includes groups of 4 loci, wherein each loci is in close proximity to a one of three fiducial marks having a plurality of lines, wherein the line weight is listed in Table 10. Each fiducial design is in the shape of a plus 805. One of the test regions includes a plurality of fiducial marks in close proximity 810.











TABLE 10





Design
Width w (um)
Pitch p (um)







1
0.2
0.4


2
0.3
0.6


3
0.4
0.8









Each loci is coated with an silane that binds the surface and couples to nucleoside phosphoramidite. Oligonucleic acid synthesis is perform on the structure and measurements are taken using the fiducial marks to calibrate align the surface with other components of a system.


Example 15
Global Alignment Marks

A silicon dioxide structure is prepared having a 16×16 array of clusters. Global alignment marks are used to aligning the surface 900 with other components of a system. Global alignment marks 905, 910, 915, 920, 935, and 940 are located at positions on a substantially planar substrate portion of the surface 900 and near an edge of the structure. Detailed circular mark 925 and plus sign mark 945 are shown in an expanded view in FIG. 9.


Example 16
Coating a Textured Surface with Diluted Activating Agent

A structure for oligonucleic acid synthesis is manufactured. Each device is 200 mm, double-side polished. A SOI wafer has 21 chips are arranged in a 200 mm wafer. Each chip is 32 mm×32 mm in size, and comprised a 16×16 array of clusters. A total of 256 clusters are present in the array. 121 reaction sites are located in a single cluster, providing 30,976 individually addressable oligo sites per chip. Each cluster pitch is 1.125 mm. Each of the reaction sites are about 50 μm in diameter. The surface of each chip is textured by methods as describe in Example 12 to include recesses with one of the texture designs listed in Table 9.


The structures for oligonucleic acid synthesis are individually cleaned by treatment with oxygen plasma. Directly after cleaning, the surface is coated with AZ resist and then baked at 90 degrees Celsius for 7 minutes. The surface is cleaned again by treatment with oxygen plasma, fluorinated in a YES CVD system (depositing (tridecafluoro-1,1,2,2-tetrahydrooctyl)-trichlorosilane), and is stripped. The surface is treated with 100% propyltrimethoxysilane at 3.5 Torr for 15 hours at chamber temperature of 100 C degrees Celsius. The surface is treated with water for 30 minutes, followed by a second deposition step. In the second deposition step, a mixture of 0.05% GOPS and 99.95% propyltrimethoxysilane is deposited on the surface and treated at 3.5 Torr for 1.5 hours at chamber temperature of 100 degrees Celsius. Lastly, the surface is activated in water for 30 minute at room temperature. The reaction sites on the surface of each structure comprise diluted GOPS. Each of the reaction sites are surrounded by surface coated with tridecafluoro-1,1,2,2-tetrahydrooctyl)-trichlorosilane.


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.

Claims
  • 1. A method for preparing a surface for polynucleotide synthesis, the method comprising: depositing a first population of a first plurality of molecules on a surface of a solid support at a first region, wherein the first region comprises a plurality of loci, and wherein the first plurality of molecules comprises a plurality of non-coupling molecules that bind to the surface and lack a reactive group capable of binding to a nucleoside; anddepositing a mixture on the solid support at the first region, wherein the mixture comprises a second population of the first plurality of molecules and a second plurality of molecules, and wherein the second plurality of molecules comprises a plurality of coupling molecules that bind to the solid support and comprise the reactive group that is capable of binding to the nucleoside, and wherein the first plurality of molecules and the second plurality of molecules are present in the mixture in a molar ratio of 10:1 to 2500:1.
  • 2. The method of claim 1, wherein each of the plurality of coupling molecules is a silane.
  • 3. The method of claim 1, wherein each of the plurality of coupling molecules is N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane.
  • 4. The method of claim 1, wherein the second molecule each of the plurality of coupling molecules is 3-glycidoxypropyltrimethoxysilane.
  • 5. The method of claim 2, wherein the silane is an aminosilane.
  • 6. The method of claim 1, wherein each of the plurality of non-coupling molecules is propyltrimethoxysilane.
  • 7. The method of claim 1, wherein each of the plurality of non-coupling molecules is a fluorosilane.
  • 8. The method of claim 7, wherein the fluorosilane is (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane.
  • 9. The method of claim 1, wherein the mixture comprises the plurality of non-coupling molecules and the plurality of coupling molecules present in a molar ratio of about 100:1 to about 2500:1.
  • 10. The method of claim 1, wherein the mixture comprises the plurality of non-coupling molecules and the plurality of coupling molecules present in a molar ratio of about 2000:1.
  • 11. The method of claim 1, wherein the plurality of coupling molecules lacks a free hydroxyl, amino, or carboxyl group.
  • 12. The method of claim 1, wherein the mixture or the first population is in a gaseous state when deposited on the surface.
  • 13. The method of claim 1, wherein the surface comprises a layer of silicon oxide.
  • 14. The method of claim 1, further comprising synthesizing a plurality of polynucleotides, wherein each polynucleotide of the plurality of polynucleotides comprises at least 10 bases in length.
  • 15. The method of claim 14, wherein the plurality of polynucleotides encode sequences with an aggregate deletion error rate of less than 1 in 1500 bases compared to predetermined sequences.
  • 16. The method of claim 15, wherein the aggregate deletion error rate is achieved without correcting errors.
  • 17. The method of claim 14, wherein the plurality of polynucleotides synthesized encode sequences with an aggregate error rate of less than 1 in 1500 bases compared to predetermined sequences without correcting errors.
  • 18. The method of claim 14, wherein each polynucleotide of the plurality of polynucleotides is 50 to 120 bases in length.
  • 19. The method of claim 14, wherein the plurality of polynucleotides comprises at least 5,000 polynucleotides.
  • 20. The method of claim 14, wherein the plurality of polynucleotides comprises at least 30,000 polynucleotides.
CROSS REFERENCE

This application claims the benefit of U.S. Provisional Application No. 62/112,083 filed Feb. 4, 2015, which is herein incorporated by reference in its entirety.

