High efficiency, small volume nucleic acid synthesis

Abstract

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes).

Description

HIGH EFFICIENCY, SMALL VOLUME NUCLEIC ACID SYNTHESIS FIELD OF THE INVENTION

[0001] The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules, as disclosed in U.S. Application No. 13 / 627,819. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes). BACKGROUND

[0002] Production of nucleic acid molecules can be fairly simple or complex depending on factors such as the type of nucleic acid molecules to be produced. For example, historically, short single stranded nucleic acid molecules such as primers have been typically generated by chemical synthesis (see, e.g., U.S. Patent No. 5,837,858. Further, longer nucleic acid molecules have typically been generated by polymerase chain reaction (PCR). One disadvantage of PCR is that generally template nucleic acid is required.

[0003] Many nucleic acid synthesis methods have limited capabilities for the generation of large de novo nucleic acid molecules. One aspect of the current disclosure is to address this limitation.

[0004] Furthermore, nucleic acid molecules that are used for gene synthesis are usually produced using expensive automated machines with limited throughput. For this reason, alternative approaches are being investigated, such as the use of microarrays as a source for nucleic acid molecules for gene synthesis. Microarrays can have hundreds of thousands of different nucleic acid molecules on a small surface and can be fabricated at very low cost.

[0005] However, approaches to nucleic acid molecule synthesis that are based on microarrays may suffer from drawbacks such as low amounts of nucleic acid molecules produced per spot. This may not be problematic for the use of hybridization assays, the application for which two-dimensional microarrays were initially developed. However, when two-dimensional microarrays are used for gene synthesis (i.e., nucleic acid molecules are fabricated on a planar surface) they may lack at least two important features. First, the quantity of nucleic acid molecules may not be large enough to assemble a fragment, such as a fragment that can be used for gene assembly. In certain instances only attomoles may be obtained. For this reason, the nucleic acid molecules generated typically need to be copied by PCR to reach quantities that are useful for fragment assembly. Moreover, the quantity of nucleic acid molecules may be further reduced by synthesis reagents, such as acids, that can act to degrade the nucleic acid molecules after synthesis.

[0006] Second, it is difficult to release synthesized nucleic acid molecules from microarrays individually, or in pools needed for the assembly of one fragment. Rather, the nucleic acid molecules are typically released together, often resulting in complex pools of thousands of different nucleic acid molecules that may not be amenable for gene synthesis without post-processing. Complicated processes like dial-out PCR or sequencing for identification and amplification of the desired nucleic acid molecules are thus often needed to make use of these pools. SUMMARY OF THE INVENTION

[0007] The invention relates, in part, to compositions and methods for the synthesis of nucleic acid molecules. The invention further relates to compositions and methods for the retrieval of synthesized nucleic acid molecules, as well as compositions and methods for the assembly of nucleic acid molecules to form molecules such as plasmids, chromosomes and genomes.

[0008] In some aspects, the invention relates to multiwell plates for non-template directed synthesis of nucleic acid molecules. In some embodiments, the plate comprises a bead (e.g., a magnetic bead) located in each of a plurality of wells of the plate and an electrochemically generated acid (EGA) being present in one or more of the plurality of wells. Instead of or in addition to having EGA in one or more wells, wells of the plate may contain other reagents set out elsewhere associated with the synthesis of nucleic acid molecules. In certain embodiments disclosed herein, a photogenerated acid (PGA) may be present in one or more of the plurality of wells instead of or in addition to an EGA. The EGA or PGA is used to remove the protecting group (e.g., DMT) before the next amidite is added to the nucleic acid molecule attached to the solid support. In some embodiments, at least one proton carrier, such as 2-chloro-6- methylpyridine or diphenylamine, may be present in the solution with the EGA or PGA. The at least one proton carrier may act to reduce the effect of DNA degradation by accepting protons from the EGA or PGA, thereby adjusting the acidity of the solution.

[0009] In one aspect, the PGA is used in methods of generating assembled nucleic acids, the methods comprising a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a well of a plate or a microchip, wherein the well is operably connected to a light source for the production of a PGA and optionally contains a proton carrier, such as 2-chloro-6-methylpyridine or diphenylamine; b) combining some or all of the nucleic acid molecules generated in (a) to produce a pool; c) joining some or all of the nucleic acid molecules present in the pool formed in (b) to form a plurality of larger nucleic acid molecules; d) eliminating nucleic acid molecules which contain sequence errors from the plurality of larger nucleic acid molecules formed in (c) to produce an error corrected nucleic acid molecule pool; and e) assembling the nucleic acid molecules in the error corrected nucleic acid molecule pool to form the assembled nucleic acid molecule. Bead sizes used in the practice of the invention may vary widely but include beads with diameters between 0.01 μm and 100 μm, 0.005 μm and 100 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m, 0.005 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m and 10 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m, 0.01 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m and 100 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m, 0.01 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m and 1,000 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m, between 1.0 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m and 2.0 μm, between 1.0 μm and 100 μm, between 2.0 μm and 100 μm, between 3.0 μm and 100 μm, between 0.5 μm and 50 μm, between 0.5 μm and 20 μm, between 1.0 μm and 10 μm, between 1.0 μm and 20 μm, between 1.0 μm and 30 μm, between 10 μm and 40 μm, between 10 μm and 60 μm, between 10 μm and 80 μm, or between 0.5 μm and 10 μm. In certain embodiments, the beads may have a diameter between 30 μm and 40 μm, such as a diameter of 31 μm or 32 μm or 33 μm or 34 μm or 35 μm. As one skilled in the art would recognize, when solid particles fall below a particular size, they begin to acquire attributes of fluids (e.g., form the equivalent of colloidal suspensions). Thus, in some instances (e.g., with the use of beads below about 100 nm in diameter), it may be desirable to treat the bead as a fluid. This may mean removal of a bead from a surface, a well or from a magnetic tip, for example, by agitation, washing, or with the use of a surfactant.

[0010] In specific embodiments of the invention, the bead size may be chosen depending on the size of the well to allow only one single bead to occupy a well. In other embodiments, more than one bead (or nucleic acid synthesis substrates of other shapes) may be in some or all of the wells. In some instances, the number of beads per well may be one, between two and twenty, between two and thirty, between two and ten, between four and twenty, between four and ten, between four and fifty, etc.

[0011] In certain embodiments, each well of the multiwell plate for synthesizing nucleic acids is configured to accommodate a monodisperse bead having a diameter of about 35-40 µm, or about 35 µm or about 29 to 33 µm. In certain embodiments, the monodisperse bead is composed of a synthetic polymer, such as polystyrene.

[0012] The number of wells may also vary widely and is limited by factors such as the amount of nucleic acid to be produced, time constraints, economic factors, and technical factors such as manufacturability and mechanic factors related to use (e.g., the lower size limit of (magnetic) bead extractors). Thus, depending on various factors, the desired synthetic scale may vary and the number of wells can be adjusted to accommodate the desired synthetic scale. In any event, the number of wells may be in number, for example, between 10 and 10,000,000, between 10 and 5,000,000, between 10 and 2,000,000, between 10 and 1,000,000, between 10 and 800,000, between 10 and 650,000, between 10 and 500,000, between 500 and 500,000, between 500 and 200,000, between 10 and 50,000, between 1,000 and 500,000, between 10,000 and 500,000, between 20,000 and 500,000, between 1,000 and 50,000, between 10 and 50,000, between 10 and 25,000, between 10 and 10,000, between 10 and 5,000, between 10 and 2,500, between 100 and 50,000, between 100 and 25,000, between 100 and 10,000, between 100 and 5,000, between 100 and 2,500, between 350 and 50,000, between 350 and 25,000, between 350 and 10,000, between 350 and 5,000, between 350 and 2,500, between 1000 and 50,000, between 1000 and 25,000, between 1000 and 10,000, between 1000 and 5,000, between 1000 and 2,500, between 1,500 and 50,000, between 1,500 and 25,000, between 1,500 and 10,000, between 1,500 and 5,000, between 1500 and 2,500, between 20,000 and 50,000, between 20,000 and 40,000, or about 35,000. In certain embodiments, the number of wells may be, for example, 35,440. In certain other exemplary embodiments, the number of wells may be, for example, about 196,000, such as 196,160, or, for example, about 9,000, such as 9020. Further, multiwell surfaces have been prepared with wells numbering in the range of 10 million. Thus, under some instances, the number of wells may be less than 5 million, 10 million, 20 million, etc.

[0013] The total volume of each well is another item which may vary and may be, for example, between <semantics>1.0×10−9μl<annotation encoding="application / x-tex">1.0 \times 10^{-9} \mu l< / annotation>< / semantics> and <semantics>50μl<annotation encoding="application / x-tex">50 \mu l< / annotation>< / semantics>, between <semantics>1.0×10−9μl<annotation encoding="application / x-tex">1.0 \times 10^{-9} \mu l< / annotation>< / semantics> and <semantics>10μl<annotation encoding="application / x-tex">10 \mu l< / annotation>< / semantics>, between <semantics>1.0×10−9μl<annotation encoding="application / x-tex">1.0 \times 10^{-9} \mu l< / annotation>< / semantics> and 1.0 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l, between 1.0 x 10-9 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l and 0.1 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l, between 1.0 x 10-9 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l and 1.0 x 10-2 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l, between 1.0 <semantics>×10−9μl<annotation encoding="application / x-tex">\times 10^{-9} \,\mu l< / annotation>< / semantics> and <semantics>1.0×10−3μl<annotation encoding="application / x-tex">1.0 \times 10^{-3} \,\mu l< / annotation>< / semantics>, between <semantics>1.0×10−9μl<annotation encoding="application / x-tex">1.0 \times 10^{-9} \,\mu l< / annotation>< / semantics> and <semantics>1.0×10−4μl<annotation encoding="application / x-tex">1.0 \times 10^{-4} \,\mu l< / annotation>< / semantics>, between <semantics>1.0×10−9μl<annotation encoding="application / x-tex">1.0 \times 10^{-9} \,\mu l< / annotation>< / semantics> and <semantics>50<annotation encoding="application / x-tex">50< / annotation>< / semantics> <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l, between 1.0 x 10-5 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l and 1.0 x 10-6 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l, between 1.0 x 10-9 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l and 1.0 x 10-7 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l, between 2.5 x <semantics>10−9<annotation encoding="application / x-tex">10^{-9}< / annotation>< / semantics> μl and <semantics>1.0×10−2<annotation encoding="application / x-tex">1.0 \times 10^{-2}< / annotation>< / semantics> μl, between <semantics>2.5×10−9<annotation encoding="application / x-tex">2.5 \times 10^{-9}< / annotation>< / semantics> μl and <semantics>1.0×10−3<annotation encoding="application / x-tex">1.0 \times 10^{-3}< / annotation>< / semantics> μl, between <semantics>2.5×10−9<annotation encoding="application / x-tex">2.5 \times 10^{-9}< / annotation>< / semantics> μl and <semantics>1.0×10−9<annotation encoding="application / x-tex">1.0 \times 10^{-9}< / annotation>< / semantics> μl and <semantics>1.0×10−9<annotation encoding="application / x-tex">1.0 \times 10^{-9}< / annotation>< / semantics> μl and <semantics>1.0×10−9<annotation encoding="application / x-tex">1.0 \times 10^{-9}< / annotation>< / semantics> μl and <semantics>1.0×10−9<annotation encoding="application / x-tex">1.0 \times 10^{-9}< / annotation>< / semantics> μl and <semantics>1.0×10<annotation encoding="application / x-tex">1.0 \times 10< / annotation>< / semantics> <semantics>10−4<annotation encoding="application / x-tex">10^{-4}< / annotation>< / semantics> μl, between <semantics>2.5×10−9<annotation encoding="application / x-tex">2.5 \times 10^{-9}< / annotation>< / semantics> μl and <semantics>1.0×10−5<annotation encoding="application / x-tex">1.0 \times 10^{-5}< / annotation>< / semantics> μl, between <semantics>2.5×10−9<annotation encoding="application / x-tex">2.5 \times 10^{-9}< / annotation>< / semantics> μl and <semantics>1.0×10−6<annotation encoding="application / x-tex">1.0 \times 10^{-6}< / annotation>< / semantics> μl, between <semantics>1.0×10−8μl<annotation encoding="application / x-tex">1.0 \times 10^{-8} \mu l< / annotation>< / semantics> and <semantics>1.0×10−6μl<annotation encoding="application / x-tex">1.0 \times 10^{-6} \mu l< / annotation>< / semantics>, between <semantics>1.0×10−8μl<annotation encoding="application / x-tex">1.0 \times 10^{-8} \mu l< / annotation>< / semantics> and <semantics>1.0×10−5μl<annotation encoding="application / x-tex">1.0 \times 10^{-5} \mu l< / annotation>< / semantics>, between <semantics>1.0×10−7μl<annotation encoding="application / x-tex">1.0 \times 10^{-7} \mu l< / annotation>< / semantics> and <semantics>1.0×10−5μl<annotation encoding="application / x-tex">1.0 \times 10^{-5} \mu l< / annotation>< / semantics>, between <semantics>1.0×10−7μl<annotation encoding="application / x-tex">1.0 \times 10^{-7} \mu l< / annotation>< / semantics> and <semantics>1.0×10−4μl<annotation encoding="application / x-tex">1.0 \times 10^{-4} \mu l< / annotation>< / semantics>, between <semantics>1.0×10−7μl<annotation encoding="application / x-tex">1.0 \times 10^{-7} \mu l< / annotation>< / semantics> and <semantics>1.0×10−3μl<annotation encoding="application / x-tex">1.0 \times 10^{-3} \mu l< / annotation>< / semantics>, between <semantics>1.0×10−7μl<annotation encoding="application / x-tex">1.0 \times 10^{-7} \mu l< / annotation>< / semantics> and <semantics>1.0×10−2μl<annotation encoding="application / x-tex">1.0 \times 10^{-2} \mu l< / annotation>< / semantics>, between <semantics>0.1μl<annotation encoding="application / x-tex">0.1 \mu l< / annotation>< / semantics> and <semantics>50μl<annotation encoding="application / x-tex">50 \mu l< / annotation>< / semantics>, between <semantics>0.01μl<annotation encoding="application / x-tex">0.01 \mu l< / annotation>< / semantics> and <semantics>50μl<annotation encoding="application / x-tex">50 \mu l< / annotation>< / semantics>, between 0.01 µl and 25 µl, between 0.01 µl and 15 µl, between 0.01 µl and 10 µl, between 0.001 μl and 50 μl, between 0.001 μl and 5 μl, between 0.001 μl and 1 μl, between 0.001 μl and 0.01 μl, between 0.001 μl and 1 μl, between 1 μl and 50 μl, between 1 μl and 25 μl, between 1 μl and 10 μl, between 10 μl and 50 μl, between 10 μl and 25 μl, or between 15 μl and 25 μl. In certain exemplary embodiments, the total volume of each well may be between 1 x 10-6 µl and 1 x 10-4 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l, between 1 x 10-5 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l and 1 x 10-4 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l, such as about 6.3 x 10-5 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l.

[0014] In many instances, multiwell plates of the invention or multiwell plates suitable for use with the invention will be operably connected to either one electrode or a set (e.g., one or several pairs) of electrodes. As discussed elsewhere herein, these electrodes can be used to generate a microenvironment associated with catalysis of one or more chemical reactions (e.g., EGA for nucleotide deprotection or EGB for cleavage of a nucleic acid molecule from solid support). Further, these electrodes, when arranged at or near a bottom portion of an individual well, can be used to generate a mechanism for bead removal for the wells, e.g., electrolysis, as discussed further below.

[0015] In certain instances, the multiwell plate disclosed herein may be operably connected to at least one light source, such as a fiber optic device. As disclosed herein, the at least one light source can be used to selectively generate a photogenerated acid (PGA) in one or more wells of the multiwell plate. The PGA is used in turn to selectively deprotect the terminal nucleotide attached to a bead or other suitable solid support in the one or more wells of the multiwell plate.

[0016] In some embodiments, multiwell plates of the invention or multiwell plates suitable for use with the invention will be connected to microfluidic channels for the introduction and removal of reagents. This allows for efficient and automated controlling of reagents.

[0017] In some embodiments, the invention provides for methods of retrieving nucleic acids linked to solid supports by a base-cleavable linker comprising: a) generating an electrochemically generated base; b) cleaving the nucleic acid from the solid support with the electrochemically generated base in an aqueous or organic solution; c) contacting the cleaved nucleic acid with a material for retaining the nucleic acid; and d) eluting the nucleic acid with an agent for removing a protecting group from the nucleic acid, optionally wherein the eluting may occur in a small volume to concentrate and / or enrich the nucleic acids. For example, elution may occur in a volume of about 1 to about 10 µl, such as e.g. about 5 µl. Alternatively, step d) may comprise eluting the nucleic acid in a solvent without removing the protecting groups and may be followed by an additional step e) comprising removing protecting groups from the nucleic acid.

[0018] The invention further provides methods for retrieving nucleic acid molecules from multiwell plates for non-directed synthesis of nucleic acid molecules, the method comprising: a) synthesizing a first copy of a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a well of a first multiwell plate in an average amount of from about 50 femtomoles to about 15,000 femtomoles; b) synthesizing a second copy of the same plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a well of a second multiwell plate in an average amount of from about 50 femtomoles to about 15,000 femtomoles; c) deprotecting and cleaving the first copy of the plurality of nucleic acid molecules from the first multiwell plate; d) deprotecting the second copy of the plurality of nucleic acid molecules from the second multiwell plate; e) contacting the first copy of the plurality of nucleic acid molecules with the second copy of the plurality of nucleic acid molecules under hybridizing conditions to generate hybridized nucleic acid molecules; f) denaturing the hybridized nucleic acid molecules by adding a denaturing solution to the second multiwell plate; and g) retrieving the denatured nucleic acid molecules from the second multiwell plate.

[0019] In some embodiments, the invention provides for methods of cleaving nucleic acids linked to solid supports by photocleavable linkers comprising generating lightwaves from a light source and cleaving the nucleic acid from the solid support with the lightwaves. In other exemplary embodiments, the invention provides for methods of cleaving nucleic acids linked to solid supports by reductive cleavable linkers comprising generating an electrochemically reduced compound and cleaving the nucleic acid from the solid support with the electrochemically reduced compound. After the nucleic acids have been cleaved from the solid support, they may be retrieved by the methods disclosed herein. As is also disclosed herein, in certain embodiments, the nucleic acids together with the solid supports may first be retrieved before cleavage.

[0020] The invention also provides methods for the generation of assembled nucleic acid molecules formed from smaller chemically synthesized nucleic acid molecules. In some embodiments, such methods may comprise one or more of the following steps: (a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a microquantity in the well of a plate; (b) combining the nucleic acid molecules generated in (a), or a portion thereof, to produce a pool; (c) joining some or all of the nucleic acid molecules present in the pool formed in (b) to form a plurality of larger nucleic acid molecules; (d) eliminating nucleic acid molecules which contain sequence errors from the plurality of larger nucleic acid molecules formed in (c) to produce an error corrected nucleic acid molecule pool; and (e) assembling the nucleic acid molecules in the error corrected nucleic acid molecule pool to form the assembled nucleic acid molecule.

[0021] In some embodiments, the joining of nucleic acid molecules present in the pool will be mediated by polymerase chain reaction (PCR).

[0022] In some embodiments step (b) may further comprise combining nucleic acid molecules generated in (a) with nucleic acid molecules obtained by other means to form a pool, wherein said other means include PCR, restriction enzyme digest, exonuclease treatment, or template-independent synthesis using a nucleotidyl transferase enzyme. In some instances, the assembled nucleic acid molecule generated in (c) and / or (e) may be assembled and introduced into a vector (e.g., a cloning vector, a destination vector, etc.).

[0023] The number of nucleic acid molecules assembled by methods of the invention can vary and, when appropriate, will correlate with the number of pooled nucleic acid molecules. In any event, nucleic acid molecules assembled in methods of the invention may be composed of at least five other (e.g., smaller) nucleic acid molecules (e.g., from about five to about five thousand, from about five to about twenty thousand, from about five to about one hundred thousand, from about fifty to about five thousand, from about fifty to about twenty thousand, from about fifty to about one hundred thousand, from about one hundred to about five thousand, from about one hundred to about one hundred thousand, from about five hundred to about five thousand, from about five hundred to about one hundred thousand, etc. nucleic acid molecules).

[0024] Nucleic acid molecules assembled by methods of the invention may vary greatly and include molecules of at least 20 kilobases (e.g., between from about 0.5 kilobase and to about 10 megabases, between from about 0.5 kilobase and to about 5 megabases, between from about 0.5 kilobase and to about 1 megabase, between from about 0.5 kilobase and to about 500 kilobases, between from about 0.5 kilobase and to about 100 kilobases, between from about 0.5 kilobase and to about 10 megabases, between from about 0.5 kilobase and to about 1 kilobase, between from about 1 kilobase and to about 10 megabases, between from about 10 kilobases and to about 5 megabases, between from about 1 kilobase and to about 5 megabases, between from about 1 kilobase and to about 2 megabases, between from about 1 kilobase and to about 1 megabase, between from about 1 kilobase and to about 500 kilobases, between from about 10 kilobases and to about 1 megabases, between from about 10 kilobase and to about 500 kilobases, between from about 10 kilobase and to about 100 kilobases, etc.).

[0025] Nucleic acid molecule assembled by methods of the invention may be, for example, single stranded, partly single stranded or double stranded, closed, circular (e.g., a plasmid); nicked, circular; or linear (e.g., a plasmid, a chromosome, etc.). Further, methods of the invention may be performed such that two or more (e.g., two, three, four, five, six, ten, twenty, etc.) assembled nucleic acid molecules are simultaneously formed in the same reaction mixture.