US Referenced Citations (917)
Number Name Date Kind
3549368 Robert 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
5299491 Kawada Apr 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
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
5739386 Holmes Apr 1998 A
5750672 Kempe May 1998 A
5780613 Letsinger et al. Jul 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
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
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 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
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 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 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
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 Staehler 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 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
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 Staehler 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 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, III 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 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 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 Dec 2017 B2
9834774 Carstens Dec 2017 B2
9839894 Banyai Dec 2017 B2
9879283 Ravinder et al. Jan 2018 B2
9889423 Banyai Feb 2018 B2
9895673 Peck Feb 2018 B2
9925510 Jacobson et al. Mar 2018 B2
9932576 Raymond et al. Apr 2018 B2
9981239 Banyai May 2018 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 et al. 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
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
20030138782 Evans Jul 2003 A1
20030143605 Lok et al. Jul 2003 A1
20030148291 Robotti Aug 2003 A1
20030148344 Rothberg et al. Aug 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
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
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
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
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
20060003381 Gilmore 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
20070141557 Raab et al. Jun 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
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
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
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
20090263802 Drmanac Oct 2009 A1
20090285825 Kini et al. Nov 2009 A1
20090324546 Notka et al. Dec 2009 A1
20100004143 Shibahara 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
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
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
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
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
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
20130323722 Carr et al. Dec 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
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
20150120265 Amirav-Drory Apr 2015 A1
20150159152 Allen et al. Jun 2015 A1
20150183853 Sharma 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
20160090592 Banyai 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
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
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
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
20170249345 Malik et al. Aug 2017 A1
20170253644 Steyaert et al. Sep 2017 A1
20170320061 Venter et al. Nov 2017 A1
20170327819 Banyai Nov 2017 A1
20170355984 Evans et al. Dec 2017 A1
20170357752 Diggans Dec 2017 A1
20170362589 Banyai Dec 2017 A1
20180029001 Banyai Feb 2018 A1
20180051278 Cox 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 May 2018 A1
20180142289 Zeitoun May 2018 A1
20180171509 Cox Jun 2018 A1
20180236425 Banyai et al. Aug 2018 A1
20180326388 Banyai et al. Nov 2018 A1
20190060345 Harrison et al. Feb 2019 A1
20190118154 Marsh et al. Apr 2019 A1
20190240636 Peck et al. Aug 2019 A1
Foreign Referenced Citations (157)
Number Date Country
1771336 May 2006 CN
102159726 Aug 2011 CN
103907117 Jul 2014 CN
104734848 Jun 2015 CN
10260805 Jul 2004 DE
0090789 Oct 1983 EP
0126621 Nov 1984 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
2002538790 Nov 2002 JP
2006503586 Feb 2006 JP
2009294195 Dec 2009 JP
WO-9015070 Dec 1990 WO
WO-9210092 Jun 1992 WO
WO-9210588 Jun 1992 WO
WO-9309668 May 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-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-0156216 Aug 2001 WO
WO-0210443 Feb 2002 WO
WO-0156216 Mar 2002 WO
WO-0220537 Mar 2002 WO
WO-0224597 Mar 2002 WO
WO-0227638 Apr 2002 WO
WO-0233669 Apr 2002 WO
WO-02072791 Sep 2002 WO
WO-03040410 May 2003 WO
WO-03046223 Jun 2003 WO
WO-03054232 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-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-2005014850 Feb 2005 WO
WO-2005051970 Jun 2005 WO
WO-2005059096 Jun 2005 WO
WO-2005059097 Jun 2005 WO
WO-2006023144 Mar 2006 WO
WO-2006076679 Jul 2006 WO
WO-2006116476 Nov 2006 WO
WO-2007120627 Oct 2007 WO
WO-2007137242 Nov 2007 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-2008109176 Sep 2008 WO
WO-2010025310 Mar 2010 WO
WO-2010025566 Mar 2010 WO
WO-2010027512 Mar 2010 WO
WO-2010089412 Aug 2010 WO
WO-2010141433 Dec 2010 WO
WO-2010141433 Apr 2011 WO
WO-2011053957 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
WO-2013101896 Jul 2013 WO
WO-2013177220 Nov 2013 WO
WO-2014004393 Jan 2014 WO
WO-2014008447 Jan 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-2014151696 Sep 2014 WO
WO-2014160004 Oct 2014 WO
WO-2014160059 Oct 2014 WO
WO-2015021280 Feb 2015 WO
WO-2015040075 Mar 2015 WO
WO-2015054292 Apr 2015 WO
WO-2015081142 Jun 2015 WO
WO-2015090879 Jun 2015 WO
WO-2015120403 Aug 2015 WO
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-2016172377 Oct 2016 WO
WO-2016173719 Nov 2016 WO
WO-2016183100 Nov 2016 WO
WO-2017049231 Mar 2017 WO
WO-2017053450 Mar 2017 WO
WO-2017059399 Apr 2017 WO
WO-2017095958 Jun 2017 WO
WO-2017118761 Jul 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-2018156792 Aug 2018 WO
WO-2018170164 Sep 2018 WO
Non-Patent Literature Citations (479)
Entry
Assembly manual for the POSaM: The ISB Piezoelelctric Oligonucleotide Synthesizer and Microarrayer, The Institute for Systems Biology, May 28, 2004 (50 pages).
Eroshenko et al.: Gene Assembly from Chip-Synthesized Oligonucleotides; Current Protocols in Chemical biology 4: 1-17 (2012).
International Application No. PCT/US2017/026232 International Preliminary Report on Patentability dated Feb. 26, 2019.
International Application No. PCT/US2017/045105 International Preliminary Report on Patentability dated Feb. 5, 2019.
International Application No. PCT/US2018/050511 International Search Report and Written Opinion dated Jan. 11, 2019.
International Application No. PCT/US2019/012218 International Search Report and Written Opinion dated Mar. 21, 2019.
Jacobus et al. Optimal cloning of PCR fragments by homologous recombination in Escherichia soli. PLoS One 10(3):e0119221 (2015).
Lee: Covalent End-Immobilization of Oligonucleotides onto Solid Surfaces; Thesis, Massachusetts Institute of Technology, Aug. 2001 (315 pages).
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.
Douthwaite et al.: Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class a GPCR formyl-peptide receptor 1; mAbs, vol. 7, Iss. 1, pp. 152-166 (Jan. 1, 2015).
Jo et al.: Engineering therapeutic antibodies targeting G-protein-coupled receptors; Experimental & Molecular Medicine; 48; 9 pages (2016).
Lausted et al.: POSaM: a fast, flexible, open-source, inkjet oligonucleotide synthesizer and microarrayer; Genome Biology 2004, 5:R58.
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).
PCT/IL2012/000326 International Preliminary Report on Patentability dated Dec. 5, 2013.
PCT/IL2012/000326 International Search Report dated Jan. 29, 2013.
PCT/US2016/064270 International Preliminary Report on Patentability dated Jun. 14, 2018.
PCT/US2018/037152 International Search Report and Written Opinion dated Aug. 28, 2018.
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/US2018/056783 International Search Report and Written Opinion of the International Searching Authority dated Dec. 20, 2018.
Puigbo. Optimizer: a web server for optimizing the codon usage of DNA sequences. Nucleic Acid Research, 35(14):126-131, 2007.
Sharan et al. Recombineering: a homologous recombination-based method of genetic engineering. Nat Profile 4(2):1-37 (originally pp. 206-223) (2009).
U.S. Appl. No. 15/860,445 Final Office Action dated Dec. 13, 2018.
U.S. Appl. No. 15/187,714 Office Action dated Apr. 4, 2019.
U.S. Appl. No. 14/241,874 Final Office Action dated Jan. 28, 2019.
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. 14/885,965 Office Action dated Aug. 28, 2018.
U.S. Appl. No. 15/156,134 Office Action dated Apr. 4, 2019.
U.S. Appl. No. 15/187,714 Restriction Requirement dated Sep. 17, 2018.
U.S. Appl. No. 15/268,422 Final Office Action dated Mar. 1, 2019.
U.S. Appl. No. 15/268,422 Restriction Requirement dated Oct. 4, 2018.
U.S. Appl. No. 15/377,547 Final Office Action dated Feb. 8, 2019.
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. 15/729,564 Final Office Action dated Dec. 13, 2018.
U.S. Appl. No. 15/151,316 Final Office Action dated Feb. 21, 2019.
U.S. Appl. No. 15/816,995 Restriction Requirement dated Apr. 4, 2019.
Wu, et al. “Sequence-Specific Capture of Protein-DNA Complexes for Mass Spectrometric Protein Identification” PLoS ONE. Oct. 20, 2011, vol. 6, No. 10.
Zhou, et al. “Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane” Scientific Reports May 9, 2014, vol. 4, No. 4912.
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.
Crick. On protein synthesis. Symp Soc Exp Biol12:138-163,1958.
Doudna et al. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1258096-1 through 1258096-9, 2014.
Genomics 101. An Introduction to the Genomic Workflow. 2016 edition, 64 pages. Available at: http://www.frontlinegenomics.com/magazine/6757/genomics-101/.
Hughes et al. Principles of early drug discovery. Br J Pharmacol 162(2):1239-1249, 2011.
Liu et al., Comparison of Next-Generation Sequencing Systems. Journal of Biomedicine and Biotechnology, 11 pages, 2012.
PCT Patent Application No. PCT/US2016/028699 International Search Report and Written Opinion dated Jul. 29, 2016.
PCT Patent Application No. PCT/US2016/031674 International Search Report and Written Opinion dated Aug. 11, 2016.
Pirrung. How to make a DNA chip. Angew. Chem. Int. Ed., 41:1276-1289, 2002.
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.
Taylor et al., Impact of surface chemistry and blocking strategies on DNA microarrays. Nucleic Acids Research, 31(16):e87, 19 pages, 2003.
U.S. Appl. No. 14/885,965 Office Action dated Jul. 7, 2016.
U.S. Appl. No. 14/452,429 Notice of Allowance dated Jun. 7, 2016.
U.S. Appl. No. 14/885,962 Office Action dated Sep. 8, 2016.
U.S. Appl. No. 14/885,963 Notice of Allowance dated May 24, 2016.
Acevedo-Rocha et al. Directed evolution of stereoselective enzymes based on genetic selection as opposed to screening systems. J. Biotechnol. 191:3-10 (2014).
Arand et al. Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site. EMBO J. 22:2583-2592 (2003).
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.
Bonini and Mondino, Adoptive T-cell therapy for cancer: The era of engineered T cells. European Journal of Immunology, 45:2457-2469, 2015.
Borovkov et al., High-quality gene assembly directly from unpurified mixtures of microassay-synthesized oligonucleotides. Nucleic Acid Research, 38(19):e180, 10 pages, 2010.
CeGaT. Tech Note available at https://www.cegat.de/web/wp-content/uploads/2018/06/Twist-Exome-Tech-Note.pdf (4 pgs.) (2018).
Chilamakuri et al. Performance comparison of four exome capture systems for deep sequencing. BMC Genomics 15(1):449 (2014).
Co-pending U.S. Appl. No. 15/902,855, filed Feb. 22, 2018.
Co-pending U.S. Appl. No. 15/921,479, filed Mar. 14, 2018.
Co-pending U.S. Appl. No. 15/921,537, filed Mar. 14, 2018.
Co-pending U.S. Appl. No. 16/006,581, filed Jun. 12, 2018.
Cruse et al. Atlas of Immunology, Third Edition. Boca Raton: CRC Press (pp. 282-283) (2010).
De Silva et al. New Trends of Digital Data Storage in DNA. BioMed Res Int. 2016:8072463 (2016).
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.
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).
Gao et al. A method for the generation of combinatorial antibody libraries using pIX phage display. PNAS 99(20):12612-12616 (2002).
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).
Han et al. Linking T-cell receptor sequence to functional phenotype at the single-cell level. Nat Biotechnol 32(7):684-692 (2014).
Imgur: The magic of the internet. Uploaded May 10, 2012, 2 pages, retrieved from: https://imgur.com/mEWuW.
Jager et al. Simultaneous Humoral and Cellular: Immune Response against Cancer—Testis Antigen NY-ES0-1: Definition of Human Histocompatibility LeukocyteAntigen (HLA)-A2—binding Peptide Epitopes. J. Exp. Med. 187(2):265-270 (1998).