[0026] The invention further provides methods for producing product nucleic acid molecules. In some instances such the methods comprise: (a) designing a product nucleic acid molecule of between 10 kilobases and 500 kilobases in size (e.g., between 500 bases and 500 kilobases, between 500 bases and 100 kilobases, between 500 bases and 1 kilobase, between 500 bases and 800 bases between 2 kilobases and 100 kilobases, between 2 kilobases and 50 kilobases, between 2 kilobases and 5 kilobases, between 10 kilobases and 500 kilobases, between 10 kilobases and 300 kilobases, between 10 kilobases and 200 kilobases, between 10 kilobases and 100 kilobases, between 10 kilobases and 50 kilobases, etc.), wherein the product nucleic acid molecule is defined by nucleotide sequence; (b) synthesizing a plurality of individual nucleic acid molecules which differ in nucleotide sequence, wherein each individual nucleic acid molecule is synthesized to prepare a quantity of between 1,000 and <semantics>1.0×109<annotation encoding="application / x-tex">1.0 \times 10^9< / annotation>< / semantics> copies and wherein the individual nucleic acid molecules are capable of hybridizing with one or more of the other individual nucleic acid molecules; (c) combining the individual nucleic acid molecules synthesized in (b) under conditions which allow for hybridization of the individual nucleic acid molecules under conditions which allow for the formation of at least one larger nucleic acid molecule; and (d) combining the at least one larger nucleic acid molecule formed in (c) with one or more additional nucleic acid molecules to form the product nucleic acid molecule, wherein the product nucleic acid molecule contains less than one sequence error per kilobase.

[0027] In many instances, an error correction process is employed during generation of product nucleic acid molecules. One place in the above work flow where an error correction process may be performed is after step (b). Error correction processes are described elsewhere herein and will often include the use of one or more mis-match repair endonucleases.

[0028] The number of individual nucleic acid molecules synthesized as part of the preparation of product nucleic acid molecules may vary greatly but include between 1,000 and <semantics>1.0×1013<annotation encoding="application / x-tex">1.0 \times 10^{13}< / annotation>< / semantics> copies, between 1,000 and <semantics>1.0×1012<annotation encoding="application / x-tex">1.0 \times 10^{12}< / annotation>< / semantics> copies, between 1,000 and <semantics>1.0×1011<annotation encoding="application / x-tex">1.0 \times 10^{11}< / annotation>< / semantics> copies, between 1,000 and <semantics>1.0×1010<annotation encoding="application / x-tex">1.0 \times 10^{10}< / annotation>< / semantics> copies, between 1,000 and <semantics>1.0×109<annotation encoding="application / x-tex">1.0 \times 10^9< / annotation>< / semantics> copies, between <semantics>2.0×109<annotation encoding="application / x-tex">2.0 \times 10^9< / annotation>< / semantics> and <semantics>1.0×1013<annotation encoding="application / x-tex">1.0 \times 10^{13}< / annotation>< / semantics> copies, between <semantics>5.0×109<annotation encoding="application / x-tex">5.0 \times 10^{9}< / annotation>< / semantics> and <semantics>1.0×1013<annotation encoding="application / x-tex">1.0 \times 10^{13}< / annotation>< / semantics> copies, between <semantics>7.0×109<annotation encoding="application / x-tex">7.0 \times 10^{9}< / annotation>< / semantics> and <semantics>1.0×1013<annotation encoding="application / x-tex">1.0 \times 10^{13}< / annotation>< / semantics> copies, between <semantics>2.0×109<annotation encoding="application / x-tex">2.0 \times 10^9< / annotation>< / semantics> and <semantics>8.0×1012<annotation encoding="application / x-tex">8.0 \times 10^{12}< / annotation>< / semantics> copies, between <semantics>2.0×109<annotation encoding="application / x-tex">2.0 \times 10^9< / annotation>< / semantics> and <semantics>5.0×1012<annotation encoding="application / x-tex">5.0 \times 10^{12}< / annotation>< / semantics> copies, between <semantics>5.0×106<annotation encoding="application / x-tex">5.0 \times 10^6< / annotation>< / semantics> and <semantics>1.0×1011<annotation encoding="application / x-tex">1.0 \times 10^{11}< / annotation>< / semantics> copies, between <semantics>1.0×107<annotation encoding="application / x-tex">1.0 \times 10^7< / annotation>< / semantics> and <semantics>1.0×1011<annotation encoding="application / x-tex">1.0 \times 10^{11}< / annotation>< / semantics> copies, between <semantics>1.0×109<annotation encoding="application / x-tex">1.0 \times 10^9< / annotation>< / semantics> and <semantics>1.0×1011<annotation encoding="application / x-tex">1.0 \times 10^{11}< / annotation>< / semantics> copies, etc.

[0029] In many instances, polymerase chain reactions may be used to amplify the at least one larger nucleic acid molecule formed in step (c) in the above product nucleic acid molecule preparation processes.

[0030] Plate formats for the synthesis of nucleic acid molecules are described elsewhere herein and they may be used in the above product nucleic acid molecule preparation processes. Further, when individual nucleic acid molecules are synthesized on beads, wherein each bead may be contained in a well. Further, beads used in this aspect of the invention, as well as other aspects of the invention may be, for example of sizes such as between 1 µm and 100 µm in diameter, between 5 µm and 50 µm in diameter, between 3 µm and 100 µm in diameter, between 5 μm and 100 μm in diameter, between 20 μm and 100 μm in diameter, between 5 μm and 60 μm in diameter, between 10 µm and 100 µm in diameter, etc. In some embodiments beads may be of a size of about 30 μm in diameter (e.g., between 28 and 32 μm). In some embodiments, beads may range from about 30 μm to about 40 μm in diameter such as about 35 μm in diameter.

[0031] The invention also includes methods for producing nucleic acid molecule in small amounts and with high sequence fidelity. In some aspects, the invention includes a method for generating a nucleic acid molecule, the method comprising synthesizing the nucleic acid molecule in a total amount of between <semantics>1×1011<annotation encoding="application / x-tex">1 \times 10^{11}< / annotation>< / semantics> and <semantics>1×1013<annotation encoding="application / x-tex">1 \times 10^{13}< / annotation>< / semantics> molecules, wherein the number of sequence errors is between 1 in 100 to 1 in 2,500 (e.g., from about 1 in 200 to about 1 in 2,500, from about 1 in 500 to about 1 in 2,500, from about 1 in 1,000 to about 1 in 2,500, from about 1 in 1,500 to about 1 in 2,500, from about 1 in 1,000 to about 1 in 2,000, etc.). As discussed elsewhere herein, error correction can be used to lower the number of sequence errors.

[0032] The invention thus includes methods for the generation of collections of nucleic acid molecules, including methods comprising: (a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a microquantity; (b) combining the nucleic acid molecules generated in (a) to produce a pool; (c) joining some or all of the nucleic acid molecules present in the pool formed in (b) to form a plurality of larger nucleic acid molecules; and (d) assembling the plurality of larger nucleic acid molecules to form the collection of nucleic acid molecules, wherein the collection of nucleic acid molecules from bioinformatic information selected from the group consisting of: (1) a copy DNA (cDNA) library containing only DNA corresponding to messenger RNA (mRNA) molecules; (2) a partial cDNA library containing DNA molecules corresponding to less than the full complement of mRNA molecules found in the cell type that the bioinformatic information was derived from; and (3) a collection of nucleic acid molecules in which some or all of the nucleic acid molecules are codon altered variants of nucleic acid molecules found in the cell type that the bioinformatic information was derived from.

[0033] The invention also provides method for the generation of self-replicating nucleic acid molecules formed from smaller chemically synthesized nucleic acid molecules. In some embodiments, such method may comprise one or more of the following steps: (a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a microquantity; (b) combining the nucleic acid molecules generated in (a) to produce a pool; (c) joining some or all of the nucleic acid molecules present in the pool formed in (b) to form a plurality of larger nucleic acid molecules; and (d) assembling the plurality of larger nucleic acid molecules to form the self- replicating nucleic acid molecule.

[0034] The invention also includes methods for synthesizing and assembling nucleic acid molecules which encode more than one expression product, the methods comprising: (a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a microquantity; (b) combining the nucleic acid molecules generated in (a) to produce a pool (c) joining some or all of the nucleic acid molecules present in the pool formed in (a) to form a plurality of larger nucleic acid molecules; and (d) assembling the plurality of larger nucleic acid molecules to form the nucleic acid molecules which encode more than one expression product.

[0035] In various aspects of the invention, the more than one expression products may be proteins involved in the same biological pathway. In more specific aspects, the more than one expression products may be proteins involved in the same biological pathway are enzymes that catalyze a series of chemical reactions in the biological pathway. Further, such chemical reactions in the same biological pathway may be sequential reactions in the sense that one chemical reaction follows another either directly (directly sequential) or after one or more intervening reaction has occurred.

[0036] As one skilled in the art would understand, many aspects of the invention are well suited for automation. Automated systems are often driven by software which may perform repetitive tasks, especially when integrated with hardware designed for micromanipulation of components and reagent flows. Thus, according to various embodiments described herein, methods of assembling and synthesizing nucleic acids may be implemented on a computing system. Further, according to various embodiments described herein, processor-executable instructions for assembling and synthesizing nucleic acids are disclosed. Thus, in some aspects the invention includes non-transitory computer-readable storage media encoded with instructions, executable by a processor, for generating assembled nucleic acid molecules, the instructions comprising instructions for: (a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a microquantity in the well of a plate or a microchip; (b) combining the nucleic acid molecules generated in (a) to produce a pool; (c) joining some or all of the nucleic acid molecules present in the pool formed in (b) to form a plurality of larger nucleic acid molecules; (d) eliminating nucleic acid molecules which contain sequence errors from the plurality of larger nucleic acid molecules formed in (c) to produce an error corrected nucleic acid molecule pool; and (e) assembling the nucleic acid molecules in the error corrected nucleic acid molecule pool to form the assembled nucleic acid molecule.

[0037] The invention also includes systems for generating assembled nucleic acid molecules, the system comprising: a processor; and a memory encoded with processor-executable instructions for: (a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a microquantity in the well of a plate or a microchip; (b) combining the nucleic acid molecules generated in (a) to produce a pool; (c) joining some or all of the nucleic acid molecules present in the pool formed in (b) to form a plurality of larger nucleic acid molecules; (d) eliminating nucleic acid molecules which contain sequence errors from the plurality of larger nucleic acid molecules formed in (c) to produce an error corrected nucleic acid molecule pool; and (e) assembling the nucleic acid molecules in the error corrected nucleic acid molecule pool to form the assembled nucleic acid molecule.

[0038] In other aspects, methods for removing beads from fluid-filled wells of a microchip for synthesizing nucleic acid molecules, wherein one or more nucleic acid molecules are attached to the bead, are disclosed. These methods can comprise providing a voltage between a first electrode that is arranged at a bottom of the fluid-filled well and a second electrode, wherein the voltage is sufficient to cause fluid in the fluid-filled well to undergo electrolysis producing one or more bubbles in the fluid to rise to a top of the fluid-filled well along with the bead or lift the bead to the top of the fluid-filled well. In other embodiments, beads can be removed from the synthetic microchip using other techniques, including, but not limited to micro-manipulation such as micropipetting, optical forces, such as optical tweezers; generation of gas bubbles; acoustic force, gravity-fed techniques, magnetic techniques (e.g., magnetic beads), dielectrophoresis, valving, or using a weir structure on the microchip.

[0039] The first electrode can be composed of platinum and in certain embodiments, the voltage is about 0.1V to about 10,000V, about 1V to about 1000V, about 2V to about 100V, or about 5V to about 15V. The second electrode can be arranged above the first electrode. A third (reference) electrode can be used in certain embodiments, as discussed further below.

[0040] The oligonucleotide synthesis microchip can comprise a lid operable to be formed on a top surface of the microchip and operable to provide a fluid flow path into and out of the well. In some examples, the second electrode can be formed in the lid.

[0041] In certain embodiments, each well of the microchip used for nucleic acid synthesis can have a depth of about 45 to 60 μm, including about 50 μm, about 55 μm, or about 60 μm. The skilled person will understand that the well sizes may depend on the dimension and / or density (i.e., the number of wells) of a microchip as described elsewhere herein. In some examples, each well of the microchip is individually addressable by a controller. In some examples, the microchip is a complementation metal-oxide-semiconductor ("CMOS") chip.

[0042] The bead can be composed of: a synthetic polymer, a modified naturally occurring polymer, glass, controlled pore glass, magnetic controlled pore glass, magnetic beads, ceramics, or one or more metals. In certain embodiments, the bead is composed of a synthetic polymer such as polystyrene. In certain embodiments, at least 50% of the oligonucleotide synthesized on the bead is between 2-200 or 50-100 base pairs in length. In certain embodiments, the bead is monodisperse as described herein, having, for example, a bead diameter that is smaller than the diameter of each well by about 5% to about 20%, about 8% to 15%, about 10 to about 30% or about 12.5%. In certain embodiments, the diameter of the monodisperse bead varies less than 10%. In certain embodiments, the diameter of the monodisperse bead is between 30-40 μm. In certain embodiments, the diameter of the monodisperse bead is about 35 µm or about 32 µm, the diameter of each well is about 40 µl or about 42 µm or between about 40 to about 45 µm, and the depth of each well is about 50 μm or about 55 μm or between about 45 μm to about 55 μm. In certain instances, the diameter of a bead used in aspects of the invention will depend on the size of the well, whereas the size of a well may be defined by the dimension and / or density of a microchip. For example, a microchip of a given size may have a higher number of smaller wells or a lower number of larger wells. Thus, a microchip of higher well density with smaller wells will require beads of smaller sizes than a microchip of lower density with larger wells, as illustrated, e.g., in the table in FIG. 35.

[0043] In certain embodiments, the monodisperse bead has a pore volume of 0.1 to 2.5 ml / g of polymer. In certain embodiments, the monodisperse bead has a surface area of 100-500 m2 / g or 350-400 m2 / g (e.g., 380 m2 / g). In certain embodiments, the monodisperse bead is coated with a reactive group, such as an amino group. In certain embodiments, the amount of amine group in the coating ranges from 0.1% to 5% (weight % nitrogen per gram of beads). In certain embodiments, the monodisperse bead has a linker loading capacity of the oligonucleotide synthesis substrate within a range of 10 to 500 µmol / g, 30 to 100 µmol / g, or 40 to 80 µmol / g. In certain embodiments, the monodisperse bead carries a universal linker such as, e.g., a UNYLINKERTM.

[0044] After displacing beads from the microchip, the methods can further comprise collecting and concentrating the beads that have been displaced from the microchip using a microfluidic or bead-collection device. The bead-collection device can transfer the collected beads to a suitable container, such as the well of a first multiwell collection plate. In some examples, the bead collection device can be in fluid communication with the fluid flow path and comprise a first channel to allow for the bead to move a first direction and a second channel to allow for fluid to move in a second direction different than the first direction, for example in an opposite direction or an orthogonal direction. The bead collection device can comprise an acoustic module that is controllable by a controller to facilitate movement of the bead in the first channel, the fluid in the second channel, or both.

[0045] In certain embodiments, the first multiwell collection plate can comprise a plurality of well structures and a fluid-permeable structure formed on a top surface of or within the plurality of well structures. The fluid-permeable structure can be a material that is semipermeable to fluids, but not to the beads and can include, but is not limited to, a mesh, a filter, a porous material, or a membrane, e.g., ion-track etched membrane. For example, the material that is semipermeable to fluids may be a thin foil, (such as, e.g., a polypropylene foil) with holes or pores that are small enough to retain beads of sizes described elsewhere herein. Such holes or pores may be generated, e.g., by laser micro milling (e.g., Oxford Lasers can drill 10 μm holes into 100 μm polypropylene foils) or by other methods known in the art. In certain embodiments, the first multiwell collection plate can comprise a plurality of well structures and further comprise a second multiwell collection plate, wherein the second multiwell collection plate comprises a plurality of well structures and a fluid-permeable structure formed on a bottom surface of the plurality of well structures, wherein the second multiwell collection plate is placed on top of the first multiwell collection plate such that the plurality of well structures in the second multiwell collection plate are aligned with the plurality of well structures in the first multiwell collection plate.

[0046] In certain embodiments, the bead collection device can comprise a needle structure that is operable to 1) place the bead from the nucleic acid molecule synthesis microchip into a well of the first multiwell collection plate by puncturing the fluid-permeable structure, and / or 2) remove fluid from the well in which the bead was placed. The needle structure can comprise a first lumen that is operable to place the bead from the nucleic acid molecule synthesis microchip into a well of the multiwell collection plate by puncturing the fluid-permeable structure and a second lumen that is operable to remove fluid from the well in which the bead was placed.

[0047] In other embodiments, rather than using a needle to puncture the fluid-permeable structure, pressure is applied to the top of a selected well in the multiwell collection plate, the pressure being sufficient to rupture the fluid-permeable structure and deposit one or more beads into the selected well of the multiwell collection plate.

[0048] In other embodiments the bead collection device comprises a needle structure that is operable to place the bead from the nucleic acid molecule synthesis microchip into a well of the first multiwell collection plate, and / or 2) deliver fluid to the well of the first multiwell collection plate in which the bead was placed. The needle structure can comprise a first lumen that is operable to place the bead from the nucleic acid molecule synthesis microchip into a well of the multiwell collection plate and a second lumen that is operable to deliver fluid to the well in which the bead was placed.

[0049] In other embodiments, the oligonucleotides synthesized on the microchip can be pooled, concentrated, cleaved and deprotected on a fluid-permeable structure arranged on a top surface of or within a multiwell collection plate. The cleaved oligonucleotides can then be eluted into the well of a multiwell collection plate without having to puncture or otherwise rupture the fluid-permeable structure.

[0050] The methods of collecting the beads from the microchip can comprise moving the bead collection device in one or more degrees of freedom to deliver the beads into the plurality of wells of the first multiwell collection plate. The microchip can be programmed to extract the bead from a specific well of interest from the microchip and deliver the bead via the bead collection device to an addressable well in the plurality of wells in the first multiwell collection plate.

[0051] When beads are flushed out of the synthesis chip, they disperse into a larger volume, as additional fluid is used to transport the beads out of the microchip. The bead- collection device is able to pool beads containing the same oligonucleotides and / or beads containing other oligonucleotides required for the assembly of larger nucleic acid fragments and concentrate them from a first larger volume into a second smaller volume in and into a suitable collection container, such as a multiwell collection plate. Thus methods of pooling and concentrating beads, as disclosed herein, comprise transferring in a first volume of fluid one or more beads from a plurality of well structures formed on the microchip to a second volume of fluid in a well of a first multiwell collection plate, wherein a nucleic acid that has been synthesized on the microchip is attached to the one or more beads, wherein the one or more beads are transferred using a bead collection device (e.g., a microfluidic device or a device comprising a needle structure), wherein the bead collection device is in fluid connection with the microchip and the first multiwell collection plate, wherein the first multiwell collection plate comprises a plurality of wells, and wherein the second volume of fluid in the well of the first multiwell collection plate is less than the first volume of fluid, thereby concentrating the nucleic acid molecule synthesized on the microchip. In certain instances the concentrating comprises reducing the first volume of fluid by a factor of about 5 to about 50, about 10 to about 100, about 10 to about 1,000, about 100 to about 10,000. In certain embodiments, the total volume of each well of the first multiwell collection plate can be between 1 and about 200 µl, between 50 and about 200 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l, between 1 and 50 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l, between 1 and 25 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l, between 3 and 12 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l or about 10 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l.

[0052] According to the present disclosure, systems for synthesis of nucleic acid molecules are disclosed. These systems can comprise one or more microchips comprising a plurality of well structures formed thereon, each well of the plurality of well structures sized to accommodate a bead for synthesis of the nucleic acid molecule, wherein each well has formed therein a first electrode at a bottom of the well that is individually controllable by a controller; and a lid member arranged on top of the microchip and comprising a fluidic channel formed therein to provide fluid path for the bead, wherein the lid member comprises a second electrode, wherein the controller is operable to provide a voltage between the first electrode and the second electrode that is sufficient to cause fluid in the well to undergo electrolysis producing one or more bubbles in the fluid to rise to a top of the well along with the bead.

[0053] These systems can further comprise a bead-collection device operable to collect and concentrate the beads that are removed from the synthesis chip. These systems can further comprise a first multiwell collection plate operable to receive one or more beads that were collected using the bead-collection device.

[0054] The bead collection device can be in fluid communication with the fluid path for the bead and comprises a first channel to allow for the bead to move a first direction toward a first multiwell collection device and a second channel to allow for fluid to move in a second direction different than the first direction, for example in opposite directions, orthogonal, or any suitable direction. The bead collection devices can comprise an acoustic module that is controllable by a controller to facilitate movement of the bead in the first channel, the fluid in the second channel, or both.

[0055] In these systems, the first electrode can be composed of platinum and in certain embodiments, the voltage is about 0.1V to about 10,000V, about 1V to about 1000V, about 2V to about 100V, or about 5 to about 15 volts. The second electrode can be arranged above the first electrode. A third (reference) electrode can be used in certain embodiments, as discussed further below.

[0056] The bead collection devices can be caused to move, by a controller, in one or more degrees of freedom to deliver the beads that are collected in the bead collection device into the plurality of wells of the first multiwell collection plate. The microchip or other computer system can be programmed to extract the bead from a specific well of interest on the microchip and deliver the bead via the bead collection device to an addressable well in the plurality of wells in the first multiwell collection plate.