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”).
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.
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).
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.
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).
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.
Mulligan. Commercial Gene Synthesis Technology PowerPoint presentation. BlueHeron® Biotechnology. Apr. 5, 2006 (48 pgs).
Organick et al., Random access in large-scale DNA data storage. Nature Biotechnology, Advance Online Publication, 8 pages, 2018.
PCT/US2016/016459 International Preliminary Report on Patentability dated Aug. 17, 2017.
PCT/US2016/016636 International Preliminary Report on Patentability dated Aug. 17, 2017.
PCT/US2016/028699 International Preliminary Report on Patentability dated Nov. 2, 2017.
PCT/US2016/031674 International Preliminary Report on Patentability dated Nov. 23, 2017.
PCT/US2016/052336 International Preliminary Report on Patentability dated Mar. 29, 2018.
PCT/US2016/052916 International Preliminary Report on Patentability dated Apr. 5, 2018.
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 Search Report and Written Opinion dated Oct. 20, 2017.
PCT/US2017/052305 International Search Report and Written Opinion dated Feb. 2, 2018.
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/US2018/022487 International Search Report and Written Opinion dated Aug. 1, 2018.
PCT/US2018/022493 International Search Report and Written Opinion dated Aug. 1, 2018.
PCT/US2018/19268 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.
Plesa et al., Multiplexed gene synthesis in emulsions for exploring protein functional landscapes. Science, 10.1126/science.aao5167, 10 pages, 2018.
Rogozin et al., Origin and evolution of spliceosomal introns. Biology Direct, 7:11, 2012.
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.
Sharpe et al., Genetically modified T cells in cancer therapy: opportunities and challenges. Disease Models and Mechanisms, 8:337-350, 2015.
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.
Srivannavit et al., Design and fabrication of microwell array chips for a solution-based, photogenerated acid-catalyzed parallel oligonuclotide 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.
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).
Twist Bioscience | White Paper. DNA-Based Digital Storage. Retrieved from the internet, Twistbioscience.com, Mar. 27, 2018, 5 pages.
U.S. Appl. No. 14/885,962 Notice of Allowance dated Nov. 8, 2017 and Sep. 29, 2017.
U.S. Appl. No. 14/885,965 Office Action dated Aug. 30, 2017.
U.S. Appl. No. 14/885,965 Office Action dated Jan. 4, 2018.
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 Office Action dated Jun. 7, 2018.
U.S. Appl. No. 15/233,835 Notice of Allowance dated Oct. 4, 2017.
U.S. Appl. No. 15/233,835 Office Action dated Jul. 26, 2017.
U.S. Appl. No. 15/377,547 Office Action dated Jul. 27, 2018.
U.S. Appl. No. 15/377,547 Office Action dated Nov. 30, 2017.
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 Sep. 21, 2017.
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. 15/682,100 Office Action dated Jan. 2, 2018.
U.S. Appl. No. 15/682,100 Restriction Requirement dated Nov. 8, 2017.
U.S. Appl. No. 15/729,564 Office Action dated Jan. 8, 2018.
U.S. Appl. No. 15/729,564 Office Action dated Jun. 6, 2018.
U.S. Appl. No. 15/860,445 Office Action dated May 30, 2018.
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).
Wagner et al., Nucleotides, Part LXV, Synthesis of 2′-Deoxyribonucleoside 5′-Phosphoramidites: New Building Blocks for the Inverse (5′-3′)-0iigonucleotide Approach. Helvetica Chimica Acta, 83(8):2023-2035, 2000.
Warr et al. Exome Sequencing: current and future perspectives. G3: (Bethesda) 5(8):1543-1550 (2015).
Wiedenheft et al., RNA-guided genetic silencing systems in bacteria and archaea. Nature, 482:331-338, 2012.
Xu et al., Design of 240,000 orthogonal 25mer DNA barcode probes. PNAS, 106(7):2289-2294, 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).
Adessi, et al., Solid phase DNA amplification: characterisation of primer attachment and amplification mechanisms. Nucleic Acids Res. Oct. 15, 2000;28(20):E87.
Alexeyev, et al., Gene synthesis, bacterial expression and purification of the Rickettsia prowazekii ATP/ADP translocase, Biochimica et Biophysics Acta, vol. 1419, 299-306 (1999).
Al-Housseiny et al., Control of interfacial instabilities using flow geometry. Nature Physics, 8:747-750 (2012); Published online at: DOI:10.1038/NPHYS2396.
Amblard, Francois et al., A magnetic manipulator for studying local rheology and micromechanical properties of biological systems, Rev. Sci.Instrum., vol. 67, No. 3, 818-827, Mar. 1996.
Arkles, et al. The Role of Polarity in the Structure of Silanes Employed in Surface Modification. Silanes and Other Coupling Agents. 2009; 5:51-64.
Assi, Fabiano et al., Massive-parallel adhesion and reactivity-measurements using simple and inexpensive magnetic tweezers, J. Appl. Phys., vol. 92, No. 9, 5584-5586, Nov. 1, 2002.
Au, Lo-Chun et al. Gene synthesis by a LCR-based approach : high level production of Leptin-L54 using synthetic gene in Escherichia coli, Biochemical and Biophysical Reasearch Communications, vol. 248, 200-203, 1998.
Baedeker, Mathias et al., Overexression of a designed 2.2kb gene of eukaryotic phenylalanine ammonialyase in Escherichia coli. FEBS Letters, vol. 457, 57-60 (1999).
Barbee, et al. Magnetic Assembly of High-Density DNA Arrays for Genomic Analyses. Anal Chem. Mar. 15, 2008; 80(6): 2149-2154.
Beaucage, et al. Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron. 1992, 48:2223-2311.
Beaucage, et al. Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 1981, 22(20):1859-1862.
Beaulieu, et al., “PCR candidate region mismatch scanning adaptation to quantitative, highthroughput genotyping”, Nucleic Acids Research, vol. 29, No. 5, 1114-1124 (2001).
Beigelman, et al. Base-modified phosphoramidite analogs of pyrimidine ribonucleosides for RNA structure-activity studies. Methods Enzymol. 2000;317:39-65.
Biswas, et al., “Identification and characterization of a thermostable MutS homolog from Thennus aquaticus”, The Journal of Biological Chemistry, vol. 271, No. 9, 5040-5048 (Mar. 1, 1996).
Biswas, et al., “Interaction of MutS/crotein with the major and minor grooves of a heteroduplex DNA”, The Journal of Biological Chemistry, vol. 272, No. 20, 13355-13364 (May 1, 1997).