[0057] According to the present disclosure, non-transitory computer-readable storage media encoded with instructions, executable by a processor, for removing one or more beads from a fluid-filled well of a microchip for synthesizing nucleic acid molecules, where nucleic acid molecules are attached to the bead, are disclosed. Also disclosed are systems comprising a processor and a memory encoded with processor-executable instructions for removing one or more beads from a fluid-filled well of a microchip for synthesizing nucleic acid molecules, where the nucleic acid molecules are attached to the bead. Instructions for the computer- readable storage media or the systems can comprise instructions for providing a voltage between a first electrode that is arranged at a bottom of the fluid-filled well and a second electrode, wherein the voltage is sufficient to cause fluid in the fluid-filled well to undergo electrolysis producing one or more bubbles in the fluid to rise to a top of the fluid-filled well along with the bead.

[0058] According to the present disclosure, methods for selectively removing one or more beads from a microchip for synthesizing nucleic acid molecules having a plurality of fluid-filled wells, wherein each of the plurality of wells comprises an electrode formed at the bottom of the well and each bead of the one or more beads occupies a single well on the microchip are disclosed. These methods can comprise: identifying one or more wells that contain one or more beads to be removed; providing a voltage between a first electrode in the one or more wells that have been identified and a second electrode, wherein the voltage is sufficient to cause fluid in the one or more wells to undergo electrolysis producing one or more bubbles to rise to a top of the one or more wells along with the one or more beads contained within the one or more wells; collecting the one or more beads that have risen to the top of the fluid-filled well with a bead- collection device; and transferring the one or more beads that were collected to one or more wells of a first multiwell collection plate or other suitable collection container where the beads can be concentrated and further processed (e.g., cleavage and deprotection) before assembly into longer nucleic acid fragments. BRIEF DESCRIPTION OF THE FIGURES

[0059] FIG. 1 is a general description of aspects of work flows of the invention. The work flow is broken into four sections, referred to as "modules" for ease of description. The work flow on the right side of the figure shows some specific step included in some aspects of methods of the invention.

[0060] FIGs. 2A and 2B are schematic representations of a row of wells according to an embodiment of the invention. The darker area in well 1 indicates the presence of a reagent (e.g., EGA) not present at a given time in the other wells.

[0061] FIG. 3 shows a nucleic acid assembly scheme. The thick ends on the assembled nucleic acid molecule shown at the bottom of the figure represent regions added by external primers, also referred to as terminal primers.

[0062] FIG. 4 shows a second nucleic acid assembly scheme. Dotted lines with arrows show PCR based synthesis direction and area.

[0063] FIG. 5 shows the assembly of two DNA fragments that do not share any homology into a vector using stitching nucleic acid molecules. The 69 base pair double-stranded stitching nucleic acid molecules, shown in bold in the lower portion of the figure, share 30-bp homology with each adjacent fragment (Fragments 1 and 2). These stitching nucleic acid molecules are used to insert 9 bp at the junction of the adjacent fragments. The insertion bases are shown underlined.

[0064] FIG. 6 is a flow chart of an exemplary process for synthesis of error-minimized nucleic acid molecules.

[0065] FIG. 7 is a work flow chart of an exemplary process for synthesis of error- minimized nucleic acid molecules. Different strands of a double-stranded nucleic acid molecule are represented by thicker and thinner line. "MME" refers to mis-match endonuclease. Small circles represent sequence errors.

[0066] FIG. 8 generally illustrates methods for assembly and cloning of nucleic acid segments in yeast. In some embodiments of the invention, a number of nucleic acid segments (one of which is a vector) are co-transforming the fragments into a yeast host cell, where they are assembled by homologous recombination to form, for example, a closed, circular nucleic acid molecule.

[0067] FIG. 9 is a drawing of an electrical coil that may be used in the practice of the invention.

[0068] FIG. 10 is cross-sectional view of one embodiment of a fluid reagent delivery system suitable for use with the invention.

[0069] FIG. 11 shows a library of linear, nucleic acid molecules (top) generated by methods of the invention and a vector (bottom) designed to accept library members. The upper portion of the figure shows a series of lines representing four members of the library. The lower open circular line represents a vector. The blocks on each end of the nucleic acid molecule represent nucleic acid segments which facilitate joining (e.g., GATEWAY® sites, regions of homology, etc.). The numbers and the termini of the nucleic acid molecules indicate compatible ends.

[0070] FIGs. 12A and 12B shows a series of variant nucleic acid molecules that may be prepared by methods of the invention and their encoded amino acid sequences. FIG. 12A shows variant nucleic acid molecules that encode different amino acid sequences. FIG. 12B shows variant nucleic acid molecules that use different codons but encode the same amino acid sequence.

[0071] FIG. 13 shows two different fluid removal options for microwell plate embodiments of synthesis platforms.

[0072] FIGs. 14A and 14B shows two different views of a nucleic acid molecules synthesis platform designed to generate identical nucleic acid molecules in each row 1401. FIG 14A is a top view and FIG 14B is a side view. Shown in the figure are fluidic channels 1401, two electrodes associated with each channel / row of wells 1402 and a series of wells containing nucleic acid synthesis substrates (e.g., individual beads) located in wells 1400. In some embodiments, the wells will be spaced 300 µm apart and will be cylindrical in shape with a diameter of 40 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m and a depth of 35 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m.

[0073] FIG. 15 is a block diagram that illustrates a computing system, upon which embodiments of the present teachings may be implemented.

[0074] FIG. 16 is a schematic of automated system for performing methods of the invention.

[0075] FIG. 17 is a top view schematic of a channel "chip".

[0076] FIG. 18 shows a simplified example of a selective bead removal process from a synthesis chip using electrolysis according to methods of the invention.

[0077] FIG. 19 shows an example bead collection process including a microfluidic or bead- collection device and a multiwell collection plate according to methods of the invention.

[0078] FIG. 20 shows the microfluidic or bead-collection device of FIG. 19 in greater detail.

[0079] FIG. 21 shows another example multiwell collection plate according to the invention. FIG. 21 also shows a bead collection method involving the use of a needle structure to puncture a fluid-permeable structure (e.g., micromesh) for placing one or more beads from the synthesis chip into a well of a multiwell collection plate or for removing fluid from one or more wells of a multiwell collection plate.

[0080] FIG. 22 shows an example process for bead removal from a synthesis chip, bead concentration in a multiwell collection plate comprising two tiers separated by a fluid-permeable structure, cleavage and deprotection of nucleic acids in the multiwell collection plate using, for example, ammonia atmosphere, and eluting the cleaved and deprotected nucleic acids into the bottom well of the multiwell collection plate using a system according to the invention.

[0081] FIGs. 23A and 23B show another example of an oligonucleotide synthesis microchip according to the invention.

[0082] FIGs. 24A, 24B, 24C, and 24D show example side-views and top-view of the fluidic lid for the microchip according to the invention.

[0083] FIG. 25 illustrates an example cleavage reaction of a bead-bound nucleic acid molecule with an electrochemically generated base.

[0084] FIG. 26 illustrates an exemplary microfluidic chip for administering a nucleic acid molecule denaturing solution and optionally a buffer to a microarray of synthesized nucleic acid molecules.

[0085] FIG. 27 shows an example work flow diagram of the nucleic acid synthesis (Module 1) and the pooling, cleavage and deprotection steps (Module 2).

[0086] FIGs. 28A, 28B and 28C show an example work flow diagram of synthesis preparation.

[0087] FIGs. 29A and 29B shows an example work flow diagram of synthesis planning.

[0088] FIG. 30 shows an example work flow diagram of microchip synthesis.

[0089] FIG. 31 shows an exemplary o-nitrobenzyl photocleavable linker linking a solid support bead to an oligonucleotide.

[0090] FIG. 32 shows an exemplary electrochemical reaction wherein reduction of 2,2'- disulfane diylbis(ethane-1-ol) to a monosulfide results in cleavage of an oligonucleotide and a disulfide reductive cleavable linker.

[0091] FIGs. 33A, 33B, and 33C shows an example time series of images depicting the selective removal of beads from a multiwell synthesis chip using electro-generated gas according to the invention.

[0092] FIG. 34 shows a chromatogram of a 40-mer test oligonucleotide synthesized according to the invention.

[0093] FIG. 35 shows variations of numbers of wells and various physical parameters.

[0094] FIG. 36 shows a workflow schematic for the assembly of oligonucleotides and double error correction. Nine line numbers are labeled in the workflow for reference in the specification.

[0095] FIG. 37 shows a table of error types and rates found in chemically synthesized oligonucleotides prior to error correction and after two rounds of error corrections using T7 endonuclease I. These data were generated as set out in Example 9.

[0096] FIG. 38 shows the remaining errors, by type, found in chemically synthesized oligonucleotides after two rounds of error corrections using T7 endonuclease I. These data were generated as set out in Example 9.

[0097] FIG. 39 shows a schematic representation of a workflow employing bead bound mismatch repair binding proteins for the separation of nucleic acid molecules that contains mismatches from those that do not contain mismatches. NMM refers to a non-mismatched nucleic acid molecule and MM refers to a mismatched nucleic acid molecule.

[0098] FIGs. 40A and 40B show the modification of porous amine functionalized particles with linker molecules.

[0099] FIGs. 41A and 41B show the oligonucleotides used for assembly of a lacZ gene. FIG. 41A is an rpHPLC chromatogram of ten different oligonucleotides sequenced simultaneously on a microfluidic chip. FIG. 41B shows the order and arrangement of the oligonucleotides for assembly of the lacZ gene.

[00100] FIG. 42 shows alternative workflow schematics for the assembly of oligonucleotides comprising Exonuclease I treatment. In a first variation, double error correction is performed using one type of endonuclease (workflow on the left). In a second variation, double error correction is performed using one or more endonucleases in the first round and a mismatch binding protein in the second round (workflow on the right). Nine line numbers are labeled in the workflow for reference in the specification.

[00101] FIGs. 43A, 43B and 43C show exemplary embodiments of how a metal layer of an electrode may be connected to the top metal of the CMOS part of a microfluidic chip.

[00102] FIG. 44 (Panels A, B, C and D) shows the selective removal of beads from a microfluidic synthesis chip using electrolysis as described in Example 6.

[00103] FIGs. 45A, 45B and 45C show a coaxial needle assembly and a filter plate comprising a fluid-permeable micromesh for use in a bead collection device.

[00104] FIG. 46 shows representative embodiments of alternative fluidics configurations used in an oligonucleotide synthesis instrument.

[00105] FIG. 47 (Panels A, B, C, D, E and F) shows the principle of using a solid support proton scavenger to prevent protons from contaminating adjacent reaction sites.

[00106] FIGs. 48A and 48B show examples of how base-coated scavenger beads can be used to protect synthesis positions in the vicinity of a reaction side with acidic conditions. A 32- µm porous synthesis support was loaded into each well. The experiment of FIG. 48A was performed in the absence of scavenger beads, whereas the experiment of FIG. 48B was performed in the presence of smaller scavenger beads filled into each well.

[00107] FIG. 49 shows in improved seamless workflow for micro-processing of biological samples comprising alternate steps of liquid handling and thermocycling. DETAILED DESCRIPTION OF THE INVENTION Definitions:

[00108] Microchip, chip, synthesis chip, synthesis microchip, array, microarray: As used herein, the terms microchip, chip, synthesis chip, synthesis microchip, array, microarray or similar variations thereof will refer to an electronic computer chip on which oligonucleotide synthesis can occur.

[00109] Multiwell plate, microplate, microwell plate, plate: As used herein, the term multiwell plate, microplate, microwell plate, plate or similar variations thereof will refer to a two-dimensional array of multiple wells located on a substantially flat surface. Multiwell plates can comprise any number of wells of any width or depth. In certain instances, a multiwell plate may be configured as a microchip. For example, when a material with well-like structures is overlaid onto a microchip.

[00110] Solid Support: As used herein, the term solid support refers to a porous or non- porous material on which polymers such as nucleic acid molecules can be synthesized and / or immobilized. As used herein "porous" means that the material contains pores which may be of non-uniform or uniform diameters (for example in the nm range). Porous materials include paper, synthetic filters etc. In such porous materials, the reaction may take place within the pores. The solid support can have any one of a number of shapes, such as pin, strip, plate, disk, rod, fiber, bends, cylindrical structure, planar surface, concave or convex surface or a capillary or column. The solid support can be a particle, including bead, microparticles, nanoparticles and the like. The solid support can be a non-bead type particle (e.g., a filament) of similar size. The support can have variable widths and sizes. For example, sizes of a bead (e.g., a magnetic bead) which may be used in the practice of the invention are described elsewhere herein. The support can be hydrophilic or capable of being rendered hydrophilic and includes inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers such as filter paper, chromatographic paper or the like. The support can be immobilized at an addressable position of a carrier. The support can be loose (such as, e.g., a resin material or a bead in a well) or can be reversibly immobilized or linked to the carrier (e.g. by cleavable chemical bonds or magnetic forces etc.).

[00111] In some embodiments, solid support may be fragmentable. Solid supports may be synthetic or modified naturally occurring polymers, such as nitrocellulose, carbon, cellulose acetate, polyvinyl chloride, polyacrylamide, cross linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF) membrane, glass, controlled pore glass, magnetic controlled pore glass, magnetic or non- magnetic beads, ceramics, metals, and the like; either used by themselves or in conjunction with other materials.

[00112] In some embodiments, the support can be in a chip, array, microarray or microwell plate format. In many instances, a support generated by methods of the invention will be one where individual nucleic acid molecules are synthesized on separate or discrete areas to generate features (i.e., locations containing individual nucleic acid molecules) on the support.

[00113] In some embodiments, the size of the defined feature is chosen to allow formation of a microvolume droplet or reaction volume on the feature, each droplet or reaction volume being kept separate from each other. As described herein, features are typically, but need not be, separated by interfeature spaces to ensure that droplets or reaction volumes or between two adjacent features do not merge. Interfeatures will typically not carry any nucleic acid molecules on their surface and will correspond to inert space. In some embodiments, features and interfeatures may differ in their hydrophilicity or hydrophobicity properties. In some embodiments, features and interfeatures may comprise a modifier. In one embodiment of the invention the feature is a well or microwell or a notch.

[00114] Nucleic acid molecules may be covalently or non-covalently attached to the surface or deposited or synthesized or assembled on the surface.

[00115] In one embodiment of the invention, Module 1 can involve the use of more than one solid support. In some embodiments, two or more solid supports may be arranged on a plate. Any arrangement of the solid supports could be employed such as rows or columns or a combination thereof. For example, rows can be aligned and / or the columns can be aligned. In other embodiments, rows and / or columns are equally spaced and staggered. Spacing between rows and / or between columns can be variable. The number of the solid supports comprised in, for example, a plate may be variable. In some embodiments, a plate may contain up to 1,536 (or more) solid supports.

[00116] Nucleic Acid Molecule: As used herein the term "nucleic acid molecule" refers to a covalently linked sequence of nucleotides or bases (e.g., ribonucleotides for RNA and deoxyribonucleotides for DNA but also include DNA / RNA hybrids where the DNA is in separate strands or in the same strands) in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester linkage to the 5' position of the pentose of the next nucleotide. Nucleic acid molecule may be single- or double-stranded or partially double-stranded. Nucleic acid molecule may appear in linear or circularized form in a supercoiled or relaxed formation with blunt or sticky ends and may contain "nicks". Nucleic acid molecule may be composed of completely complementary single strands or of partially complementary single strands forming at least one mismatch of bases. Nucleic acid molecule may further comprise two self- complementary sequences that may form a double-stranded stem region, optionally separated at one end by a loop sequence. The two regions of nucleic acid molecule which comprise the double-stranded stem region are substantially complementary to each other, resulting in self- hybridization. However, the stem can include one or more mismatches, insertions or deletions.

[00117] Nucleic acid molecules may comprise chemically, enzymatically, or metabolically modified forms of nucleic acid molecules or combinations thereof. Chemically synthesized nucleic acid molecules may refer to nucleic acids typically less than or equal to 150 nucleotides long (e.g., between 5 and 150, between 10 and 100, between 15 and 50 nucleotides in length) whereas enzymatically synthesized nucleic acid molecules may encompass smaller as well as larger nucleic acid molecules as described elsewhere in the application. Enzymatic synthesis of nucleic acid molecules may include stepwise processes using enzymes such as polymerases, ligases, exonucleases, endonucleases or the like or a combination thereof. Thus, the invention provides, in part, compositions and combined methods relating to the enzymatic assembly of chemically synthesized nucleic acid molecules.

[00118] Nucleic acid molecule also refers to short nucleic acid molecules, often referred to as, for example, primers or probes. Primers are often referred to as single-stranded starter nucleic acid molecules for enzymatic assembly reactions whereas probes may be typically used to detect at least partially complementary nucleic acid molecules. A nucleic acid molecule has a "5'-terminus" and a "3'-terminus" because nucleic acid molecule phosphodiester linkages occur between the 5' carbon and 3' carbon of the pentose ring of the substituent mononucleotides. The end of a nucleic acid molecule at which a new linkage would be to a 5' carbon is its 5' terminal nucleotide. The end of a nucleic acid molecule at which a new linkage would be to a 3' carbon is its 3' terminal nucleotide. A terminal nucleotide or base, as used herein, is the nucleotide at the end position of the 3'- or 5'-terminus. A nucleic acid molecule sequence, even if internal to a larger nucleic acid molecule (e.g., a sequence region within a nucleic acid molecule), also can be said to have 5'- and 3'-ends.

[00119] Transition: As used herein, the term "transition", when used in reference to the nucleotide sequence of a nucleic acid molecule refers to a point mutation that changes a purine nucleotide to another purine <semantics>(A↔G)<annotation encoding="application / x-tex">(A \leftrightarrow G)< / annotation>< / semantics> or a pyrimidine nucleotide to another pyrimidine <semantics>(C↔T)<annotation encoding="application / x-tex">(C \leftrightarrow T)< / annotation>< / semantics>.

[00120] Transversion: As used herein, the term "transversion", when used in reference to the nucleotide sequence of a nucleic acid molecule refers to a point mutation involving the substitution of a (two ring) purine for a (one ring) pyrimidine or a (one ring) pyrimidine for a (two ring) purine.

[00121] Indel: As used herein, the term "indel", refers to the insertion or deletion of one or more bases in a nucleic acid molecule. Overview:

[00122] The invention relates, in part, to compositions and methods for the preparation of nucleic acid molecules. While the invention has numerous aspects and variations associated with it, some of these aspects and variations are set out in FIG. 1 in outline form.

[00123] One advantage of the invention is that for many applications, small amounts of synthesized nucleic acid are suitable for achieving an intended purpose (e.g., preparation of microarrays, construction of a plasmid which contains a selectable marker, etc.). In some instances, small amounts of nucleic acid are suitable for working with due to factors such as enzymatic (e.g., PCR) and intracellular amplification.

[00124] The left side of FIG. 1 shows four general "modules" representing different portions of some embodiments of the invention. Thus, in some aspects, the invention involves one or more of the following: (1) nucleic acid molecule synthesis, (2) pooling of nucleic acid molecules, (3) assembly of a plurality of nucleic acid molecules, and / or (4) transfer of assembled nucleic acids (e.g., transfer to a cell).

[00125] In relation to more specific embodiments of the invention, the right side of FIG. 1 shows some additional details related to the modules shown on the left side of the figure. Above a number of the text blocks are bolded terms such as "ENZYMATIC" and "CELLULAR". These terms indicate exemplary general means by which the process referred to can be performed. As one skilled in the art would understand, some processes can be performed, for example, either chemically, enzymatically, or in a cell.

[00126] Module 1, as shown in FIG. 1 refers to a single process termed "Microscale Parallel Nucleic Acid Molecule Synthesis". As set out elsewhere herein, this process will typically involve several steps which will vary with how the process is performed. In many embodiments, the general function of Module 1 will be the generation of a plurality of nucleic acid molecules. These nucleic acid molecules may be designed as a group to be joined to form one or more larger nucleic acid molecule or when contacted with additional nucleic acid molecules (e.g., "stitching" nucleic acid molecules).

[00127] Module 2, as shown in FIG. 1 refers to processes termed "Pooling of Solid Supports", "Nucleic Acid Molecule Cleavage", and "Deprotection". The general function of Module 2 will be the preparation of nucleic acid molecules for participation in one or more process referred to in Module 3. This will often mean combining nucleic acid molecules which differ in sequence and the removal of any chemical groups which are either not necessary or not desirable for the performance of one or more processes referred to in Module 3.

[00128] Using Module 2 as an example, as one skilled in the art would recognize, FIG. 1 shows general embodiments of the invention. More specifically, Module 2 refers to the pooling of solid supports. These supports will typically contain nucleic acid molecules. In some embodiments, nucleic acid molecules may be obtained in a form free of solid supports, then pooled.

[00129] Module 3, as shown in FIG. 1 refers to the processes termed "Fragment Amplification and Assembly", "Error Correction", and "Final Assembly". The general function of Module 3 processes is the generation of assembled nucleic acid molecules with high sequence fidelity, with comparison to the sequence of nucleic acid molecules which were sought to be produced.

[00130] Module 4, as shown in FIG. 1 refers to the processes of termed "Recipient Cell Insertion". As one skilled in the art would understand, introduction of nucleic acid molecules generated by methods of the invention into cells is only one application. In most instances, a nucleic acid molecule assembled according to methods of the invention will be designed for a specific application. Applications vary widely and include biofuel production, bioremediation, and chemical precursor production.

[00131] In some embodiments, monodispersed particles obtained by methods as described in U.S. Patent No. 6,335,438, may be used in the practice of the invention. For example, in certain embodiments disclosed herein and as disclosed in U.S. Patent No. 6,335,438, use may be made of support matrices comprising a polyvinyl backbone and having amino groups that are optionally acylated. The support matrices may be obtained by polymerizing at least one monovinyl monomer with at least one di-, tri-, or polyvinyl monomer. At least one of the monomers is a vinyl aromatic monomer, and the polymerization reaction may occur in the presence of at least one amino vinyl aromatic monomer, such as aminostyrene. For example, in certain embodiments disclosed herein, the solid support may be aminostyrene beads made from the polymerization of at least one monovinyl monomer and at least one di-, tri-, or polyvinyl monomer in the presence of amino vinyl aromatic monomers. In alternative embodiments, an amino vinyl aliphatic monomer may be used instead of a vinyl aromatic monomer to modify or adapt the reactivity and / or amine content of a bead as disclosed elsewhere herein. For example instead of aminostyrene, vinylbenzyl-chloride may be used.

[00132] In certain other embodiments disclosed herein, solid support beads may be prepared by the methods recited in Lewandowski, K. et al., "Preparation of Macroporous, Monodisperse, Functionalized Styrene-divinylbenzene Copolymer Beads: Effect of the Nature of the Monomers and Total Porogen Volume on the Porous Properties," J. App. Polymer Science, 67: 597-607 (1998). For example, in certain embodiments, monodisperse beads may be used that have been prepared from the polymerization of mixtures of styrene and substituted styrene monomers, such as 4-methylstyrene, 4-aminostyrene, 3-aminostyrene, 4- acetoxystyrene, and 4-tert-butoxycarbonyl oxystyrene, with divinylbenzene. The monodisperse beads may be prepared in the presence of various porogens. Module 1

[00133] In the invention, the nucleic acid molecules may be attached to solid supports, such as particles or beads (e.g., controlled pore glass beads or polystyrene beads). In one embodiment, magnetic or non-magnetic microbeads are used as solid supports. In many instances, porous µm-size microbeads with large surface to volume ratios may be used in the current invention. The uniform nature of such monodispersed particles generally provides for uniform reaction rates particularly suited to synthesis in automated chemical synthesizers (e.g., nucleic acid molecule synthesizers). These beads may be obtained with various chemical activation groups suitable for use for different applications.

[00134] In some aspects, the invention relates to a multiwell plate for non-template directed synthesis of nucleic acid molecules (e.g., chemical synthesis). In a preferred embodiment, the multiwell plate comprises a plurality of wells, wherein each well is configured to accommodate one or more monodisperse beads. In some instances, each well of the multiwell plate may be configured to accommodate one monodisperse bead.

[00135] A well according to the invention may be defined by its depth. A minimal depth of a well should at least slightly exceed the diameter of a bead to guarantee that the bead can be covered (e.g., homogenously covered) with reagents / EGA. The depth of a well may be, for example, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or less than 200% of the bead diameter. However, the maximum depth of a well will typically be configured such that only a defined number of beads (e.g., only a single bead) can be placed in the well. For example, the depth of a well will typically not exceed about 150% of the bead diameter to ensure that a second bead cannot be placed in a well already occupied by a first bead.

[00136] In certain instances a bead may be of a size or diameter to tightly fit into a well. For example, a bead with a diameter of about 35 to 40 µm may be placed in a well that has an inner diameter of 40 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m and a depth of 55 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m. This would result in the bead fitting into the well with narrow tolerance. As explained elsewhere herein, due to certain aspects of structural heterogeneity of beads that may be used in the practice of the invention, fairly tight bead / well tolerances fall within the scope of the invention. For example, beads may be smaller than wells by 20% or less (e.g., from about 1% to about 10%, from about 2% to about 10%, from about 4% to about 10%, from about 5% to about 10%, from about 2% to about 8%, from about 5% to about 10%, from about 4% to about 8%, from about 1% to about 15%, from about 5% to about 15%, from about 8% to about 15%, from about 5% to about 20%, from about 10% to about 20%, from about 15% to about 20%, etc.) in terms of bead diameter compared to well diameter. For purposes of illustration, if a bead with a diameter of about 35 µm is placed in a well with a diameter of about 40 µm, then the bead is smaller than the well by 12.5%.

[00137] In instances where a well is configured to accommodate a single bead, the diameter of the bead may be approximately 80-90% of the inner diameter of a well to ensure that the bead surface will be homogenously brought in contact with and / or covered by reagents and / or EGA and the entire bead surface area will be used equally efficient for synthesis of nucleic acid molecules. By using a bead with a diameter of less than 100% of the inner well diameter it can also be ensured that heat developed in the well can freely pass off the well / can escape from the well. For example, the distance between the bead surface and the inner wall of a well may be about 2 to 10% of the bead diameter, such as, for example, about 5% of the bead diameter. In many instances the bead will be spherical and the well will have a round or cylindrical shape. However, other forms and shapes are also possible. In certain instances the well may, e.g., have a rectangular, square, octagonal or filamentous shape. For example, in a setting where a bead is sized to essentially exactly fit into a square-shaped well, heat could escape from the "corners" of the well. Well size and shape is preferably chosen to allow efficient fluid exchange when switching reagents.

[00138] In a preferred aspect of the invention, the bead is a monodisperse / monosized porous bead. In some embodiments the bead may be a magnetic bead or a non-magnetic bead. The use of monodisperse beads in wells of defined size as described above helps in ensuring that all beads are (1) equally and homogenously contacted by reagents / EGA in the well and (2) provides equal loading capacity and (3) equal amounts of reactive synthesis positions per well, therefore assisting in the preparation of equal starting conditions for parallel reactions.

[00139] The term monodisperse is used herein to characterize a population of particles or beads with low heterogeneity and a homogenous size distribution. The size distribution of a bead may be defined by the percentage CV (coefficient of variation). The CV for a plurality of beads may for example be within a range of 50 to 100%. Monodisperse beads used in aspects of the invention are characterized by a low CV which may be within a range of 1 to 10%, 1 to 20%, 1 to 30%, 1 to 30%, 3 to 20%, 5 to 15%, 2 to 10%, 10 to 25%, less than 10%, preferably less than 5% or less than 3%. For example, a monodisperse bead population may have more than 90%, preferably more than 95% of the beads with sizes within their mean diameter of <semantics>±<annotation encoding="application / x-tex">\pm< / annotation>< / semantics> 5%. Monodisperse beads for use in embodiments of the invention may be obtained according to a process as described, for example, in Patent Publication No. WO 2000 / 061647.

[00140] With many oligonucleotide synthesis systems, synthesis efficiency decreases once nucleic acid molecules reach certain lengths. Further, the lengths at which synthesis efficiency decreases can vary with synthesis parameters. One reason that synthesis efficiency is believed to decrease is due to steric hindrance effects. In particular, oligonucleotides are three dimensional compounds that occupy space. Thus, once the local space surrounding an oligonucleotide chain becomes limited, steric hindrance effects interfere with the addition of new bases to the molecule. The invention includes compositions and methods for providing synthesis parameters to lessen decreases in oligonucleotide related length synthesis efficiency.

[00141] A number of parameters may be adjusted to lessen decreases in oligonucleotide related length synthesis efficiency. Further, different parameters may be adjusted in relation to each other. The relation of well size to bead size as described above in combination with bead- specific parameters (bead surface area, pore volume, amine content, linker loading capacity, etc.) may be adjusted. To increase yields of correctly synthesized longer oligonucleotides, a bead with (1) a larger pore volume to surface ratio and (2) lower linker loading may be advantageous. These parameters present a situation where increased yield of longer oligonucleotides of the desired sequence in terms of percentage of "correct" oligonucleotides produced is partially the result of synthesizing fewer oligonucleotides per unit area of bead. In particular, the lessening of steric hindrance effects typically requires that oligonucleotides being synthesized be provided sufficient space to prevent them from "bumping" into each other during the synthesis process. This requires a lower loading capacity on the bead than is technically feasible. The net result is that fewer oligonucleotides are produced per unit area of bead surface as compared to that which is technically possible. Depending on the desired length, quality and yield, adjustment of these parameters can be used to achieve an optimal result as further described below. Thus, in some aspects, the invention includes compositions and methods employing beads having a lower linker loading capacity than that which is technically feasible. Data related to linker loading capacity and efficiency of oligonucleotide synthesis is set out elsewhere herein.

[00142] As used herein, the term "long oligonucleotides" refers to oligonucleotides of a length where most standard commercially used and available oligonucleotide synthesis platforms begin to exhibit increased synthesis error rates. In many instances, these errors result from increases in the percentage of oligonucleotide molecules exhibit "missing" bases. In other words, the rate at which bases are not added to oligonucleotide molecules being synthesized increases significantly. For example, in some instances the addition of each base, also referred to as "coupling", is approximately 99% efficient. In such instances, when an oligonucleotide of a certain length n is to be synthesized, only 0.99n-1 of the oligonucleotides will be correct based on the accumulation of inefficient coupling events. Also, other factors such as side reactions or the concentration or ratio of reactive compounds may contribute to less efficient coupling thereby adding to the overall error rate with increasing length of the oligonucleotide. For purposes of illustration, assume that an oligonucleotide is synthesized where there is a 1.5% error rate of failure to add a base for each base addition, for each oligonucleotide being synthesized. Further, this "failure to add" error rate remains constant from base 2 to base 35, then increases to 2% at base 36, 3% for base 37 and 5% for base 38 and 8% for base 39. In such an instance, oligonucleotides over 35 bases in length would be considered to be "long oligonucleotides". In most instances, long oligonucleotides are oligonucleotides of more than 35, more than 40, more than 45, more than 50, more than 55, more than 60, more than 80, more than 100, more than 120, more than 150, up to about 200 etc., bases in length.

[00143] Errors such as deletions, insertions or mutations may occur for various reasons. In some instances, errors may result from inefficient or incomplete deblocking events (e.g., incomplete removal of the 5'-DMT protecting groups). If deblocking is not achieved quantitatively, added bases cannot be coupled to all oligonucleotides synthesized on a bead. The protecting group of one or more oligonucleotides that has not been removed in a given synthesis cycle may, however, be removed in subsequent synthesis cycles followed by coupling of further bases. In this case, one or more oligonucleotides may carry one or more deletions. Whereas certain conditions during synthesis may result in oligonucleotides having deletions, other conditions may lead to the incorporation of one or more wrong bases resulting in insertions or mutations. In the latter case, an oligonucleotide may have the intended length but be of incorrect total sequence. The error rate of a specific oligonucleotide (e.g., a population of oligonucleotides intended to have the same sequence) may be determined by various means. The error rate may be determined based on a subset of oligonucleotides comprising the desired length (e.g., HPLC) purified) and may not take into account any smaller fragments or truncated oligonucleotides generated as byproducts. One way of determining the error rate (the total amount of insertions, deletions and mutations) of oligonucleotides of a certain length is by direct sequencing. Another way to determine the error rate or the "quality" of oligonucleotides may be based on the efficiency of assembling one or more sets of oligonucleotides into larger nucleic acid molecules of correct sequence. Whether determined by direct or indirect means, a population of oligonucleotides of rather low quality may exhibit an error rate of 2 to 3% such as, for example, 2.5% which would represent about one error in 40 bases. Oligonucleotides of higher quality may exhibit an error rate of less than 1%, less than 0.5%, less than 0.25% or less than 0.1% (1 error in 1000 bases). Therefore, one aspect of the invention is to provide means and methods to synthesize long oligonucleotides of high quality (i.e., with low error rate).

[00144] As indicated above, a portion of oligonucleotides synthesized on a support may be truncated (i.e., have a length less than the desired length). A truncated oligonucleotide may, for example, be generated where a 5'-DMT protecting group has been removed but no base coupling has occurred resulting in an unreacted 5'-hydroxyl group which may be subject to additional coupling in a subsequent synthesis step. To avoid accumulation of deletions with each successive cycle a "capping step" is used to block any unreacted 5'-hydroxyl groups. This will, however, result in shorter oligonucleotides as no further bases can be added to capped oligonucleotides in subsequent reactions. Such truncations accumulate with increasing length of oligonucleotides being synthesized. It is therefore desired to generate a large portion or high yield of oligonucleotides having the desired full-length. The yield may be defined as the percentage of oligonucleotides generated per well / bead having the desired length or the amount of full-length oligonucleotides generated per well / bead. For example, a 35 µm bead with a linker loading capacity of 300 femtomole may carry 30 to 50% of full-length oligonucleotides resulting in an amount of 90 to 150 femtomole of oligonucleotide having the desired length. One aspect of the invention is therefore to provide means and methods to synthesize long oligonucleotides at high yield, such as, for example, between 30% and 50%, between 40% and 60%, between 50% and 70% or more than 50% of the total amount of oligonucleotides synthesized in a well / on a bead. Alternatively, the amount of full-length oligonucleotides synthesized in a well / on a bead may be between 90 to 150 femtomole, between 100 and 200 femtomole, between 150 and 300 femtomole, more than 300 femtomole, etc.

[00145] It may be desirable to quantify the amounts of nucleic acid molecules (e.g., oligonucleotides) at various points during the synthesis and / or assembly. In some instances, oligonucleotides may be detected on the surfaces of supports (e.g., beads). This detection may be relatively non-quantitative or quantitative. Non-quantitative detection may be of interest, for example, when it is desirable to detect synthesis failure on a bead (e.g., a bead present in a well). In such instances, beads would typically be scored for a minimum amount of nucleic acid present. Quantitative detection may be of interest, for example, when it is desirable to have an estimate of the number of nucleic acid molecules present.

[00146] Most quantification methods do not provide for "exact" quantification of a nucleic acid being measured. Variations often occur due to variables such as nucleotide sequence, etc. By quantification it is meant that the measured value is within 85% of the actual amount of nucleic acid present.

[00147] In many instances, it will be desirable to be able to detect and / or quantify nucleic acid molecules in a "non-destructive" manner. By this is meant that the detection / quantification method and / or reagent does not damage the DNA molecules being identified and does not interfere with "downstream" uses of detected / quantified nucleic acid molecules. Fluorophores are one type of molecule that may be used in detection / quantification methods. In many instances, fluorophores, as well as other detectable labels, may bind to nucleic acid molecules non-covalently.

[00148] Fluorophores, as well as other detectable labels, that may be used in the practice of the invention may bind preferentially to DNA over RNA, preferentially to RNA over DNA, and / or preferentially to single-stranded (ss) nucleic acids over double-stranded (ds) nucleic acids. Thus, fluorophores that may be used in the practice of the invention may bind preferentially to ssDNA over dsRNA.

[00149] One desirable feature of fluorophores, as well as other detectable labels, that may be used in the practice of the invention is that they not exhibit substantial labeling activity when associated with solid supports (e.g., beads) and / or chemical groups (e.g., linkers) used for nucleic acid synthesis. In other words, labels will typically be chosen such that solid supports used for nucleic acid synthesis generate little or no signal prior to nucleic acid synthesis.

[00150] One example of a fluorophore that may be used to detect and / or quantify nucleic acid molecules IS QUANT-IT OLIGREEN® ss DNA Reagent (Thermo Fisher Scientific Inc.). This reagent binds non-covalently to nucleic acid molecules and may be used for quantification, wherein the quantity and / or length of a given nucleic acid molecule can be determined as a function of fluorescence intensity. Further, this fluorophore preferentially binds to single- stranded DNA and exhibits increased fluorescence when bound to nucleic acid. Also, QUANT-IT OLIGREEN® ss DNA Reagent is non-destructive and thus detected and / or quantified nucleic acid molecules may be used in later processes (e.g., error correction reactions, nucleic acid assembly reactions, etc.).

[00151] Additional fluorophores that may be used in the practice of the invention include ones that comprise a cyanine dye, a phenanthridinium dye, a bisbenzimide dye, a bisbenzimidazole dye, an acridine dye, or a chromomycinone dye. In some instances, the fluorophore is QUANT-IT OLIGREEN®, PICOGREEN®, SYBR GREEN®, SYBR GREEN II®, SYBR GOLD®, SYBR SAFE® DNA gel stain, or CYQUANT GR® dye, all of which are available from Thermo Fisher Scientific, Inc. Other fluorophores that may be used include EVAGREEN® (Biotium, Inc.), DAPI (4',6-diamidino-2-phenylindole), ethidium bromide, propidium iodide, dihydroethidium, hexidium iodide, QUANTIFLUOR® ssDNA dye (Promega Corp.), or QUANTIFLUOR® dsDNA dye (Promega Corp.).

[00152] The invention thus includes methods comprising the generation of beads comprising bound (e.g., covalently bound) nucleic acid molecules and the detection and / or quantification of the bound nucleic acid molecules.

[00153] Related methods include those for determining whether failure of nucleic acid synthesis has occurred in one or more wells of a multiwell plate. Synthesis failure may occur for any number of reasons. One such reason, when EGA based deprotection is used, is that electric current is not supplied to one or more wells at the necessary times. The invention thus includes methods for assessing the functionality of an electronic synthesis chip of a type such as that shown in FIG. 24D. The invention thus also includes methods in which a chip is used for nucleic acid synthesis and, after the addition of a number of synthesis cycles for base addition (e.g., from about 6 to about 15 cycles), beads in the wells of the chip are contacted with a dye (e.g., a fluorophore) to determine whether nucleic acid synthesis has occurred in each well. Further, wells where synthesis has not occurred, if any, may be identified. The number of wells in which synthesis does not occur and their location in the chip may be identified. The operator (or computer with preset parameters) may then choose to take actions such as directing synthesis only in functioning wells or rejecting the chip due to the number of non-functional wells.

[00154] The invention also provides methods for determining the length of nucleic acid molecules associated with solid support. This is based upon the principle that detectable labels (e.g., QUANT-IT OLIGREEN®) may be selected such that they labeled nucleic acid molecules in a manner where detectable labeling activity is proportional to the length of nucleic acid molecules being detected.

[00155] In a preferred embodiment of the invention, monodisperse beads are used and they are porous beads and characterized by a specific pore volume, wherein, for example, 1 ml of pore volume per 1 gram of polymer is equal to 50% porosity. For example, a bead with a pore volume of 2.2 ml / g polymer has a porosity of 70%. Bead porosity depends on the polymer used and may be an important factor in achieving synthesis of nucleic acid molecules of a certain length. In certain instances the pore volume of a bead suitable for aspects of the invention may be within a range of 0.1 to 2.5 ml / g polymer. Exemplary percent porosities for crosslinked polystyrene having a density of 1.1 g / ml are indicated in Table 1 below. However, porosities may also be defined on a volume basis. Bead porosities that may be useful for certain aspects of the invention may be within a range of from about 50% to about 70%, from about 60% to about 80%, from about 65% to about 75%, such as e.g. about 70%. [Image disponible dans le document PDF, Image available in the PDF document]

[00156] A monodisperse bead may further comprise a linker as defined elsewhere herein and may be characterized by its linker loading capacity. The linker loading capacity of a bead suitable for aspects of the invention may be within a range of 10 to 500 µmol / g, 50 to 350 μmol / g, 70 to 200 μmol / g, 30 to 100 μmol / g, 10 to 40 μmol / g, 20 to 50 μmol / g, 50 to 70 μmol / g or 40 to 80 µmol / g such as, for example, about 60 µmol / g. The length of a nucleic acid molecule that can be synthesized on a bead in quantitative amounts as described elsewhere herein may depend on the linker loading capacity. The higher the linker loading of a support, the higher the overall yield of synthesized oligonucleotides will be. Historically, higher linker loading capacities have been sought to increase the number of oligonucleotides that could be produced per unit area of support. However, for long oligonucleotides a very high yield may not be desirable as it may compromise the quality of the oligonucleotides as discussed supra. Thus, the adaptation of the linker loading capacity of a bead in view of the length of an oligonucleotide to be synthesized is one aspect addressed by methods and compounds of the invention to generate oligonucleotides of high quality with sufficient yields and desired length. Linker loading capacities that may be optimal to achieve synthesis of nucleic acid molecules of a certain length are indicated in Table 2. [Image disponible dans le document PDF, Image available in the PDF document]

[00157] A monodisperse bead may be further defined by its surface area. For example, the surface area of a bead may be within a range of 10 to 1000 m2 / g, between 100 and 500 m2 / g, between 200 and 400 m2 / g such as, for example, around 380 m2 / g. The surface area of porous monodisperse beads can, for example, be determined according to a method developed by Brunauer, Emmett and Teller referred to as the BET method which is based on the physical adsorption of a vapor or gas onto the surface of a solid (Brunauer, S., Emmett, P. and Teller, E., J. Amer. Chem. Soc. 60 (1938), p. 309). This method uses dry beads for testing so, for accurate measurement, the pores should be of stable volume when exposed to solvents as compared to when dry. In certain embodiments the surface area may be determined for beads dried at 60°C for 2 hours from tetrahydrofuran or methanol.