Bjornson, Keith P. et al., “Differential and simultaneous adenosine Di- and Tri˜hosphate binding by MutS”, The Journal of Biological Chemistry, vol. 278, No. 20, 18557-18562 (May 16, 2003).
Blanchard, et al. High-Density Oligonucleotide Arrays. Biosens. & Bioelectronics. 1996; 11:687-690.
Blanchard, in:Genetic Engineering, Principles and Methods, vol. 20, Ed. J. Sedlow, New York: Plenum Press, p. 111-124, 1979.
Butler, et al. In situ synthesis of oligonucleotide arrays by using surface tension. J Am Chem Soc. Sep. 19, 2001;123(37):8887-94.
Carr, et al. Protein-mediated error correction for de novo DNA synthesis. Nucleic Acids Res. Nov. 23, 2004;32(20):e162.
Caruthers, Gene synthesis machines: DNA chemistry and its uses. Science. Oct. 18, 1985;230(4723):281-5.
Caruthers, Chemical synthesis of deoxyoligonucleotides by the phosphoramidite method. In Methods in Enzymology, Chapter 15, 154:287-313 1987.
Casmiro, Danilo R. et al., “PCR-based gene synthesis and protein NMR spectroscopy”, Structure, vol. 5, ⋅No. 11, 1407-1412 1997.
Cello, et al. Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science. Aug. 9, 2002;297(5583):1016-8.
Chalmers, et al. Scaling up the ligase chain reaction-based approach to gene synthesis. Biotechniques. Feb. 2001;30(2):249-52.
Chan, et al. Natural and engineered nicking endonucleases—from cleavage mechanism to engineering of strand-specificity. Nucleic Acids Res. Jan. 2011; 39(1): 1-18.
Chen, et al. Chemical modification of gene silencing oligonucleotides for drug discovery and development. Drug Discov Today. Apr. 15, 2005;10(8):587-93.
Cheng, et al. High throughput parallel synthesis of oligonucleotides with 1536 channel synthesizer. Nucleic Acids Res. Sep. 15, 2002;30(18):e93.
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.
Cleary, et al. Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis. Nat Methods. Dec. 2004;1(3):241-8. Epub Nov. 18, 2004.
Cutler, David J. ef 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.
Deamer, David W. 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)—http://www.nature.com/nbt/journal/v27/n4/abs/nbt.1530.html.
Dietrich, Rudiger.et al., “Gene assembly based on blunt-ended double-stranded DNA-modules”, Biotechnology Techniques, vol. 12, No. 1, 49-54 (Jan. 1998).
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. 2010, 327(5961):78-81.
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.
Eadie, et al. Guanine modification during chemical DNA synthesis. Nucleic Acids Res. Oct. 26, 1987;15(20):8333-49.
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.
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.
Engler, et al. Golden gate shuffling: a one-pot DNA shuffling method based on type lls restriction enzymes. PLoS One. 2009;4(5):e5553. doi: 10.1371/journal.pone.0005553. Epub May 14, 2009.
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, Olesya D. 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).
Fodor, et al. Light-directed, spatially addressable parallel chemical synthesis. Science. Feb. 15, 1991;251(4995):767-73.
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.
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. 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, Norbert et al., “Optical tweezing electroghoresis of isolated, highly charged colloidal spheres”, Colloids and Surfaces A: Physiochem. Eng. Aspec s, vol. 195, 227-241 (2001).
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, 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.
Gosse, Charlie et al. “Magnetic tweezers: micromanipulation and force measurement at the molecular level”, Biophysical Journal, vol. 8, 3314-3329 (Jun. 2002).
Grovenor. Microelectronic materials. CRC Press. 1989; 113-123.
Haber, Charbel et al., Magnetic tweezers for DNA micromanipulation, Rev. Sci. Instrum., vol. 71, No. 12, 4561-4570 (Dec. 2000).
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.
Heckers Karl H. et al., “Error analysis of chemically synthesized polynucleotides”, BioTechniques, vol. 24, No. 2, 255-260 (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).
Hosu, Basarab G. et al., Magnetic tweezers for intracellular applications⋅, Rev. Sci. Instrum., vol. 74, No. 9, 4158-4163 (Sep. 2003).
Huang, Hayden 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 Biotechnol. Apr. 2001;19(4):342-7.
Hutchison, et al. Cell-free cloning using phi29 DNA polymerase. Proc Natl Acad Sci U S A. Nov. 29, 2005;102(48):17332-6.
Jacobs and Schar, DNA glycosylases: In DNA repair and beyond. Chromosome, 121:1-20 (2012)—http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3260424/.
Jeffrey M. 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.
Ke, Song-Hua 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, Shana, et al. Single-base mismatch detection based on charge transduction through DNA, Nucleic Acids Research, vol. 27, No. 24, 4830-4837 (1999).
Kim, Yang-Gyun et al., “Chimeric restriction endonuclease”, Proc. Natl. Acad. Sci. USA, vol. 91, 883-887 (Feb. 1994).
Kim, Yang-Gyun, “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).
Kim, Yan˜Gyun et al., “Site specific cleavage of DNA-RNA hybrids by zinc finger/Fok I cleavage domain fusions” Gene, vol. 203, 43-49 (1997).
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.
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, Martin U. et al., “Chemical amplification: continuous-flow PCR on a chip”, Science, vol. 280, 1046-1048 (May 15, 1998).
Kosuri, et al. A scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nature Biotechnology. 2010; 28:1295-1299.
Lagally, E.T. et al., “Single-molecule DNA amplification and analysis in an integrated microfluidic device” Anal. Chem., vol. 73, No. , 565-570 (Feb. 1, 2001).
Lahue, R.S. et. al., “DNA mismatch correction in a defined system”, Science, vol. 425; No. 4914, 160-164 (Jul. 14, 1989).
Lambrinakos, A. et al., “Reactivity of potassium permanganate and tetraethylammonium chloride with mismatched bases and a simple mu ation detection protoco”,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, Matthew J. 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.
Leamon, et al. A massively parallel PicoTiterPlate based platform for discrete picoliter-scale polymerase chain reactions. Electrophoresis. Nov. 2003;24(21):3769-77.
Lee, Covalent end-immobilization of oligonucleotides onto solid surfaces. Thesis submitted to the Department of Chemical Engineering in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosopy in Chemical Engineering at the Massachusetts Institute of Technology. Aug. 2001, 315 pages.