[00158] A monodisperse bead may be further functionalized or coated with reactive groups which may affect the linker loading capacity. A bead functionalized for oligonucleotide synthesis may, for example, carry amine groups and may be defined by its amine content. The amine content of a solid support may be expressed by weight % nitrogen per gram of beads and may be within a range of 0.01 and 5%, between 0.1% and 3%, between 0.15% and 0.5%, between 2% and 5%, between 0.5% and 1.5%, between 1.5% and 2%. Methods for elemental analysis to determine the weight % nitrogen and calculate amine content of solid supports (mol. amine per gram of support) are known in the art and may, for example, be calculated according to methods described by Dumas A. (1826): Annales de chimie, 33,342 or as further set forth by the US Environmental Protection Agency in method 440.0: Determination of Carbon and Nitrogen in Sediments and Particulates of Estuarine / Coastal Waters Using Elemental Analysis. In certain instances, the amine content may be about 1.8%, about 1.5%, about 1.2%, about 1.0%, about 0.8%, about 0.5%, about 0.25%, about 3%, about 3.5%, about 4% or about 5%. For example, in instances where aminostyrene is used as a monomer the amine content may be theoretically determined as indicated in Table 3 below. Table 3: Exemplary analysis of amine content for beads containing aminostyrene. [Image disponible dans le document PDF, Image available in the PDF document]

[00159] The skilled person will understand that the amine content of a solid support depends on the amine-containing compound or monomer used for polymerization. The amine content of a solid support may thus be adapted by using different amounts of an amine-containing monomer. For example, lower amounts of aminostyrene such as, e.g., less than 10 weight % or less than 5 weight % per gram of the total amount of monomers used in a polymerization mixture may be used to generate beads with a lower amine content. In certain aspects of the invention, it may be desired to decrease the amine content to limit the linker loading capacity to an amount that is optimal to achieve synthesis of oligonucleotides of a certain length. Therefore the invention also relates to monodisperse beads with a low amine content of between 0.15% and 0.5%, between 0.5% and 1.5%, between 1.5% and 2%, or an amine content of less than 3%, less than 2%, less than 1.5%, less than 1%, less than 0.5% or less than 0.25%. Alternatively, the amine content may be modified based on the selection of a different amine containing monomer or a monomer containing a functionalizable group. For example, monomers such as, vinylbenzyl-chloride may be used in the polymerization of a bead suitable for aspects of the invention. Thus, in one embodiment of the invention the amine content of a monosized bead is determined by the amount of vinylbenzyl-chloride used in polymerization.

[00160] To achieve efficient synthesis of long oligonucleotides the relation of well size to bead size, as described, above in combination with bead-specific parameters (bead surface area, pore volume, amine content, linker loading capacity) may be important parameters.

[00161] Therefore, the invention also relates to a multiwell plate for non-template directed synthesis of nucleic acid molecules, wherein the multiwell plate comprises a plurality of wells, wherein each well is configured to accommodate a monodisperse bead, wherein the diameter of beads varies by less than 10% and wherein the beads are further characterized by having a linker loading capacity of the oligonucleotide synthesis substrate is within a range of 30 to 100 µmol / g.

[00162] The invention includes compositions comprising a support for oligonucleotide synthesis, the composition comprising a solid support with a loading capacity suitable for use in the synthesis of a long oligonucleotide with a low error rate.

[00163] The invention thus includes methods for the synthesis of a long oligonucleotide with a low error rate, the methods comprising synthesizing the long oligonucleotide, wherein the long oligonucleotide is synthesized on a substrate with a linker loading capacity adjusted to allow for the production of a long oligonucleotide with a low error rate.

[00164] The invention further comprises compositions and methods wherein the long oligonucleotide synthesized is over 35 nucleotides in length.

[00165] The invention further comprises compositions and methods wherein the long oligonucleotide synthesized is of a length of from 35 nucleotides to 60 nucleotides.

[00166] The invention further comprises compositions and methods wherein the long oligonucleotide synthesized is of a length of from 50 nucleotides to 100 nucleotides and the average error rate for the synthesis of the first 35 nucleotides is within one standard deviation of the synthesis error rate of nucleotides 36 through 40.

[00167] The invention further comprises compositions and methods wherein the long oligonucleotide is synthesized with an error rate of less than 0.5%.

[00168] The invention further comprises compositions and methods wherein the long oligonucleotide is synthesized with an error rate of less than 0.5% for each of bases 3 to 50.

[00169] The invention further comprises compositions and methods wherein the amount of oligonucleotide of a certain length (e.g. more than 35, more than 40, more than 45, more than 50, more than 55, more than 60, more than 80, more than 100, more than 120, more than 150, up to about 200 bases in length) synthesized in a well / on a bead is between 30% and 50%, between 40% and 60%, between 50% and 70% or more than 50% of the total amount of oligonucleotides synthesized on a bead.

[00170] The invention further includes compositions and methods for the synthesis of an oligonucleotide on a monodisperse bead having a diameter between 25 and 40 µm (such as about 35 μm), wherein the amount of oligonucleotide of a certain length synthesized on the bead is selected from the group consisting of: 1 femtomole to 1 picomole, 10 femtomoles to 500 femtomoles, and 50 femtomoles to 250 femtomoles.

[00171] In some aspects the invention involves the use of a support such as a monodisperse bead selected for low linker loading capacity (e.g., 35-50 µmol / g), resulting in the production of smaller amounts of oligonucleotides than technically feasible. Advantages of this are that the use of low linker loading capacity allows for the production of longer oligonucleotides with low error rates (i.e. a higher percentage of correct full-length oligonucleotides), especially in the terminal region of the oligonucleotide synthesized late in the synthesis process.

[00172] The invention includes compositions and methods for the synthesis of an oligonucleotide on a support, wherein the linker loading capacity of the support is within a range selected from the group consisting of: 70 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>mol / g to 100 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>mol / g, <semantics>50μmol / g<annotation encoding="application / x-tex">50 \mu mol / g< / annotation>< / semantics> to <semantics>70μmol / g<annotation encoding="application / x-tex">70 \mu mol / g< / annotation>< / semantics>, <semantics>35μmol / g<annotation encoding="application / x-tex">35 \mu mol / g< / annotation>< / semantics> to <semantics>50μmol / g<annotation encoding="application / x-tex">50 \mu mol / g< / annotation>< / semantics>, and 15 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>mol / g to 35 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>mol / g.

[00173] The invention includes compositions and methods for the synthesis of an oligonucleotide on a support (e.g., a monodisperse bead), wherein the linker loading capacity of the support is adjusted to allow for the production of oligonucleotides between 50 and 200 nucleotides in length with an error rate of less than 0.5% and / or at a yield of between 30% and 50%, between 40% and 60%, between 50% and 70% or more than 50% of the total amount of oligonucleotides synthesized on the support.

[00174] The invention further includes compositions and methods, wherein the oligonucleotides are between 35 and 80 nucleotides in length.

[00175] The invention further includes compositions and methods, wherein the oligonucleotides are between 50 and 80 nucleotides in length.

[00176] The invention further includes compositions and methods, wherein the oligonucleotides are between 50 and 100 nucleotides in length.

[00177] The invention further includes compositions and methods, wherein the oligonucleotides are between 50 and 200 nucleotides in length.

[00178] The invention further includes a monodisperse porous bead for solid-phase synthesis of oligonucleotides of a length of between 35 and 200 bases, wherein the bead is a polystyrene bead coated with reactive groups such as amine groups or hydroxyl groups, and wherein said bead comprises: a diameter of between 10 and 100 µm or between 20 and 40 µm with a coefficient of variation of less than 10% or less than 5%, a surface area within a range of between 100 and 500 m2 / g or within a range of between 150 and 300 <semantics>m2 / g<annotation encoding="application / x-tex">m^2 / g< / annotation>< / semantics>, a porosity within a range of 60% to 80%, optionally, an amine content of between about 2% and about 8% or between about 3% and about 5%, or less than 3%, a linker loading capacity of between about 15 μmol / g to about 100 μmol / g, or between about 35 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>mol / g to about 70 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>mol / g, optionally, wherein said bead carries a linker, and wherein the linker is a universal linker.

[00179] Beads with a low amine content as discussed above will comprise a lower surface reactivity and will be occupied with a limited amount of linker molecules. However, beads loaded with linkers may still comprise reactive amine groups on their surface that may interfere with oligonucleotide synthesis. To remove such reactive groups, linker-carrying beads may be subjected to a capping process prior to oligonucleotide synthesis. For example, reagents such as acid anhydride or isocyanate may be used to inactivate reactive amines on the bead surface. Capping may be performed at room temperature for about 48 hours or alternatively at a higher temperature and reduced incubation times (such as, e.g., at 50°C for 24 hours).

[00180] Magnetic bead technology is described in U.S. Patent No. 5,512,439.

[00181] Synthesis substrates other than those composed of CPG or magnetic materials may also be used with the invention and include those composed of polystyrene (e.g., polystyrene-1% divinylbenzene, macroporous polystyrene, and poly(ethylene glycol)-polystyrene (PEG-PS)), polyamide (e.g., polyamide bonded silica gel), silica gel, and cellulose. Some of these substrates are available in resin form. In many instances, substrates that are resins may be placed in wells, instead of or in conjunction with beads, and may be used for nucleic acid synthesis.

[00182] Other nucleic acid ligation methods, and arrays which employ them, are known in the art. For example, methods are known which use an amine or a peroxide (which opens to an ether bridge) activated surface. As noted elsewhere herein, for EGA methods in the art, a hydroxyl group has been described and used to link nucleic acid to a silica bead surface. The invention includes such linking methods and compositions which contain them.

[00183] In some instances, it may also be desired to use a semi-solid support that may have a gel-like or viscous consistence or matrix instead of a solid support. The invention contemplates this and in suitable instances here where a solid support is referred to a non-solid support may be used.

[00184] Factors which determine the amount of nucleic acid which can be synthesized include surface area and size of particles upon which synthesis occurs. Thus, to some extent, support (e.g., bead) parameters can be adjusted to alter the amount of nucleic acid synthesized. Beads which may be used in the practice of the invention may vary widely in terms of size, including the following size ranges: from about 0.01 μm to about 1,000 μm, from about 0.1 μm to about 1,000 μm, from about 1.0 μm to about 1,000 μm, from about 0.01 μm to about 400 μm, from about 0.01 µm to about 200 µm, from about 0.01 µm to about 100 µm, from about 0.1 µm to about 100 μm, from about 0.1 μm to about 50 μm, from about 1.0 μm to about 600 μm, from about 1.0 μm to about 400 μm, from about 1.0 μm to about 200 μm, from about 1.0 μm to about 100 μm, from about 2.0 μm to about 400 μm, from about 2.0 μm to about 200 μm, from about 5.0 μm to about 500 μm, etc. in average diameter. In certain embodiments, the beads may have a diameter between 30 μm and 40 μm, such as a diameter of 31 μm or 32 μm or 33 μm or 34 μm or <semantics>35μm<annotation encoding="application / x-tex">35 \mu m< / annotation>< / semantics>.

[00185] Further, beads may be used which allow for an average amount of nucleic acid to be produced in the following amounts: from about 0.00001 nanomoles to about 0.0001 nanomoles, from about 0.00001 nanomoles to about 0.001 nanomoles, from about 0.00001 nanomoles to about 0.01 nanomoles, from about 0.0001 nanomoles to about 0.001 nanomoles, from about 0.0001 nanomoles to about 0.01 nanomoles, from about 0.0001 nanomoles to about 0.1 nanomoles, from about, from about 0.0001 nanomoles to about 1,000 nanomoles, from about 0.001 nanomole to about 1,000 nanomoles, from about 0.01 nanomoles to about 1,000 nanomoles, from about 10 nanomoles to about 1,000 nanomoles, from about 30 nanomoles to about 1,000 nanomoles, from about 50 nanomoles to about 1,000 nanomoles, from about 200 nanomoles to about 1,000 nanomoles, from about 1.0 nanomole to about 500 nanomoles, from about 1.0 nanomole to about 250 nanomoles, from about 10 nanomoles to about 500 nanomoles, etc. In certain exemplary embodiments, beads may be used which allow for an average amount of nucleic acid to be produced in an amount from about 90 femtomoles to about 150 femtomoles. In certain embodiments, beads may be used that allow for the number of nucleic acid molecules produced per bead to range from about <semantics>1×109<annotation encoding="application / x-tex">1 \times 10^9< / annotation>< / semantics> to about <semantics>1×1013<annotation encoding="application / x-tex">1 \times 10^{13}< / annotation>< / semantics>, such as, for example, <semantics>1×1011<annotation encoding="application / x-tex">1 \times 10^{11}< / annotation>< / semantics> nucleic acid molecules. [Image disponible dans le document PDF, Image available in the PDF document]

[00186] In many instances, the yield of nucleic acid molecules chemically synthesized decreases once a certain size has been reached. In many embodiments of the invention, chemically synthesized nucleic acid molecules will be in the range of from about 8 to about 100 nucleotides, from about 8 to about 35 nucleotides, from about 8 to about 40 nucleotides, from about 8 to about 50 nucleotides, from about 8 to about 100 nucleotides, from about 15 to about 100 nucleotides, from about 15 to about 75 nucleotides, from about 15 to about 50 nucleotides, from about 20 to about 60 nucleotides, from about 40 to about 400 nucleotides, from about 40 to about 300 nucleotides, from about 40 to about 200 nucleotides, from about 40 to about 100 nucleotides, from about 40 to about 90 nucleotides, from about 50 to about 400 nucleotides, from about 50 to about 300 nucleotides, from about 50 to about 200 nucleotides, from about 50 to about 100 nucleotides, from about 50 to about 90 nucleotides, from about 50 to about 80 nucleotides, from about 75 to about 400 nucleotides, from about 75 to about 300 nucleotides, or from about 75 to about 200 nucleotides.

[00187] As one skilled in the art would recognize, the amount of nucleic acid required to be produced will vary with, for examples, the application and the efficiency of assembly methods used. When a replicable molecule (e.g., via PCR, insertion into a cell, etc.) is generated, theoretically only one assembled nucleic acid molecule need be generated. If the number of nucleic acid molecules generated are reduced to the point where theoretically only one fully assembled nucleic acid molecule is generated, then half the time no fully assembled nucleic acid molecule will generated. Thus, one lower limit for the amount of nucleic acid to be produced using methods of the invention is based upon the number of fully assembled nucleic acid molecules which may be generated. This number will often vary with the number of synthetic nucleic acid molecules that must be combined to form the final construct. Methods of the invention will typically be designed to generate, prior to non-assembly amplification (e.g., first PCR assembly resulting in one or more replicable molecules or other final product nucleic acid molecules), from about 1 to about 500,000, from about 10 to about 500,000, from about 100 to about 500,000, from about 500 to about 500,000, from about 1 to about 1,000, from about 1 to about 500, from about 10 to about 1,000, from about 10 to about 500, from about 100 to about 1,000, from about 100 to about 500, from about 100 to about 5,000, from about 100 to about 50,000, from about 100 to about 250,000, from about 1,000 to about 50,000, etc. assembled nucleic acid molecules. As used in this paragraph, "assembled nucleic acid molecules" refers to the number of desired product nucleic acid molecules produced by direct assembly of oligonucleotides initiated by hybridization of overlapping oligonucleotides, as compared to amplification using, for example, terminal primers. Amplification after direct assembly will then result in copies being made of the assembled nucleic acid molecules. One commercial aspect of this feature of the invention is that the number of oligonucleotides synthesized can be minimized to save on cost.

[00188] As one skilled in the art would understand, nucleic acid synthesis substrate area directly reflects the number of nucleic acid molecules which may be synthesized on that substrate. Table 5 below shows bead size, surface area calculations and an estimated number of nucleic acid molecules that may be generated on the specified beads. [Image disponible dans le document PDF, Image available in the PDF document]

[00189] In some embodiments, oligonucleotide synthesis will be performed using 2.8 µm beads in a plate with one bead per well. Further, the wells may be designed as cylindrical holes or chambers that are about 4 and 3 µm deep. When well spacing of 100 µm is used, a 10 mm2 chip can accommodate 10,000 wells.

[00190] In other embodiments, oligonucleotide synthesis will be performed using 35 µm beads in a plate with one bead per well. The wells may be designed as cylindrical holes or chambers that are about 50 µm deep and about 40 µm in diameter. In certain embodiments, there may be about 30 µm spacing between wells. In certain embodiments, an 18 mm2 chip can accommodate about 35,440 addressable wells.

[00191] In many instances when plates are made by etching, the wells will be of a non-cylindrical shapes and may be pyramid, cone or quadratic shaped. In some instances, the wells may be in the shape of a reverse, truncated cone.

[00192] The number of individual nucleic acid molecules generated will also vary with the application. While costs savings can be achieved by reagent usage reductions, it will generally be desirable to generate enough nucleic acid molecules need for, for example, efficient assembly. Further, the number of nucleic acid molecules having a particular nucleotide sequence produced will generally reflect the "carrying capacity" of the synthesis substrate. For example, a 35 micron bead typically can be used to generate about 1 x 1011 nucleic acid molecules. For example, in many instances, as bead size, decreases, so will the number of nucleic acid molecules that may be produced on each bead.

[00193] Methods of the invention may be used to generate from about 100 to about <semantics>1×1013<annotation encoding="application / x-tex">1 \times 10^{13}< / annotation>< / semantics>, from about 1,000 to about <semantics>1×1013<annotation encoding="application / x-tex">1 \times 10^{13}< / annotation>< / semantics>, from about 10,000 to about <semantics>1×1013<annotation encoding="application / x-tex">1 \times 10^{13}< / annotation>< / semantics>, from about 100 to about <semantics>1×1012<annotation encoding="application / x-tex">1 \times 10^{12}< / annotation>< / semantics>, from about 1,000 to about <semantics>1×1012<annotation encoding="application / x-tex">1 \times 10^{12}< / annotation>< / semantics>, from about 10,000 to about <semantics>1×1012<annotation encoding="application / x-tex">1 \times 10^{12}< / annotation>< / semantics>, from about 100 to about <semantics>1×1011<annotation encoding="application / x-tex">1 \times 10^{11}< / annotation>< / semantics>, from about 1,000 to about <semantics>1×1011<annotation encoding="application / x-tex">1 \times 10^{11}< / annotation>< / semantics>, from about 10,000 to about <semantics>1×1011<annotation encoding="application / x-tex">1 \times 10^{11}< / annotation>< / semantics>, from about <semantics>1×1011<annotation encoding="application / x-tex">1 \times 10^{11}< / annotation>< / semantics> to about <semantics>1×1013<annotation encoding="application / x-tex">1 \times 10^{13}< / annotation>< / semantics>, from about <semantics>100<annotation encoding="application / x-tex">100< / annotation>< / semantics> to about <semantics>1×109<annotation encoding="application / x-tex">1 \times 10^{9}< / annotation>< / semantics>, from about <semantics>1,000<annotation encoding="application / x-tex">1,000< / annotation>< / semantics> to about <semantics>1×1011<annotation encoding="application / x-tex">1 \times 10^{11}< / annotation>< / semantics> 109, from about 10,000 to about 1 x 109, etc. nucleic acid molecules designed to have the same nucleotide sequence. In certain exemplary embodiments, about 1 x 1011 nucleic acid molecules may be generated per bead or well, whereas the total amount of nucleic acid molecules designed to have the same nucleotide sequence generated in a plurality of wells may be from about <semantics>1×105<annotation encoding="application / x-tex">1 \times 10^5< / annotation>< / semantics> to about <semantics>1×108<annotation encoding="application / x-tex">1 \times 10^8< / annotation>< / semantics>, from about <semantics>1×107<annotation encoding="application / x-tex">1 \times 10^7< / annotation>< / semantics> to about <semantics>1×1010<annotation encoding="application / x-tex">1 \times 10^{10}< / annotation>< / semantics>, from about <semantics>1×109<annotation encoding="application / x-tex">1 \times 10^9< / annotation>< / semantics> to about <semantics>1×1012<annotation encoding="application / x-tex">1 \times 10^{12}< / annotation>< / semantics>, from about <semantics>1×1011<annotation encoding="application / x-tex">1 \times 10^{11}< / annotation>< / semantics> to about <semantics>1×1013<annotation encoding="application / x-tex">1 \times 10^{13}< / annotation>< / semantics>, from about <semantics>1×1012<annotation encoding="application / x-tex">1 \times 10^{12}< / annotation>< / semantics> to about <semantics>1×1015<annotation encoding="application / x-tex">1 \times 10^{15}< / annotation>< / semantics>, from about <semantics>1×1011<annotation encoding="application / x-tex">1 \times 10^{11}< / annotation>< / semantics> to about <semantics>1×1016<annotation encoding="application / x-tex">1 \times 10^{16}< / annotation>< / semantics>, such as about <semantics>1×1012<annotation encoding="application / x-tex">1 \times 10^{12}< / annotation>< / semantics> to about <semantics>1×1013<annotation encoding="application / x-tex">1 \times 10^{13}< / annotation>< / semantics>.

[00194] The number of nucleic acid molecule synthesis sites (e.g., wells) can vary greatly and will be determined by a number of factors including (1) the limitations of engineering and nucleic acid molecule synthesis hardware and (2) the amount of nucleic acid which is desired (see elsewhere herein for a discussion of this factor). As examples, the number of nucleic acid molecule synthesis sites (e.g., wells) in synthesis platforms used in the practice of the invention may vary in total number between 9 and 300,000, between 9 and 100,000, between 9 and 40,000, between 9 and 1,000, between 9 and 500, between 1,000 and 200,000, between 1,000 and 400,000, between 1,000 and 500,000, between 1,000 and 1,00,000, between 1,000 and 10,000,000, between 20,000 and 1,000,000, between 50,000 and 10,000,000, between 10,000 and 5,000,000, between 1,000 and 100,000, between 2,000 and 100,000, between 5,000 and 100,000, between 10,000 and 100,000, between 20,000 and 100,000, between 30,000 and 100,000, between 1,000 and 80,000, between 1,000 and 70,000, between 1,000 and 50,000, between 1,000 and 40,000, between 1,000 and 30,000, between 1,000 and 20,000, between 1,000 and 10,000, between 1,000 and 8,000, between 1,000 and 5,000, between 5,000 and 50,000, between 10,000 and 50,000, between 5,000 and 35,000, etc. In addition, the number of nucleic acid molecule synthesis sites (e.g., wells) may vary between 1,000 and 5,000, between 1,000 and 10,000, between 1,000 and 20,000, between 1,000 and 40,000, between 2,000 and 5,000, between 2,000 and 10,000, between 4,000 and 15,000, between 100 and 1,000, between 100 and 3,000, between 100 and 5,000, between 250 and 5,000, etc. per mm2. In certain embodiments, the number of wells may be, for example, 35,440. In certain other exemplary embodiments, the number of wells may be, for example, 196,160, or, for example, 9020.

[00195] The amount of reagent space per nucleic acid molecule synthesis site (e.g., well) will vary with the size and shape of the well and, in particular, the area of the space capable of accepting reagents. This will vary with factors such as whether the nucleic acid molecule synthesis site is a flat surface (e.g., relying on surface tension to keep reagents localized over the synthesis site or a cavity (e.g., a well). Also, the amount of reagent applied may be determined by the amount of reagent necessary to cover the synthesis site, deliver the necessary amount of reactant(s), and / or dilute, remove, or wash away reagents present at the synthesis site. The amount of reagent applied (when the reagent is a liquid) and the amount of reagent space at the synthesis site may vary greatly including between 0.001 x10-15 l (femtoliter) and 100 µl, between <semantics>0.01×10−15<annotation encoding="application / x-tex">0.01 \times 10^{-15}< / annotation>< / semantics> l (femtoliter) and <semantics>100μ<annotation encoding="application / x-tex">100 \mu< / annotation>< / semantics>l, between <semantics>0.1×10−15<annotation encoding="application / x-tex">0.1 \times 10^{-15}< / annotation>< / semantics> l (femtoliter) and <semantics>100μ<annotation encoding="application / x-tex">100 \mu< / annotation>< / semantics>l, between <semantics>1.0<annotation encoding="application / x-tex">1.0< / annotation>< / semantics> <semantics>x10−15<annotation encoding="application / x-tex">x10^{-15}< / annotation>< / semantics> 1 (femtoliter) and 100 µl, between 0.1 <semantics>x10−15<annotation encoding="application / x-tex">x10^{-15}< / annotation>< / semantics> 1 (femtoliter) and 1 µl, between 0.1 <semantics>x10−15<annotation encoding="application / x-tex">x10^{-15}< / annotation>< / semantics> 1 (femtoliter) and 500 nl, between 0.1 x10-15 l (femtoliter) and 100 nl, between 0.1 x10-15 l (femtoliter) and 1 nl, between 0.1 x10-15 l (femtoliter) and 500 pl (picoliter), between 0.1 x10-15 l (femtoliter) and 100 pl, between 0.1 x10-15 l (femtoliter) and 10 pl, between 0.1 x10-15 l (femtoliter) and 1 pl, between 0.001 x10-15 l (femtoliter) and 1 pl, between 0.001 x10-15 l (femtoliter) and 1.0 <semantics>×10−15<annotation encoding="application / x-tex">\times 10^{-15}< / annotation>< / semantics> 1 (femtoliter), between 0.001 <semantics>×10−15<annotation encoding="application / x-tex">\times 10^{-15}< / annotation>< / semantics> 1 (femtoliter) and 100 <semantics>×10−15<annotation encoding="application / x-tex">\times 10^{-15}< / annotation>< / semantics> 1 (femtoliter), between <semantics>1.0×10−15<annotation encoding="application / x-tex">1.0 \times 10^{-15}< / annotation>< / semantics> l (femtoliter) and <semantics>500×10−15<annotation encoding="application / x-tex">500 \times 10^{-15}< / annotation>< / semantics> l (femtoliter), etc.

[00196] The number and size of wells used in various aspects of the invention will be determined by the overall configuration of a microchip, whereas the diameter of a bead used for synthesis of a nucleic acid molecule will in turn depend on the well dimensions. In certain aspects, the configuration of a microchip may be adapted by the variation of certain parameters. Exemplary configurations showing possible variations of (i) chip size, (ii) active area, and (iii) well diameter / distance are illustrated in the Table of FIG. 35, wherein the active area comprises the total area of a chip where the wells are contained, including the space between the wells. For example, a 18 mm2 microchip may have an active area of about 73% with 35,237 wells of 40 µm in diameter and 50 μm in depth, and with a distance between wells of about 30 μm (75% of well diameter) and a well volume of 6.28 x 10-5 µl. In instances where the wells are configured to accommodate one bead, the bead may have a diameter (measured in acetonitrile) of about 87.5% of the well diameter, which would be 35 µm in this example.

[00197] Distances between wells may be within a range of between 10% to 90% of well diameter, such as e.g. between 20% and 50%, between 30% and 70%, between 50% and 90% or between 60% and 80% such, as, e.g., 75%. Furthermore, the depth of a well may be within a range of about 100% to about 200% of the diameter of the well, such as e.g. between about 100% to about 130%, between about 110% to about 150%, between about 120% to about 170%, between about 150% to about 200%, between about 120% to about 130%, such as, e.g., about 125%. In instances, where a well is configured to accommodate a single bead, the bead diameter may be within a range of between about 55% to about 99% of the well diameter, such as, <semantics>e.g.<annotation encoding="application / x-tex">e.g.< / annotation>< / semantics>, between about 60% and about 95%, between about 65% and about 85%, between about 75% and about 90%, such as e.g. about 87%. In other instances the microchip may be configured to accommodate two or more beads of smaller sizes in a single well.

[00198] In certain embodiments, a microchip may have a shape as illustrated in FIG. 24D with a leaf-like flow chamber whereas in other embodiments the chip may have a rectangular or orthogonal shape. To arrive at an optimal chip configuration, the impact of the variation of certain parameters needs to be considered. Whereas certain parameters (such as, e.g., the number of wells or active area) may provide means for scale-up, other parameters may impact the performance or suitability of the chip for certain uses. For example, a microchip too small in size may not allow for fluidics applications and may therefore have a minimum size of not less than about 10 mm2 whereas a chip too large in size (i.e., larger than about 20 to 25 mm2) may not be compatible with high-yield manufacturing (e.g., when CMOS technology is used). Likewise, the size and dimension of a well should be such that a sufficient amount of oligonucleotides required for the assembly of larger nucleic acid molecules can be produced. For example, wells with a diameter of less than about 10 µm may be suboptimal to product sufficient amounts of oligonucleotides, whereas wells with a diameter of more than about 90 µm to about 110 µm may cause too long diffusion times for reagents, which may be problematic in certain instances, e.g., where EGA is produced.

[00199] To make the solid support material suitable for nucleic acid molecule synthesis, non-nucleosidic linkers or nucleoside succinates may be covalently attached to reactive amino groups. If necessary, however, other surface functions such as carboxyl, hydroxyl, or thiol, for example, could be used to attach a linker carrying a hydroxyl group or alternatively a 3'-attached nucleotide.

[00200] In many instances a nucleic acid molecule synthesized on a solid support such as, e.g., a bead, may be physically coupled to the support by a linker. In certain exemplary embodiments, the linker, when present, may be a chemical entity that attaches the 3'-O of the nucleic acid molecule to the solid support (e.g., a functional group on a solid support). In other exemplary embodiments, the linker, when present, may have a structure such that it allows for attachment of other functionalities in addition to the 3'-O. Such linker structures are disclosed, for example, in U.S. Patent No. 7,202,264, and may be used according to certain embodiments disclosed herein. In most cases, the linker will be stable to all the reagents used during nucleic acid molecule synthesis, but cleavable under specific conditions at the end of the synthesis process. One linker commonly used in nucleic acid molecule synthesis is the succinyl linker. Additionally, universal linkers may be used for nucleic acid molecule synthesis according to embodiments disclosed herein and discussed below. A universal linker is a linker that allows for the synthesis of nucleic acid molecules regardless of the nature of the 3'-terminal base of the first nucleotide that is to be sequenced. Different linkers with different properties are known to those skilled in the art and can be selected by the skilled person depending on the downstream process requirements.

[00201] Nucleosidic solid supports (e.g., support prederivatized with base) are widely used in nucleic acid molecule synthesis. One example of such a support is one where the 3'-hydroxy group of the 3'-terminal nucleoside residue is attached to the solid support via a 3'-O-succinyl arm. The use of nucleosidic solid supports requires usage of beads prederivatized with different types of bases (one for each base). However, the fact that a nucleosidic solid support has to be selected in a sequence-specific manner (according to the first base required for each nucleic acid molecule) reduces the throughput of the entire synthesis process due to laborious pre-selection and distribution of beads attached to a specific starter base to individual microwells.

[00202] A more convenient method for synthesis starts with a universal support where a non-nucleosidic linker is attached to the solid support material. An advantage of this approach is that the same solid support may be used irrespectively of the sequence of the nucleic acid molecule to be synthesized. One example of a universal support that can be used in the current invention is described in U.S. Patent No. 7,202,264. However, other universal linkers known by the skilled in the art may be equally appropriate to carry out the invention. For the complete removal of the linker and the 3'- terminal phosphate from the assembled nucleic acid molecule, some of the universal solid supports known in the art require gaseous ammonia, aqueous ammonium hydroxide, alcohols, aqueous methylamine or a mixture thereof. Additionally, some of the universal solid supports known in the art may be photocleavable and thus require UV lightwaves for removal. See, for example, Anderson, E. et al., "Novel photocleavable universal support for oligonucleotide synthesis," Nucleosides, Nucleotides and Nucleic Acids, vol. 22 (5-8): 1403-6 (2003), disclosing a photocleaving linker comprising a nucleophilic amine protected with a photocleavable group.

[00203] In some embodiments, supports prederivatized with a base (e.g., dU, dA, dT, dC, dG, etc.) present may be employed. For example, a synthesis chip with multiple (e.g., four) loading regions (e.g., reagent flow zones) may be used so that beads prederivatized with the same base can be loaded in the same region. Also, multiple synthesis runs could be made in which the starting base is different in each run. Thus, for example, the first run may be made with nucleic acid molecules that begin with dA, followed by dC, then dT, then dG. Another possibility would be to synthesize nucleic acid segments, wherein all of the nucleic acid molecules being synthesized begin with the same base. For example, synthesis start points could be chosen that begin with a dG, with initial dGs chosen as start points being positioned so that suitable sequence complementarity regions are generated to allow for assembly of a final product nucleic acid molecule.

[00204] A number of methods for synthesizing nucleic acid are known. Many of these methods follow a series of basic steps, such as, for example, the following, with appropriate washing steps using, for example, acetonitrile, ethylacetate or other washing reagents suitable for practicing the invention: a) the first nucleotide, which has been protected at the 5' position (or, in certain embodiments wherein synthesis proceeds in the 5' to 3' direction, the first nucleotide may be protected at the 3' position), is derivatized to a solid support, such as a polystyrene bead or controlled pore glass, or is obtained prederivatized; b) the sugar group of the first nucleotide is deprotected (e.g., via detritlyation) (a process often referred to as "Deprotection"), using, for example, an EGA, trichloroacetic acid in methylene chloride or dichloroacetic acid in toluene, which results in a colored product which may be monitored for reaction progress; c) the second nucleotide, which has the phosphorus, sugar and base groups protected, is added to the growing chain, usually in the presence of a catalyst, such as, for example, tetrazole or 4,5-dicyanoimidazole (a process often referred to as "Coupling"); d) unreacted first nucleotide is capped to avoid accumulation of deletions, using, for example, acetic anhydride and N-methylimidazole (a process often referred to as "Capping"); e) the phosphite triester is oxidized to form the more stable phosphate triester, usually using any suitable compound, for example, iodine reagents (a process often referred to "Oxidizing"); f) the process is repeated as needed depending on the desired length of the nucleic acid molecule; and g) cleavage from the solid support is done, usually using aqueous or gaseous ammonia at elevated temperatures. The skilled in the art will recognize that in certain embodiments of the invention the order of steps may vary or some of the steps including the washing steps may be repeated as appropriate according to the used protocol.

[00205] In the current invention, the state of the art phosphoramidite synthesis chemistry is further improved by modification of specific steps of the above protocol. In one embodiment organocatalysts can be used to improve, for example, the efficiency of the coupling step. Organocatalysts and some uses of such catalysts are set out in Avenier and Hollfelder, Combining Medium Effects and Cofactor Catalysis: Metal-Coordinated Synzymes Accelerate Phosphate Transfer by 108 Chem. Eur. J. 15:12371 – 12380 (2009) and Jordan et al., Asymmetric phosphorylation through catalytic <semantics>P(III)<annotation encoding="application / x-tex">P(III)< / annotation>< / semantics> phosphoramidite transfer: Enantioselective synthesis of D-myo-inositol-6-phosphate, Proc. Nat. Acad. Sci. USA, 107: 20620–20624 (2010). Electrochemically and Photogenerated Acids

[00206] In some embodiments, the invention makes use of localized chemical reactions through the production of electrochemically generated acid (EGA). In other embodiments, the invention makes use of localized chemical reactions through the production of photogenerated acid (PGA). As an example, addressable electrical or photogenerated signals may be used for the production of acid at sufficient concentration to allow deprotection of the dimethoxytrityl (DMT) protecting group from surface. (Maurer et al., "Electrochemically Generated Acid and Its Containment to 100 Micron Reaction Areas for the Production of DNA Microarrays" PLoS, Issue 1, e34 (December 2006).)

[00207] One issue with the production of EGA or PGA as part of a nucleic acid molecule synthesis protocol on a surface (e.g., a microsurface) is "splash over" to adjoining regions. "Splash over", which includes diffusion, can result in reactions occurring in unintended location (e.g., caused by diffusion of EGA or PGA). While such effects may be fairly minor when one reaction occurs, when multiple reactions occur in succession splash over effects multiple reaction cycles may result in numerous misincorporated bases. This issue can be addressed in several ways. One way is to overlay the reaction areas with a buffer (e.g., a buffer containing an organic base) which sufficiently neutralizes the acid if it moves from the local environment. Another way is through physical containment or compartmentalization. For example, if the EGA or PGA is generated in a well and catalyzes a reaction in that well, the well may be of sufficient size to prevent the acid from exiting. Containment within the well is thus a factor of the size of the well and the amount of acid generated. In some reaction formats, some acid will invariably exit the well. This should pose no problems unless a quantity sufficient to catalyze a reaction reaches another well in which that reaction is not supposed to occur. As noted above, the use of an overlaying buffer can be used to minimize such reactions.

[00208] Factors other than splash over can also result in failed or incomplete nucleic acid synthesis, including bead loss, incomplete filling of the wells with beads, cross contamination of one or more wells, defective electrodes, or incorrect or contaminated reagents. To account for splash over or these other factors, each separate nucleic acid molecule to be synthesized can be assigned to more than one well. In addition, strategic placement of these replicate nucleic acid molecules throughout the microchip can mitigate the effects of splash over or other factors that can lead to failed or incomplete nucleic acid synthesis.

[00209] The array of individually addressable electrodes associated with each well permits replicates of the same nucleic acid molecule to be spread throughout the whole microchip. In this way, the effect on any error during the synthesis step can be mitigated. For example, if an unexpected event (e.g., cross contamination of neighboring wells, defective electrodes) occurs within a specific region of the microchip, but the replicates of the same nucleic acid molecule are spread across the microchip rather than being localized in the same region, fewer replicates will be affected. This risk mitigation strategy can be accomplished by mapping the replicates of the same nucleic acid molecule to different regions of the microchip using custom programming.

[00210] The number of wells to which a nucleic acid molecule is assigned for synthesis can vary based on factors, such as, the amount of nucleic acid to be produced, how difficult it is to synthesize the nucleic acid molecule, and how many fragments need to be synthesized. The number of wells to which a nucleic acid molecule is assigned may be, for example, between 1 and 10, between 1 and 20, between 1 and 50, between 1 and 100, between 1 and 1000, between 1 and 10,000, between 1 and 34,000, between 1 and 50,000, between 1 and 100,000, between 1 and 500,000, between 5 and 10, between 10 and 20, between 10 and 50, between 10 and 100, between 100 and 1000, between 1000 and 10,000, between 10,000 and 34,000, between 10,000 and 50,000, between 34,000 and 50,000, between 50,000, and 100,000, and between 100,000 and 500,000.

[00211] By way of example, for a microchip having 35,440 wells, if each nucleic acid molecule was assigned to 10 wells, 3,544 nucleic acid molecules could be synthesized in parallel on a single microchip. Increasing the number of wells in a microchip usually results in smaller volume sizes for each well. By way of example, for a microchip having 35,440 wells, the wells typically have a volume of about <semantics>6.3×10−5μl<annotation encoding="application / x-tex">6.3 \times 10^{-5} \mu l< / annotation>< / semantics>.

[00212] Plates which may be used in the practice of the present invention include modified forms of plates described in U.S. Patent Publication No. 2010 / 0137143 A1, the disclosure of which is incorporated herein by reference, shows such a representative plate format.

[00213] FIGs. 2A and 2B are schematic representations of a row of wells 200 according to an embodiment of the invention. The embodiment of FIGs. 2A and 2B illustrates five wells each containing a magnetic bead 201 at the bottom. Beneath each well is an electrode 202 which can deliver current to the well that it is associated with. Each electrode is communicates with a current controller 203 which regulates current to the electrode. The magnetic bead may contain a linker associated with an initial building block. As an example, the bead may contain first nucleotide (with an A, T, C, G or U base, or a modified base, depending on the first base desired in the nucleic acid molecule to be synthesized). The first base may be added as part of the synthesis process (e.g., with the bead having a protected hydroxyl group) or may be prederivatized prior to insertion into the well. In either event, in most cases, a protective group will be present (e.g., at the 5' position) which must be removed before another base may be covalently connected as part of a nucleic acid molecule chain.

[00214] Microfluidic channels (not shown in FIG. 2A) may be included for efficiently addition and removal of reagents from the wells. Thus, the invention includes, in part, a microfluidic plate designed to interface with a microfluidic system for adding and removing fluids from wells of the plate. Microfluidic channels used in similar plates are described in U.S. Patent Publication No. 2010 / 0137143 A1.

[00215] The cover of the plate 204 shown in FIG. 2A contains aligned electrodes which are connected to the current controller. A larger electrode (e.g., an electrode which extends over the tops of all of the wells) may be included in the cover to "close the circuit". Thus, the cover may contain one electrode aligned with each well for which an electrochemical reaction is sought to be, one electrode in operable connection with all wells of the plate, or multiple electrodes some or all of which are in operable connection with two or more wells. In an alternative embodiment, the cover electrode for each well is replaced with one or more electrodes embedded or positioned along one or more sidewall of each well. Thus, it is not critical that electrodes be positioned in the cover. In fact, in many instances, it will be desirable (e.g., ease of manufacturing) to place the electrodes in a place other than the cover.

[00216] Reference electrodes (RE) 205 may also be included to provide a stable and pre- defined electric potential. To apply a specific potential on a working electrode (WE), the potential of the WE against the potential of the RE may be measured. Next the potential between counter electrode (CE) and WE may be adjusted until the potential between RE and CE has the correct value.

[00217] One method for deprotection may employ the oxidization of hydroquinone to benzoquinone (redox system) on the WE in order to produce protons. To set a specific pH in a well, a constant current may be applied for a specified period of time. In instances of a less active WE, a strong increase of the WE potential will occur. This can lead to unintended reactions (e.g., oxidation of the solvent or damage of WE material at high potential). To avoid this effect, the potential of the WE may be controlled.

[00218] The current controller (interchangeably, controller) may be a microprocessor or processor, such as shown in FIG. 15, for example. The controller (not shown) may comprise a conventional current control system, including, for example, a microprocessor circuit in communication with a memory circuit. The memory circuit may include instructions for directing the microprocessor circuit to energize one or more of the electrodes (e.g., energize electrodes associated with well 1 or a plurality of wells). Optionally, the memory circuit may include instructions for activating one of a pair of electrodes (e.g., activate the bottom electrode associated with well 1). In still another embodiment, the memory circuit may include instructions for gradually increasing / decreasing bias to the electrodes so as to reduce possibility of a sudden surge at the well.

[00219] In another embodiment, the current controller communicates with external processor circuit(s) such as a potentiostat circuit, input / output ("I / O") devices and displays. The circuit or circuit board enables the control of the device and may also be used to communicate with other devices (such as PC, iPad, etc.).

[00220] In a variation of the embodiment of FIG. 2A, both electrodes (the anode and the cathode) may be placed at the bottom of the well. This allows for electrical current to be generated near the bottom of the well, thereby generating a localized EGA in the area closely adjacent the bead. Depending on the method by which reagents are added to and / or removed from the wells and other factors, such configuration can be used to limit cross-talk between the wells, interference or unintended EGA contamination.

[00221] A related embodiment is shown in FIG. 2B. Here the cover contains aligned electrodes 205 which extends into the reagent portion of the well. Drainage tubes 206 are positioned at the bottom of each well. These drainage tubes serve several functions. One function is removing reagents at the completion of a chemical reaction step <semantics>(e.g.,baseaddition,<annotation encoding="application / x-tex">(e.g., base addition,< / annotation>< / semantics> washing, deprotection, etc.). Another function is lowering the fluid level for the deprotection step. In other words, fluid may be added to all of the wells, then the fluid level may be lowered through drainage tubes before biasing the wells. Lowering the well's fluid level reduces cross- spillage between wells and increases synthesis fidelity. The lowered fluid level also decreases potential cross-talk and contamination between adjacent wells. The same is true of general fluid removal through the bottle of the well. This is so because cross-well contamination with EGA can result in incorrect base incorporation. Even if EGA generated base mis-incorporation occurs in 0.5% of nucleic acid molecules being synthesized in adjoining wells, the net result could be roughly a doubling of base mis-incorporation. Thus, drawing down the fluid level in the wells and bottom of the well drainage results in increase synthesis fidelity.