Lee, C.S. et al., “Microelectromagnets for the control of magnetic nanoparticles”, Appl. Phys. Lett., vol. 79, No. 20, 3308-3310, 2001.
Lee, et al. A microfluidic oligonucleotide synthesizer. Nucleic Acids Research 2010 vol. 38(8):2514-2521. DOI: 10.1093/nar/gkq092.
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.
Lipshutz, Robert J. et al., “High density synthetic oligonucleotide arrays”, Nature Genetics Supplement, vol. 21, Jan. 20-24, 1999.
Lishanski, Alia 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. Enhanced Signals and Fast Nucleic Acid Hybridization by Microfluidic Chaotic Mixing. Angew. Chem. Int. Ed. 2006; 45:3618-3623.
Lizardi, et al. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat Genet. Jul. 1998;19(3):225-32.
Li, Lin et al., “Functional domains in Fok I restriction endonuclease”, Proc. Natl. Acad. Sci. USA, vol. 89, 4275-4279 (May 1992).
Lu, A.-Lien et al., “Methyl-directed repair of DNA base-pair mismatches in vitro”,Proc. Natl. Acad. Sci. USA, vol. 80, 4639-4643 (Aug. 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. 2012; 16:260-267.
Ma et al., Versatile surface functionalization of cyclic olefin copolymer (COC) with sputtered SiO2 thin film for potential BioMEMS applications. Journal of Materials Chemistry, DOI: 10.1039/b904663a, 11 pages (2009).
Margulies, et al. Genome sequencing in open microfabricated high-density picolitre reactors. Nature. Sep. 15, 2005;437(7057):376-80.
Matteucci, et al. Synthesis of deoxyoligonucleotides on a polymer support. J. Am. Chem. Soc. 1981; 103(11):3185-3191.
McGall, et al. Light-directed synthesis of high-density oligonucleotide arrays using semiconductor photoresists. Proc Natl Acad Sci U S A. Nov. 26, 1996;93(24):13555-60.
McGall, et al. The Efficiency of Light-Directed Synthesis of DNA Arrays on Glass Substrates. J. Am. Chem. Soc. 1997; 119(22):5081-5090.
Mendel-Hartvig. Padlock probes and rolling circle amplification. New possibilities for sensitive gene detection. Comprehensive Summaries of Uppsala Dissrtations from the Faculty of Medicine 1175. Uppsala University. 2002, 39 pages. http://www.diva-portal.org/smash/get/diva2:161926/FULLTEXT01.pdf.
Mitra, et al. In situ localized amplification and contact replication of many individual DNA molecules. Nucleic Acids Res. Dec. 15, 1999;27(24):e34.
Muller, Caroline et al. “Protection and labelling of thymidine by a fluorescent photolabile group”, Helvetica Chimica Acta, vol. 84, 3735-3741 (2001).
Nakatani, Kazuhiko et al., “Recognition of a single guanine bulge by 2-Acylamino-1 ,8-naphthyridine”, J. Am. Chem. Soc., vol. 122, 2172-2177 (2000).
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.
Ochman, et al. Genetic applications of an inverse polymerase chain reaction. Genetics. Nov. 1998;120(3):621-3.
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.
PCT Patent Application No. PCT/US14/049834 International Preliminary Report on Patentability dated Feb. 18, 2016.
PCT Patent Application No. PCT/US2015/043605 International Search Report and Written Opinion dated Jan. 6, 2016.
PCT Patent Application No. PCT/US2015/043605 Invitation to Pay Additional Fees dated Oct. 28, 2015.
PCT/US2014/049834 International Search Report and Written Opinion dated Mar. 19, 2015.
PCT/US2014/049834, Invitation to Pay Additional Fees and, where applicable, protest fee, dated Jan. 5, 2015.
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.
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, 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. 2007;132:65-85.
Pon. Solid-phase supports for oligonucleotide synthesis. Methods Mol Biol. 1993;20:465-96.
Eisen, Jonathan A., “A phylogenomic study of the MutS family of proteins”, Nucleic Acids Research, vol. 26, No. 18, 4291-4300 (1998).
Hoover et al., “DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis”, Nucleic Acids Research, vol. 30, No. 10, e43, 7 page (2002).
Jackson, Brian A. et al., “Recognition of DNA base mismatches by a rhodium intercalator”, J. Am. Chem. Soc., vol. 19, 12986⋅12987 (1997).
Prodromou, et al. Recursive PCR: a novel technique for total gene synthesis. Protein Eng. Dec. 1992;5(8):827-9.
Quan, et al. Parallel on-chip gene synthesis and application to optimization of protein expression. Nature Biotechnology. 2011; 29:449-452.
RF Electric discharge type excimer lamp. Products Catalog. Excimer lamp light source “flat excimer” (2.0 MB/PDf).—http://www.hamamatsu.com/jp/en/product/category/1001/3026/index.html, 2 pages, downloaded on Dec. 28, 2015.
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 FAQS and Ordering Guide. Roche Applied Science. Access Jan. 12, 2015, 37 pages. http://www.roche-diagnostics.ch/content/dam/corporate/roche-dia_ch/documents/broschueren/applied_science/biochemical-reagents-custom-biotech/cloning/04520599990_EN_EA_Restriction-Enzymes-FAQS-and-Ordering-Guide.pdf.
Ruminy, et al., “Long-range identification of hepatocyte nuclear factor-3 (FoxA) high and low-affinity binding Sites wit a chimeric nuclease”, J. Mol. Bioi., vol. 310, 523-535 (2001).
Saboulard, et al. High-throughput site-directed mutagenesis using oligonucleotides synthesized on DNA chips. Biotechniques. Sep. 2005;39(3):363-8.
Sacconi, L. et al., Three-dimendional 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.
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.
Schmalzing, Dieter et al., “Microchip electrophoresis: a method for high-speed SNP detection”, Nucleic Acids Research, vol. 28, No. 9, i-vi (2000).
Sierzchala, Agnieszka B. et al., “Solid-phase oligodeoxynucleotide synthesis: a two-step cycle using peroxy anion eprotection”, J. Am. Chem. Soc., vol. 125, No. 44, 13427-13441 (2003).
Singh-Gasson, Sangeet et al., Maskless fabrication of lijht-directed olxyonucleotide microarrays using a digital micromirror array, Nature Biotechnology, vol. 17, 974-978 (Oct. 1999).
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.
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, Jane et al., “Mutation detection with MutH, MutL, and MutS mismatch repair proteins”, Proc. Natl. Acad. Sci. USA, vol. 93, 4374-4379 (Apr. 1996).
Smith Jane et al., “Removal of Polymerase-Produced mutant sequences from PCR products”, Proc. Natl. Acad. Sci. USA, vol. 94, 6847-6850 (Jun. 1997).
Smith, Steven B. et al., “Direct mechanical measurements of the elasticity of single DNA molecules using magnetic beads”, Science, vol. 258, 1122-1126 (Nov. 13, 1992).