[00222] One means for removing fluid from wells is from the top of the wells. This can be done by any number of means including the use of pipette tips or the introduction of an absorbent material. In either instance, the goal would be to remove enough fluid from each well to minimize "splash over". In some instance, the only wells that fluid levels will be reduced in will be ones which undergo a reaction (e.g., the generation of EGA, resulting in deprotection). In other words, fluid level reduction can be performed only in wells where one or more reactants are generated.

[00223] The construction of the wells can be accomplished by conventional manufacturing methods, including, for example, CMOS and VLSI techniques. The wells can be formed in semiconductor or polymeric substrates. In an exemplary embodiment, the wells are configured in a semiconductor substrate using conventional etching and boring techniques. The insider surface of the wells may be coated with insulating material to reduce cross talk between adjacent wells. In corollary embodiment, well surfaces may be coated to increase conductivity thereby generating EGA more uniformly. Well surfaces may be coated with different layers to reduce cross-talk while increasing electro- or thermal-conductivity inside the well. Thus, the walls may comprise a composite of different material which while reducing cross-talk between the wells, would increase conductivity within each well for rapid EGA generation.

[00224] The top surface of the wells (the span between adjacent wells) may also be coated to provide reagent repellent surfaces. By way of example, the top surfaces may be coated with hydrophobic compositions to repel cross-contamination. Methods for reducing well to well cross-contamination are set out in U.S. Patent No. 6,444,111. This method uses a low concentrated base as proton scavenger which produces a proton gradient with a high concentration at the site of EGA generation and a lower concentration with increasing distance from the reaction center. Ideally, such gradient should allow for efficient removal of the DMT group on the active synthesis position and at the same time prevent protons from reaching a neighboring synthesis position. However, this method suffers from the difficulty of adjusting the scavenger base to a concentration that results in a 100% deprotection at the active synthesis position and 0% deprotection at the adjacent inactive synthesis positions. Whereas a base concentration that is too high would result in an incomplete deprotection reaction at the active synthesis site causing deletions in the growing nucleic acid molecule, a base concentration that is too low would allow protons to escape to neighboring sites causing deprotection and base insertions at those inactive synthesis sites.

[00225] To overcome these limitations, the inventors have developed a method which uses proton scavenger base bound to solid supports. A representative embodiment of using scavenger beads to avoid cross-talk between synthesis sites is illustrated in FIG. 47 (panels A-F). In this example, nucleic acid molecules are synthesized on a ~32 µm porous monodisperse bead ("synthesis support") in a well of a microfluidic chip via EGA generation as described elsewhere herein. Protons (H+) required to remove the temporary DMT protecting group on nucleic acid molecules growing on a synthesis support are generated at the bottom of a first well comprising an active electrode (FIG. 47, Panel A, left part), whereas no protons are produced in an adjacent second well comprising an inactive electrode (FIG. 47, Panel A, right part). Protons generated in the first well diffuse to the top of the well. To prevent protons from leaving the first well, smaller beads ("scavenger beads") coated with basic groups (e.g. alkaline amine groups such as NH2 or NEt2) are placed in the first and second wells to cover the synthesis supports. The basic groups capture protons diffusing from the first well (leading to a conversion of the NH2 group to an NH3 group in this example), such that no protons will reach a second well with an inactive electrode (FIG. 47, Panel B). In case some of the protons may escape from a first well, they can be captured by scavenger beads present in the second wells before reaching the DMT protecting groups. Finally, a restoring solution is flushed over the wells to remove the protons from the scavenger beads to restore the basic groups on the scavenger beads for the next synthesis cycle. In the example of FIG. 47, Panel C the restoring solution comprises an alkaline molecule (e.g., 5% triethylamine (NEt3) diluted in toluene). Different solutions with base restoring properties are known to those skilled in the art and can be selected by the skilled person depending on the reactivity of the converted scavenger groups.

[00226] The proton-mediated conversion and subsequent restoration of base-coated scavenger beads is further illustrated by FIG. Panels D through F.

[00227] In some embodiments, the scavenger base does not react with the phosphoramidites used as building blocks for the nucleic acid molecules growing on the synthesis support. This can be achieved by using scavenger groups that are not nucleophilic such as, e.g., triethylamine, lutidine or 1,8-Diazabicycloundec-7-ene or a diethylamine group bound to an aliphatic residue. Alternatively, the scavenger base may be selected to generate an instable product that rapidly decays upon reaction with a phosphoramidite, such as, e.g., a tetrazolium salt. Scavenger beads may be provided with various sizes but the size should be selected to allow placement of multiple scavenger beads into a well in combination with at least one synthesis support. Scavenger bead sizes used in the practice of the invention may vary and depend on the size of a well and / or the size of a synthesis support placed in a well of a given size. Typically, a scavenger bead may have a size that is smaller than the size of a synthesis support used in combination with the scavenger bead. For example, a scavenger bead may have a diameter that is about 10%, 25%, 30%, 50%, 60%, 75% or 80% of the diameter of a synthesis support. In certain embodiments, scavenger beads may include beads with diameters between 0.05 µm and 3 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m, 1 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m and 5 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m, 3 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m and 10 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m, 5 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m and 20 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m, 10 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m and 30 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>m.

[00228] In some instances, scavenger beads may fill the majority (e.g., greater than 60%) of the void in wells containing synthesis beads. In such cases, scavenger beads would often be small enough to fit into the various void spaces. One such void space is the lower portions of the wells below the synthesis beads. In some instances, scavenger beads may surround synthesis beads. In such instances, synthesis beads will typically not fill the horizontal volume of the wells.

[00229] In some instances scavenger beads may be larger than wells containing synthesis beads. In such instances, scavenger beads may be used to "cap" the wells. Such capping scavenger beads may be removed by the flow stream when reagents are exchanged. Further, capping scavenger beads may be used in conjunction with scavenger beads that are smaller than synthesis beads.

[00230] In some instances, well depth may be adjusted to allow for the addition of various depths of scavenging beads on top of synthesis beads. In some instances, scavenging beads may be the same size as synthesis beads and the wells may be of a depth that is suitable for holding two beads. In such instances, typically scavenging beads would be located at the top of the wells and synthesis beads would be located at the bottom of the wells.

[00231] As further supported by Example 11, the inventors have demonstrated that scavenger beads are capable of efficiently protecting neighboring synthesis sites by avoiding cross-talk of generated protons. Scavenger beads according to the invention can be used in any acidic environment where proton cross-talk between reaction or synthesis sites should be avoided (such as chip or array formats using electro-chemically or photogenerated acid as described elsewhere herein). The skilled person would understand that in systems where a base is generated (e.g., an electro-chemically generated base) the scavenging mechanism could be reversed to use scavenger beads carrying acidic groups on the surface. The invention thus includes a method for protecting one or more second synthesis sites in solid phase nucleic acid synthesis from contamination with protons generated at one or more adjacent first synthesis sites, wherein said method comprises (a) providing a solid support carrying basic groups on the surface, (b) placing the solid support at a first synthesis site such that the support is in fluid communication with protons diffusing from that first synthesis site, (c) allowing the basic groups on the solid support to react with the diffusing protons thereby converting the basic groups into the corresponding acid, (d) optionally restoring the basic groups on the solid support by contacting the solid support with a restoring agent.

[00232] Furthermore, the invention comprises the use of scavenger beads as specified above for EGA- or PGA-based nucleic acid synthesis platforms.

[00233] Finally, the shape of the wells may be configured to reduce cross-contamination while increasing reaction speed. For example, the wells may be configured to have cylindrical, barrel or conical shapes.

[00234] In many methods using, for example, the plate configuration of FIGs. 2A-2B, the sugar group of the first nucleotide is deprotected by activating (energizing) a chemical reaction initiated by an electrical signal (or a pulse). As noted elsewhere herein, one method for doing this is through the generation of an electrochemically generated acid (EGA). In other embodiments, the sugar group of the first nucleotide is deprotected by activating (energizing) a chemical reaction initiated by a photogenerated signal. In many cases, it will be desirable to control the amount of chemical reactant made (e.g., EGA or PGA) so as to efficiently catalyze the deprotection reaction while limiting the possibility of reactant from cross-contamination.

[00235] FIG. 17 shows a top view of a channel chip design having three electrodes. Counter electrode elements 1700 and 1702 are located at the top of the two side channels and across the bottom of the flow channel 1701. Reference electrodes 1703 surround the two wells with working electrodes 1704 are also present.

[00236] In order to limit the flow of protons a series of steps may be taken, including (1) the use of buffers which prevent significant pH shifts in the presence of small amounts of protons, (2) the use of a quinone redox system, and (3) designing the dimensions of wells and channels to maintain substantial distances between them (e.g., using well volume 150 times smaller than according channel volume).

[00237] For example, using the schematic shown in FIG. 17 for purposes of illustration, the distances between the working electrode 1704 and counter electrode elements 1700 and 1702 may be about 200µm. Further, interception of protons by base molecules may be used to decrease the number of protons that reach other wells. Also, reference electrode strips 1703 between wells having the same potential as the counter electrode elements 1700 and 1702 can be used to generate base molecules and further could prevent proton "cross-talk". Methods and components such as these, in addition to other methods set out herein, provide for high fidelity nucleic acid molecule synthesis.

[00238] For purposes of illustration, a prederivatized bead (which may be magnetic or non- magnetic) may be placed in wells 1 through 5 of FIGs. 2 and 2B with an "A" bead in wells 1 and 5, a "C' bead in well 2, a "U bead in well 3, and a "G" bead in well 4. All five wells may then be filled with an EGA reagent (e.g., a reagent containing methanol, acetonitrile, hydroquinone, anthraquinone, tetraethylamonium p-toluene sulfonate, and 2,6-lutidine). The next base to be added to chain is G and the only nucleic acid molecule of the molecules to be generated which contains a G at position 2 is in Well 1. Thus, current is applied only to Well 1. This current creates an acidic microenvironment which results in deprotection of the 5' position of the nucleotide only in Well 1. After a fixed (or variable) reaction time, all five wells are washed. A nucleotide, which has the phosphorus, sugar and a base (a T in this instance), is added to all of the wells in the presence of a catalyst (e.g., a tetrazole catalyst). After a predefined reaction time, all five wells are washed and unreacted first nucleotide may be capped to avoid accumulation of deletions, using, for example, acetic anhydride and N-methylimidazole. Again, after a predetermined reaction time, all five wells are washed and phosphite triesters formed by chemical reaction may be oxidized to form the more stable phosphate triester, using, for example, iodine containing reagents. This process is then repeated until the final base of the nucleic acid molecule has been added. Later, the synthesized nucleic acid molecules may be cleaved from the solid support. This may be done, for example, using aqueous or gaseous ammonia with heating. The cleavage method may vary, however, with factors such as the linker used.

[00239] The amount of current applied to each well and its duration will vary with parameters such as the amount of reagent to be generated and the size of the well. The applied current may be a pulse of varying shape and / magnitude. The pulse may define a series of varying amplitude pulses (frequency) or a gradual increase / decrease amplitude. The amplitude and duration of the pulse can be adjusted for the optimum generation of reagent. As an example, the current applied to a well may be adjusted for a specified period of time to generate a specified quantity of EGA. The amount of EGA intended for generation will typically be at least enough sufficient to fully catalyze deprotection of the nucleic acid molecules present.

[00240] High fidelity nucleic acid synthesis requires that almost complete deprotection of oligonucleotides occurs prior to addition of the next base in the oligonucleotide chain. Incomplete deprotection will typically result in a subset of the oligonucleotides missing a base in the "undeprotected" molecules. Highly acidic environments have been shown to depurinated nucleic acid molecules. This poses a synthesis quality issue because highly acidic conditions will result in near complete deprotection but can also result in depurination of oligonucleotides being deprotected.

[00241] Parameters may be adjusted in manner in which near complete deprotection of an oligonucleotide occurs with minimal depurination. Some of the factors that may be adjusted to achieve this goal include the concentration of EGA precursor placed in contact with the synthesis support, the amount of current (direct or alternating) applied to the solution containing the synthesis support, the length of time the current is applied to the solution, the presence of a buffering agent in the solution (including the type, concentration, and pKa of the buffering agent, when present), the number of molecules of oligonucleotide being synthesized, and the length of time that EGA is in contact with the synthesis support. The invention includes compositions and methods where one or more of these parameters, as well as other parameters are altered to adjust the fidelity of nucleic acid synthesis. In many instances, parameters will be adjusted so as to provide for high fidelity nucleic acid synthesis.

[00242] Application of current to affect EGA-based DMT deprotection: Current may be applied constantly up to 2 µA and voltage up to 10 V, such as up to 8 V or 7.5 V, is applied to an electrode in the controlled circuit for a time period of up to 30 seconds. Current may also be applied in pulse durations from 1ms to 2000ms during a time of 1ms to 60 seconds. Current may also be applied as in various pulses (e.g., from about two to about 10,000, from about ten to about 10,000, from about fifty to about 10,000, from about 100 to about 10,000, from about 1,000 to about 10,000, from about ten to about 500, etc. pulses) up to <semantics>2μA<annotation encoding="application / x-tex">2 \mu A< / annotation>< / semantics> (e.g., from about 0.02 nA to about 20,000 nA, from about 0.2 nA to about 20,000 nA, from about 0.2 nA to about 5,000 nA, from about 0.2 nA to about 2,000 nA, from about 0.2 nA to about 1,000 nA, from about 0.2 nA to about 5000 nA, from about 2.0 nA to about 20,000 nA, from about 2.0 nA to about 10,000 nA, from about 2.0 nA to about 5,000 nA, from about 2.0 nA to about 2,000 nA, from about 5.0 nA to about 20,000 nA, from about 5.0 nA to about 8,000 nA, from about 10 nA to about 20,000 nA, from about 10 nA to about 8,000 nA, from about 10 nA to about 5,000 nA, from about 20 nA to about 20,000 nA, from about 20 nA to about 8,000 nA, from about 50 nA to about 20,000 nA, from about 50 nA to about 10,000 nA, from about 50 nA to about 5,000 nA, from about 100 nA to about 10,000 nA, from about 500 nA to about 20,000 nA, from about 500 nA to about 10,000 nA, from about 500 nA to about 5,000 nA, from about 1,000 nA to about 20,000 nA, from about 1,000 nA to about 10,000 nA, etc.). In certain embodiments, the current may be applied, either constantly or in pulses, up to about 1 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>A, such as up to about 0.5 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>A or up to about 0.3 µA. In certain exemplary embodiments, the applied potential between the working electrode and the control electrode is at about 7.5 V.

[00243] In some instances, current may be pulsed for anywhere from about 1 second to about 30 seconds, from about 2 second to about 30 seconds, from about 4 second to about 30 seconds, from about 5 second to about 30 seconds, from about 5 second to about 20 seconds, from about 5 second to about 15 seconds, from about 5 second to about 10 seconds, etc. Of course, efficient deprotection and nucleic acid molecule synthesis must be determined as the exact composition and concentration of EGA reagent is influenced by the precise conductive, structural and geometric properties of the electrodes and microwells and the parameters associated with the application (current, voltage and time) of current.

[00244] Composition and concentration of EGA components: The exact composition and concentration of EGA reagent is influenced by the precise conductive, structural and geometric properties of the electrodes and microwells and the parameters associated with the application (current, voltage and time) of current to convert the EGA to its acid forms. Generally, the smaller the volume for EGA production to affect deprotection, the smaller the required current strength and / or time of current application. Since the amount of nucleic acid molecule produced in such microscale systems falls below a threshold that can be directly and accurately measured, surrogate assays, such as hybridization or product enrichment following target amplification, for nucleic acid molecule synthesis and coupling efficiency are typically required.

[00245] A number of reagents may be used for the production of electronically generated acid. Generally it will be desirable to generate acid locally in order to deprotect terminal nucleotides connected to solid supports (e.g., a bead). A number of reagents are known in the art. These reagents may contain compounds that produce protons when electrons are removed through use of an electrode, a solvent, a buffering agent, and a compound that enhances the conductivity of the reagent mixture. For example, a reagent composed of hydroquinone, quinone, acetonitrile, and tetrabutyl ammonium hexafluorphosphate is set out in PCT Publication WO 2003 / 020415. Also, a reagent composed of hydroquinone, anthraquinone, acetonitrile, methanol, tetraethyl ammonium p-toluene sulfonate, and 2,6-lutidine is set out in Mauer et al., PLoS One, Issue 1:e34 (2006). Further, a similar reagent composed of hydroquinone, benzoquinone, acetonitrile, methanol, tetraethyl ammonium p-toluene sulfonate, and 2,6-lutidine is set out in PCT Publication WO 2006 / 105037.

[00246] These reagents may contain compounds that generate protons when electrons are removed through use of an electrode, a solvent, a buffering agent, and a compound that enhances the conductivity of the reagent mixture. An exemplary reagent composed of hydroquinone, quinone, acetonitrile, and tetrabutyl ammonium hexafluorphosphate is set out in PCT Publication WO 2003 / 020415. Also, a reagent composed of hydroquinone, anthraquinone, acetonitrile, methanol, tetraethyl ammonium p-toluene sulfonate, and 2,6-lutidine is set out in Mauer et al., PLoS One, Issue 1:e34 (2006). Further, a similar reagent composed of hydroquinone, benzoquinone, acetonitrile, methanol, tetraethyl ammonium p-toluene sulfonate, and 2,6-lutidine is set out in PCT Publication WO 2006 / 105037.

[00247] Acetonitrile may be replaced with any suitable solvent capable of dissolving the components to form the deblocking solution for electrochemical deblocking of acid-labile protecting groups, so long as the solvent does not interfere with the chemical synthesis process.

[00248] Methanol is present in the deblocking solution to enhance the solubility of hydroquinone, benzoquinone and derivatives of thereof. In brief, the higher the methanol concentration, the more hydroquinone that can be added to the deblock solution. Since hydroquinone is a main proton source when the electric field is applied, the concentration of hydroquinone desirable may be adjusted to generate the desired number of protons under the selected conditions. The methanol may also act as a proton source. Methanol may be replaced with other alcohols as long as methanol function(s) are preserved in the deblocking solution. If solubility of compounds in the solvent is not an issue, then the alcohol may be omitted.

[00249] Benzoquinone is believed to react at the cathode to form a hydroquinone derivative. This compound may also be replaced, or even omitted. However, a higher potential is generally required, if no benzoquinone (or similar compound) is present in the mixture. Such higher potentials may (1) harm electrical components and other hardware, (2) induce the formation of undesirable reactants, (3) damage nucleic acid molecules being produced, and (4) cause the formation of gas bubbles.

[00250] Tetraethyl ammonium p-toluene sulfonate is a salt that provides conductivity to the deblocking solution to allow electrochemical generation of acidic reagent at active electrodes. This compound may also be replaced with a compound that performs a similar function.

[00251] 2,6-lutidine is believed to confine the electrochemically generated acidic reagent to the active electrode area by reacting with the acidic reagent as it diffuses away from the space immediately above the active electrode. This compound may also be replaced with a compound that performs a similar function. [Image disponible dans le document PDF, Image available in the PDF document]

[00252] Solvents that may be used for the generation of EGA may be aqueous or non- aqueous. An aqueous, solvent-based, EGA reagent is set out in U.S. Pat. No. 6,093,302 is composed of a sodium phosphate buffer, pH 7.2. Additional aqueous buffers disclosed therein include acetate buffers, borate buffers, carbonate buffers, citrate buffers, HEPES buffers, MOPS buffers, phosphate buffers, TRIS buffers, and KI solutions.

[00253] Non-aqueous solvents (e.g., organic solvents) that may be used for the generation of EGA. Representative examples of solvents include methylene chloride, 1,1,1-trichloroethane, 1,1,2-trichloro-1,2-diifluoroethane, 1,1,2-trichloroethane, 1,4-dichlorobenzene, 1-butanol, dimethyl sulfoxide, 1-hexene, 1-propanol, 2-(2-butoxyethoxy) ethyl acetate, 2-butoxyethanol acetate, 2-butoxyethyl acetate, 2-ethoxyethanol acetate, 2-ethoxyethanol, triethylene glycol, 2- methoxyethanol acetate, 2-methoxyethanol, 2-methylhexane, 2-nitropropane, acetone alcohol, acetone, acetonitrile, allyl alcohol, benzene, ethylbenzene, ethylene glycol, formamide, furfural, n-methoxynonafluorobutane, n-methylpyrrolidone, n-nonane, n-octane, n-octyl alcohol, n-butyl acetate, n-pentane, n-propyl acetate, n-propyl alcohol, ortho-dichlorobenzene, perchloroethene, propylene glycol diacetate, propylene glycol, t-amyl alcohol, t-butyl alcohol, tetrahydrofuran, toluene, trans-1,2-dichloroethylene, trichloroethene, trichloroethylene, trichloromethane, vinyl choloridediethylene glycol, dimethyl formamide, furfuryl alcohol, heptafluorocyclopentane, heptafluoropropyl methyl ether, heptane, hexachlorocyclohexane, hexane, isoamyl alcohol, isobutyl acetate, isobutyl alcohol, isobutyl isobutyrate, isomethoxynonafluorobutane, iso- methoxynonafluorobutane, isophorone, isopropyl acetate, iso-propyl alcohol, methanol, methoxy propyl acetate, methyl amyl ketone, methyl chloride, methyl chloroform, methyl ethyl ketone, methyl glycol acetate methyl isobutyl ketone, nitrobenzene, nitromethane, dipropylene glycol, ethanol, ethyl acetate, ethyl benzene, ethyl ether, ethyl glycol acetate, ethyl glycol, benzyl chloride, biphenyl, diacetone alcohol, dibromomethane, dichlorodiphenyltrichloroethane, dichloroethene, diethyl ether, cycloheptane, cyclohexane, cyclohexanol, cyclohexanone, cyclononane, cyclooctane, cyclopentane, chlorobenzene, chlorobromomethane, methyl propyl ketone, monochlorotoluene, n-amyl alcohol, n-butyl acetate, n-butyl alcohol, n-decane, cyclodecane, and xylene, and combinations thereof.