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.
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.
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, M. et al., “Magnetic trapping of multicomponent nanowires”, The Johns Hopkins University, Baltimore, Maryland, p. 1-3 (Jun. 25, 2001).
Tian, et al. Accurate multiplex gene synthesis from programmable DNA microchips. Nature. Dec. 23, 2004;432(7020):1050-4.
Unger, et al. Monolithic microfabricated valves and pumps by multilayer soft lithography. Science. Apr. 7, 2000;288(5463):113-6.
U.S. Appl. No. 14/452,429 Office Action dated Apr. 9, 2015.
PCT Patent Application No. PCT/US2016/016459 International Search Report and Written Opinion dated Apr. 13, 2016.
U.S. Appl. No. 14/452,429 Office Action dated Oct. 21, 2015.
U.S. Appl. No. 14/885,962 Restriction Requirment dated Mar. 1, 2016.
U.S. Appl. No. 14/885,963 Office Action dated Feb. 5, 2016.
U.S. Appl. No. 14/885,965 Office Action dated Feb. 18, 2016.
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.
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. 1998;67:99-134.
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 Joel 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.
Wah, David A. et al., “Structure of Fok I has implications for DNA cleavage”, Proc. Natl. Acad. Sci. USA, vol. 95, 10564-10569 (Sep. 1998).
Wah, David A. 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.
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, Adrian 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).
Wirtz, Denis, “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, Chrislaine 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, Richard D. 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.
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. Specificity of the nick-closing activity of bacteriophage T4 DNA ligase. Gene. 1989;76(2):245-54.
Wu, Xing-Zheng 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).
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. Jul. 7, 2004;32(12):e98.
Xiong, et al. Non-polymerase-cycling-assembly-based chemical gene synthesis: Strategies, methods, and progress. Biotechnology Advances. 2008; 26(2):121-134.
Yang, et al “Purification, cloning, and characterization of the CEL I nuclease”, Biochemistry, vol. 39, No. 13, 3533-351 (2000).
Youil, Rima et al., “Detection of 51 of 51 known mouse P.Giobin promoter mutations with T4 Endonuclease VII. The EMC Method”, Genomics, vol. 32, 431-435 (1996).
Young, et al. Two-step total gene synthesis method. Nucleic Acids Res. Apr. 15, 2004;32(7):e59.
Zheleznaya, et al. Nicking endonucleases. Biochemistry (Mosc). Dec. 2009;74(13):1457-66.
Arkles, Hydrophobicity, Hydrophilicity. Reprinted with permission from the Oct. 2006 issue of Paint & Coatings Industry magazine, Retrieved from the internet on Mar. 19, 2016, 10 pages.
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.
Fullwood et al., Next-generation DNA sequencing of paired-end tags [PET] for transcriptome and genome analysis Genome Research, 19:521-532, 2009.
Karagiannis and Ei-Osta, RNA interference and potential therapeutic applications of short interfering RNAs Cancer Gene Therapy, 12:787-795, 2005.
Kinde et al., Detection and quantification of rare mutations with massively parallel sequencing PNAS, 108(23):9530-9535, 2011.
Kosuri et al., A scalable gene synthesis platform using high-fidelity DNA microchips Nat. Biotechnol., 28(12):1295-1299, 2010.
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.
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.
Mei et al., Cell-free protein synthesis in microfluidic array devices Biotechnol. Prog. 23(6):1305-1311, 2007.
Meyers and Friedland, Knowledge-based simulation of genetic regulation in bacteriophage lambda Nucl. Acids Research, 12(1):1-16, 1984.
Nishikura, A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst Cell, 107:415-418, 2001.
PCT Patent Application No. PCT/US2016/016636 International Search Report and Written Opinion dated May 2, 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.
Tsai et al., Dimeric CRISPR RNA-guided Fokl nucleases for highly specific genome editing Nat. Biotechnol., 32(6):569-576, 2014.
Yehezkel et al., De novo DNA synthesis using single molecule PCR Nucleic Acids Research, 36(17):e107, 2008.
Zhou et al., Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences Nucleic Acids Research, 32(18):5409-5417, 2004.
Saaem et al., In situ synthesis of DNA microarray on functionalized cyclic olefin copolymer substrate ACS Applied Materials & Interfaces, 2(2):491-497, 2010.
Andoni and Indyk, Near-Optimal Hashing Algorithms for Approximate Nearest Neighbor in High Dimensions, Communications of the ACM, 51(1):117-122, Jan. 2008.
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.
Barton et al., A desk electrohydrodynamic jet printing system. Mechatronics, 20:611-616, 2010.
Blanchard, et al., “High-Density Oligonucleotide Arrays,” Biosensors & Bioelectronics, 11(617):687-690, 1996.
Blawat et al., Forward error correction for DNA data storage. Procedia Computer Science, 80:1011-1022, 2016.
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.
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.
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. [Accessed: May 25, 2017], 10 pages.
Caruthers, The Chemical Synthesis of DNA/RNA: Our Gift to Science. J. Biol. Chem., 288(2):1420-1427, 2013.
Chen et al., Programmable chemical controllers made from DNA, Nat. Nanotechnol., 8(10):755-762, 2013.
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 n s tu oligonucleotide synthesis,” Nature Methods, 1(13):241-248, 2004.
Cohen et al., Human population: The next half century. Science, 302:1172-1175, 2003.
Elsik et al., The Genome sequence of taurine cattle: A window of ruminant biology and evolution. Science, 324:522-528, 2009.
Erlich and Zielinski, DNA fountain enables a robust and efficient storage architecture. Science, 355(6328):950-054, 2017.
European Patent Application No. 14834665.3 extended European Search Report dated Apr. 28, 2017.
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.
GeneArt Seamless Cloning and Assembly Kits. Life Technologies Synthetic Biology. 8 pages, available online Jun. 15, 2012.
Gibson Assembly. Product Listing. Application Overview. 2 pages, available online Dec. 16, 2014.
Goldman et al., Towards practical, high-capacity, low-maintenance information storage in synthesized DNA, Nature, 494(7435):77-80, 2013.
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.
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.
In-Fusion Cloning: Accuracy, Not Background. Cloning & Competent Cells, ClonTech Laboratories, 3 pages, available online Jul. 6, 2014.
Jinek et al., A Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337:816-821, 2012.
Kim et al., High-resolution patterns of quantum dots formed by electrohydrodynamic jet printing for light-emitting diodes. Nano Letters, 15:969-973, 2015.
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.
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.
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.gov/pmc/articles/PMC507883/.
Leproust et al., “Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process,” Nucleic Acids Research, 35(8):2522-2540, 2010.
Lewontin and Harti, Population genetics in forensic DNA typing. Science, 254:1745-1750, 1991.
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.
Liu et al., Rational design of CXCR4 specific antibodies with elongated CDRs. JACS, 136:10557-10560, 2014.
McBride & Caruthers, “An investigation of several deoxynucleoside phosphoramidites useful for synthesizing deoxyoligonucleotides.” Tetrahedron Lett. 24: 245-248, 1983.
Milo and Phillips, Numbers here reflect the number of protein coding genes and excludes tRNA and non-coding RNA. Cell Biology by the Numbers, p. 286, 2015.
Neiman M.S,. Negentropy principle in information processing systems. Radiotekhnika, 1966, Nº11, 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.
Organick et al., Scaling up DNA data storage and random access retrieval, bioRxiv, preprint first posted online Mar. 7, 2017, 14 pages.
PCT Patent Application No. PCT/US2015/043605 International Preliminary Report on Patentability dated Feb. 16, 2017.
PCT Patent Application No. PCT/US2016/052336 International Search Report and Written Opinion dated Dec. 7, 2016.
PCT Patent Application No. PCT/US2016/052916 International Search Report and Written Opinion dated Dec. 30, 2016.
PCT/US2016/064270 International Search Report and Written Opinion dated Apr. 28, 2017.
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.
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.
Rastegari, et al., XNOR-Net: ImageNet Classification Using Binary Convolutional Neural Networks, in ECCV 2016, Part IV, LNCS 9908, p. 525-542, 2016.
Sargolzaei et al., Extent of linkage disequilibrium in Holstein cattle in North America. J.Dairy Science, 91:2106-2117, 2007.
Seelig, et al., Enzyme-Free Nucleic Acid Logic Circuits, Science 314(5805):1585-1588, 2006.
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.
The Hood Laboratory, “Beta Group.” Assembly Manual for the POSaM: The ISB Piezoelelctric Oligonucleotide Synthesizer and Microarrayer, Inkjet Microarrayer Manual Version 1.2, 50 pages, May 28, 2004.
The SLIC, Gibson, CPEC and SLiCE assembly methods (and GeneArt Seamless, In-Fusion Cloning). 5 pages, available online Sep. 2, 2010.
U.S. Appl. No. 14/241,874 Office Action dated Feb. 27, 2017.
U.S. Appl. No. 14/885,962 Office Action dated Dec. 16, 2016.
U.S. Appl. No. 14/885,965 Office Action dated Feb. 10, 2017.
U.S. Appl. No. 15/154,879 Notice of Allowance dated Feb. 1, 2017.
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 Office Action dated Feb. 8, 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. 15/377,547 Office Action dated Mar. 24, 2017.
Van Tassell et al., SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries. Nature Methods, 5:247-252, 2008.
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.
Wijshoff, Herman. Structure and fluid-dynamics in Piezo inkjet printheads. Thesis. Venio, The Netherlands, published 2008, p. 1-185.
Wright and Church, An open-source oligomicroarray standard for human and mouse. Nature Biotechnology, 20:1082-1083, 2002.
Xiong et al., Chemical gene synthesis: Strategies, softwares, error corrections, and applications. FEMS Microbiol. Rev., 32:522-540, 2008.
Yazdi, et al., A Rewritable, Random-Access DNA-Based Storage System, Scientific Reports, 5, Article No. 14138, 27 pages, 2015.
Zhang and Seelig, Dynamic DNA nanotechnology using strand-displacement reactions, Nat. Chem., 3(2):103-113, 2011.
Zhirnov et al., Nucleic acid memory. Nature Materials, 15:366, 2016.
European Patent Application No. 16871446.7 European Search Report dated Apr. 10, 2019.
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).
International Application No. PCT/US2017/052305 International Preliminary Report on Patentability dated Apr. 30, 2019.
International Application No. PCT/US2017/062391 International Preliminary Report on Patentability dated May 21, 2019.
International Application No. PCT/US2018/019268 International Preliminary Report on Patentability dated Sep. 6, 2019.
International Application No. PCT/US2018/057857 International Search Report and Written Opinion dated Mar. 18, 2019.
International Application No. PCT/US2019/032992 International Search Report and Written Opinion dated Oct. 28, 2019.
International Application No. PCT/US2019/032992 Invitation to Pay Additional Fees dated Sep. 6, 2019.
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).
U.S. Appl. No. 15/151,316 Office Action dated Oct. 4, 2019.
U.S. Appl. No. 15/187,714 Final Office Action dated Sep. 17, 2019.
U.S. Appl. No. 15/268,422 Final Office Action dated Oct. 3, 2019.
U.S. Appl. No. 15/602,991 Office Action dated May 31, 2019.
U.S. Appl. No. 15/603,013 Final Office Action dated Nov. 6, 2019.
U.S. Appl. No. 15/619,322 Office Action dated Aug. 14, 2019.
U.S. Appl. No. 15/709,274 Notice of Allowance dated Apr. 3, 2019.
U.S. Appl. No. 15/729,564 Office Action dated May 30, 2019.
U.S. Appl. No. 15/816,995 Office Action dated Sep. 20, 2019.
U.S. Appl. No. 15/835,243 Restriction Requirement dated Sep. 10, 2019.
U.S. Appl. No. 15/844,395 Restriction Requirement dated May 17, 2019.
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. 15/960,319 Office Action dated Aug. 16, 2019.
U.S. Appl. No. 16/006,581 Office Action dated Sep. 25, 2019.
U.S. Appl. No. 16/239,453 Office Action dated Nov. 7, 2019.
U.S. Appl. No. 16/409,608 Office Action dated Sep. 9, 2019.
U.S. Appl. No. 16/530,717 Office Action dated Sep. 6, 2019.
U.S. Appl. No. 15/603,013 Office Action dated Jun. 26, 2019.
Related Publications (1)
Number Date Country
20160229884 A1 Aug 2016 US
Provisional Applications (1)
Number Date Country
62112083 Feb 2015 US