[00254] In some instances, EGA reagents of the invention may contain an alcohol and / or a glycol. Alcohols and glycols that may be used in such reagents include 2-ethoxyethanol acetate, 2-ethoxyethanol, 2-methoxyethanol acetate, 2-methoxyethanol, 1-butanol, 2-butoxyethanol acetate, dimethyl ethanol amine, dipropylene glycol, ethanol, ethyl glycol acetate, ethyl glycol, ethylene glycol, methanol, ethanol, propanol, isobutanol, acetone alcohol, allyl alcohol, cyclohexanol, diacetone alcohol, diethanol amine, diethylene glycol, furfuryl alcohol, isoamyl alcohol, isopropyl alcohol, n-amyl alcohol, n-butyl alcohol, n-octyl alcohol, n-propyl alcohol, propylene glycol diacetate, propylene glycol, t-amyl alcohol, t-butyl alcohol, triethanolamine, and triethylene glycol. In some instances, EGA reagents may contain two or more alcohols or glycols.

[00255] In some instances, EGA reagents of the invention may contain one or more salt (e.g., an organic salt). Salts that may be used in such reagents include 1,3-dimethyl-imidazolium bis(pentafluoroethyl)phosphinate, 1,3-dimethyl-imidazolium methyl sulfate, 1,3-dimethyl- imidazolium trifluoromethanesulfonate, 1-butyl-3-methyl-imidazolium 2-(2- methoxyethoxy)ethyl sulfate, 1-butyl-3-methyl-imidazolium bis(trifluoromethyl)imide, 1-butyl- 3-methyl-imidazolium cobalt tetracarbonyl, 1-butyl-3-methyl-imidazolium dicyanamide, 1- butyl-3-methyl-imidazolium hexafluorophosphate, 1-butyl-3-methyl-imidazolium methyl sulfate, 1-butyl-3-methyl-imidazolium octylsulfate, 1-butyl-3-methyl-imidazolium tetrafluoroborate, 1- butyl-3-methyl-imidazolium tosylate, 1-benzyl-3-methyl-imidazolium hexafluoroantimonate, 1- benzyl-3-methyl-imidazolium hexafluorophosphate, 1-benzyl-3-methyl-imidazolium methylsulfate, 1-benzyl-3-methyl-imidazolium tetrafluoroborate, 1-benzyl-3-methyl- imidazolium trifluoromethanesulfonate, 1-butyl-1-methyl-pyrrolidinium bis(trifluoromethylsulfonyl)imide, 1-ethyl-2,3-dimethyl-imidazolium trifluoromethanesulfonate, 1-ethyl-3-methyl-imidazolium bis(pentafluoroethyl)phosphinate, 1-ethyl-3-methyl-imidazolium bis(pentafluoroethylsulfonyl)imide, 1-ethyl-3-methyl-imidazolium bis(trifluoromethyl)imide, 1- ethyl-3-methyl-imidazolium bis(trifluoromethylsulfonyl)imide, 1-butyl-1-methyl-pyrrolidinium dicyanamide, tetrabutylammonium hexafluorophosphate, tetraethylammonium p- toluenesulfonate, 1,1-dibutyl-pyrrolidinium bis(trifluoromethylsulfonyl)imide, 1,1-dimethyl- pyrrolidinium tris(pentafluoroethyl)trifluorophosphate, 1,1-dipropyl-pyrrolidinium bis(trifluoromethylsulfonyl)imide, 1,2-dimethyl-3-propylimidazolium bis(trifluoromethylsulfonyl)imide, 1,2-dimethyl-3-propylimidazolium tris(trifluoromethylsulfonyl)methide, 1-butyl-1-methyl-pyrrolidinium hexafluoroantimonate, 1- butyl-1-methyl-pyrrolidinium hexafluorophosphate, 1-butyl-1-methyl-pyrrolidinium methylsulfate, 1-butyl-1-methyl-pyrrolidinium tetracyanoborate, 1-butyl-1-methyl-pyrrolidinium tetrafluoroborate, 1-butyl-1-methyl-pyrrolidinium trifluoromethanesulfonate, 1-butyl-1-methyl- pyrrolidinium tris(pentafluoroethyl)trifluorophosphate, 1-butyl-2,3-dimethyl-imidazolium hexafluoroantimonate, 1-butyl-2,3-dimethyl-imidazolium hexafluorophosphate, 1-butyl-2,3- dimethyl-imidazolium methylsulfate, 1-butyl-2,3-dimethyl-imidazolium tetrafluoroborate, 1- butyl-2,3-dimethyl-imidazolium tosylate, 1-butyl-2,3-dimethyl-imidazolium trifluoromethanesulfonate, 1-butyl-3-ethyl-imidazolium trifluoromethanesulfonate, 1-butyl-3- methyl-imidazolium trifluoroacetate, 1-butyl-3-methyl-imidazolium trifluoromethane sulfonate, 1-butyl-3-methyl-pyridinium bis(trifluormethylsulfonyl)imide, 1-butyl-4-methyl-pyridinium hexafluorophosphate, 1-butyl-4-methyl-pyridinium tetrafluoroborate, 1-butyl-imidazolium hexafluorophosphate, 1-butyl-imidazolium tetrafluoroborate, 1-butyl-imidazolium tosylate, 1- butyl-imidazolium trifluoromethanesulfonate, 1-ethyl-1-methyl-pyrrolidinium bis(trifluoromethyl)imide, 1-ethyl-1-methyl-pyrrolidinium hexafluoroantimonate, 1-ethyl-1- methyl-pyrrolidinium hexafluorophosphate, 1-ethyl-1-methyl-pyrrolidinium methylsulfate, 1- ethyl-1-methyl-pyrrolidinium tetrafluoroborate, 1-ethyl-1-methyl-pyrrolidinium trifluoromethanesulfonate, 1-ethyl-2,3-dimethyl-imidazolium hexaflluoroantimonate, 1-ethyl-3- methyl-imidazolium tosylate, 1-ethyl-3-methyl-imidazolium trifluoroacetate, 1-ethyl-3-methyl- imidazolium trifluoromethanesulfonate, 1-ethyl-3-methyl-imidazolium trifluoromethyltrifluoroborate, 1-hexyl-1-methyl-pyrrolidinium bis(trifluoromethylsulfonyl)imide, 1-hexyl-1-methyl-pyrrolidinium dicyanamide, 1-hexyl-2,3- dimethyl-imidazolium tetrafluoroborate, 1-hexyl-2,3-dimethyl-imidazolium trifluoromethanesulfonate, 1-hexyl-3-methyl-imidazolium bis(trifluoromethylsulfonyl)imide, 1- hexyl-3-methyl-imidazolium bis(trifluoromethylsulfonyl)methane, 1-hexyl-3-methyl- imidazolium dicyanamide, 1-hexyl-3-methyl-imidazolium hexafluoroantimonate, 1-hexyl-3- methyl-imidazolium hexafluorophosphate, 1-hexyl-3-methyl-imidazolium methylsulfate, 1- hexyl-3-methyl-imidazolium tetracyanoborate, 1-ethyl-2,3-dimethyl-imidazolium hexaflluorophosphate, 1-ethyl-2,3-dimethyl-imidazolium methylsulfate, 1-ethyl-2,3-dimethyl- imidazolium tetrafluoroborate, 1-ethyl-2,3-dimethyl-imidazolium tosylate, 1-ethyl-3-methyl- imidazolium bis[1,2-benzenediolato(2-)-O,O']-borate, 1-ethyl-3-methyl-imidazolium bis[oxalato(2-)]-borate, 1-ethyl-3-methyl-imidazolium cobalt tetracarbonyl, 1-ethyl-3-methyl- imidazolium dicyanamide, 1-ethyl-3-methyl-imidazolium hexafluoroantimonate, 1-ethyl-3- methyl-imidazolium hexafluorophosphate, 1-ethyl-3-methyl-imidazolium nitrate, 1-ethyl-3- methyl-imidazolium tetrafluoroborate, 1-hexyl-3-methyl-imidazolium tris(heptafluoropropyl)trifluorophosphate, 1-hexyl-3-methyl-imidazolium tris(pentafluoroethyl)trifluorophosphate, 1-hexyl-3-methyl-imidazolium tris(pentafluoroethyl)trifluorophosphate, 1-hexyl-3-methyl-imidazolium tetrafluoroborate, 1- hexyl-3-methyl-imidazolium trifluoromethanesulfonate, 1-methyl-3-(3,3,4,4,5,5,6,6,7,7,8,8,8- tridecafluoroctyl)-imidazolium-hexa- -fluorophosphate, 1-methyl-3-octyl-imidazolium tetrafluoroborate, 1-methyl-imidazolium hexafluorophosphate, 1-octyl-3-methyl-imidazolium bis(trifluoromethylsulfonyl)imide, 1-octyl-3-methyl-imidazolium bis(trifluoromethylsulfonyl)methane, 1-octyl-3-methyl-imidazolium hexafluoroantimonate, 1- pentyl-3-methyl-imidazolium tris(nonafluorobutyl)trifluorophosphate, 1-pentyl-3-methyl- imidazolium tris(pentafluoroethyl)trifluorophosphate, 1-phenylpropyl-3-methyl-imidazolium hexafluoroantimonate, 1-phenylpropyl-3-methyl-imidazolium trifluoromethanesulfonate, 1- tetradecyl-3-methyl-imidazolium tetrafluoroborate, 3-ethyl-N-butyl-pyridinium hexafluoroantimonate, 3-ethyl-N-butyl-pyridinium hexafluorophosphate, 3-ethyl-N-butyl- pyridinium tetrafluoroborate, 3-ethyl-N-butyl-pyridinium trifluoromethanesulfonate, 1-methyl- imidazolium tetrafluoroborate, 1-methyl-imidazolium tosylate, 1-methyl-imidazolium trifluoromethanesulfonate, 1-octadecyl-3-methyl-imidazolium bis(trifluoromethylsulfonyl)imide, 1-octadecyl-3-methyl-imidazolium hexafluorophosphate, 1-octyl-1-methyl-pyrrolidinium bis(trifluoromethylsulfonyl)imide, 1-octyl-3-methyl-imidazolium hexafluorophosphate, 1-octyl- 3-methyl-imidazolium methylsulfate, 1-octyl-3-methyl-imidazolium tetrafluoroborate, 3-methyl- 1-propyl-pyridinium bis(trifluormethylsulfonyl)imide, 3-methyl-N-butyl-pyridinium hexafluoroantimonate, 3-methyl-N-butyl-pyridinium hexafluorophosphate, 3-methyl-N-butyl- pyridinium methylsulfate, 3-methyl-N-butyl-pyridinium tetrafluoroborate, 3-methyl-N-butyl- pyridinium trifluoromethanesulfonate, 4-methyl-N-butyl-pyridinium hexafluorophosphate, 4- methyl-N-butyl-pyridinium tetrafluoroborate, benzyl triphenyl-phosphonium bis(trifluoromethyl)imide, bis(trifluoromethylsulfonyl)imide, bis-tetramethyl ammonium oxalate, butyl dimethyl imidazolium hexafluorophosphate, dimethyl distearyl ammonium bisulfate, dimethyl distearyl ammonium methosulfate, ethyl triphenyl phosphonium acetate, N-butyl- pyridinium trifluoromethanesulfonate, N-hexyl-pyridinium bis(trifluoromethylsulfonyl)imide, guanidinium trifluoromethanesulfonate, guanidinium tris(pentafluoroethyl)Trifluorophosphate, hexamethyl-guanidinium trifluoromethanesulfonate, hexamethyl-guanidinium tris(pentafluoroethyl)trifluorophosphate, N,N,N',N',N''-pentamethyl-N''-isopropyl-guanidinium trifluoromethanesulfonate, N,N,N',N'-tetramethyl-N"-ethyl-guanidinium trifluoromethanesulfonate, N,N,N',N'-tetramethyl-N"-ethyl-guanidinium tris(pentafluoroethyl)trifluorophosphate, N-butyl-pyridinium bis(trifluoromethyl)imide, N-butyl- pyridinium hexafluoroantimonate, N-butyl-pyridinium hexafluorophosphate, N-butyl-pyridinium methylsulfate, N-butyl-pyridinium tetrafluoroborate, N,N,N',N',N''-pentamethyl-N''-isopropyl- guanidinium tris(pentafluoroethyl)trifluorophosphate, N,N,N',N',N''-pentamethyl-N''-propyl- guanidinium trifluoromethanesulfonate, N-hexyl-pyridinium bis(trifluoromethylsulfonyl)methane, O-ethyl-N,N,N',N'-tetramethyl-isouronium trifluoromethanesulfonate, O-ethyl-N,N,N',N'-tetramethyl-isouronium tris(pentafluoroethyl)trifluorophosphate, O-methyl-N,N, N',N'-tetramethyl-isouronium trifluoromethanesulfonate, tetraethyl ammonium tris(pentafluoroethyl)trifluorophosphate, tetramethyl ammonium bis(trifluoromethyl)imide, tetramethyl ammonium bis(trifluoromethylsulfonyl)imide, tetramethyl ammonium bis[oxalato(2-)]-borate, tetramethyl ammonium bis[salicylato(2-)]borate, tetramethyl ammonium hexafluorophosphate, tetramethyl ammonium tetrafluoroborate, tetramethyl ammonium tris(pentafluoroethyl)trifluorophosphate, tributylethyl ammonium ethylsulfate, trihexyl(tetradecyl)-phosphonium bis(2,4,4- trimethylpentyl)phosphinate, trihexyl(tetradecyl)-phosphonium bis(trifluoromethylsulfonyl)imide, trihexyl(tetradecyl)-phosphonium bis(trifluoromethylsulfonyl)methane, N-hexyl-pyridinium hexafluorophosphate, N-hexyl- pyridinium tetrafluoroborate, N-hexyl-pyridinium trifluoromethanesulfonate, N-octyl-pyridinium bis(trifluoromethylsulfonyl)imide, N-octyl-pyridinium tris(trifluoromethylsulfonyl)methane, O- methyl-N,N,N',N'-tetramethyl-isouronium tris(pentafluoroethyl)trifluorophosphate, S-ethyl- N,N,N',N'-tetramethyl isothiouronium trifluoromethanesulfonate, S-ethyl-N,N,N',N'- tetramethylisothiouronium tris(pentafluoroethyl)trifluorophosphate, S-ethyl-N,N,N',N'- tetramethylthiouronium tetrafluoroborate, tetrabutyl ammonium bis(trifluoromethyl)imide, tetrabutyl ammonium bis(trifluoromethylsulfonyl)imide, tetrabutyl ammonium hydrogen sulfate, tetrabutyl ammonium hexafluorophosphate, tetrabutyl ammonium nitrate, tetraethyl ammonium bis(trifluoromethyl)imide, tetraethyl ammonium bis(trifluoromethylsulfonyl)imide, tetrabutyl ammonium perchlorate, tetrabutyl ammonium sulfate, tetrabutyl ammonium tetracyanoborate, tetrabutyl ammonium tetrafluoroborate, tetrabutyl ammonium tris(pentafluoroethyl)trifluorophosphate, tetrabutyl phosphonium acetate, tetrabutyl phosphonium bis(trifluoromethyl)imide, tetrabutyl phosphonium bis[1,2-benzenediolato(2-)- O,O']-borate, tetrabutyl phosphonium bis[oxalato(2-)]-borate, tetrabutyl phosphonium tetracyanoborate, tetrabutyl phosphonium tris(pentafluoroethyl)trifluorophosphate, tetraethyl ammonium bis[1,2-benzenediolato(2-)-O,O']-borate, tetraethyl ammonium bis[2,2'- biphenyldiolato(2-)-O,O']-borate, tetraethyl ammonium bis[malonato(2-)]-borate, tetraethyl ammonium bis[salicylato(2-)]-borate, tetraethyl ammonium hexafluorophosphate, tetraethyl ammonium hydrogen maleate, tetraethyl ammonium tetrafluoroborate, tetraethyl ammonium tosylate, trihexyl(tetradecyl)-phosphonium bis[1,2-benzenediolato(2-)-O,O']-borate, trihexyl(tetradecyl)-phosphonium decanoate, trihexyl(tetradecyl)-phosphonium dicyanamide, trihexyl(tetradecyl)-phosphonium hexafluorophosphate, trihexyl(tetradecyl)-phosphonium tetracyanoborate, trihexyl(tetradecyl)-phosphonium tetrafluoroborate, trihexyl(tetradecyl)- phosphonium, tris(pentafluoroethyl)trifluorophosphate, and tri-iso-butyl(methyl)-phosphonium tosylate, and combinations thereof.

[00256] Buffering agents may comprise, for example, an organic base and may be present at concentration of from about 0.0001 mM to about 200 mM. Representative examples of organic bases include N,N-diisopropylethylamine, lutidine(dimethylpyridine isomers), [Image disponible dans le document PDF, Image available in the PDF document]

[00257] R1, R2, and R3 are independently selected from the group consisting of hydrogen, and substituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group, and halo, amide, carboxy, amino, secondary amino, tertiary amino, hydrazino, azido, alkazoxy, cyano, isocyano, cyanato, thiocyanato, fulminato, selenocyanato, carboxyamido, acylimino, nitroso, aminooxy, hydrazonoyl, oxime, acylhydrazino, amidino, sulfide, thiosulfoxide, sulfone, thiosulfone, thiosulfate, hydroxyl, formyl, hydroperoxy, carbamoyl, trimethyl silyl, nitro, nitroso, oxamoyl, pentazolyl, sulfamoyl, sulfenamoyl, sulfeno, sulfinamoyl, sulfino, sulfo, sulfoamino, hydrothiol, tetrazolyl, thiocarbamoyl, thiocarbazono, thiocarbodiazono, thiocarbonohydrazido, thiocarboxy, thioformyl, thioacyl, thiocyanato, thiosemicarbazido, thiosulfino, thiosulfo, thioureido, triazano, triazeno, triazinyl, trithiosulfo, and phosphoric acid ester.

[00258] R4, R5, R6, R7, R8, R9, and R10 are independently selected from the group consisting of hydrogen, and substituted and unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic ring, and polycyclic group, and halo, amide, alkoxy, acyl, acyloxy, oxycarbonyl, alkoxycarbonyloxy, carboxy, amino, secondary amino, tertiary amino, hydrazino, azido, alkazoxy, cyano, isocyano, cyanato, thiocyanato, fulminato, selenocyanato, carboxyamido, acylimino, nitroso, aminooxy, carboximidoyl, hydrazonoyl, oxime, acylhydrazino, amidino, sulfide, sulfoxide, thiosulfoxide, sulfone, thiosulfone, thiosulfate, hydroxyl, formyl, hydroxyperoxy, hydroperoxy, peroxy acid, carbamoyl, trimethyl silyl, nitro, nitroso, oxamoyl, pentazolyl, sulfamoyl, sulfenamoyl, sulfeno, sulfinamoyl, sulfino, sulfo, sulfoamino, hydrothiol, tetrazolyl, thiocarbamoyl, thiocarbazono, thiocarbodiazono, thiocarbonohydrazido, thiocarboxy, thioformyl, thioacyl, thiocyanato, thiosemicarbazido, thiosulfino, thiosulfo, thioureido, triazano, triazeno, triazinyl, trithiosulfo, and phosphoric acid ester.

[00259] Acid generators and base generators, when present, may be any number of compounds, including quinone and non-quinone compounds. In some embodiments, hydroquinone and benzoquinone may be replaced with thiophenol, 1,4-butanedithiol, 1,3- propanedithiol, methylthiophene or another thiol. EGA reagents may contain from about 0.1 mM to about 2.0 M of thiophenol, 1,4-butanedithiol, 1,3-propanedithiol, methylthiophene, or other thiol, or a combination thereof. Other compounds, such as various quinone compounds may also be used. Examples of potentially useful compunds are 2-methylhydroquinone, methylhydroquinone, 2-t-butylhydroquinone, 2,5-di-t-butylhydroquinone, 2,6-di-t- butylhydroquinone, 2,6-dimethylhydroquinone, 2,3,5-trimethylhydroquinone, amylquinone, amyloxyhydroquinone, naphthoquinone, anthraquinone, 4-t-butylcatechol, 4,6-di-t-butylcatechol, 2,3,5,6-tetrachloro-1,4-benzoquinone, methylbenzoquinone, 2,6-dimethylbenzoquinone and various quinhydrones.

[00260] EGA reagents, also including hydroquinone and benzoquinone, with tetrabutylammonium hexafluorophosphate or tetraethylammonium paratoluolsulfanate in anhydrous acetonitrile are used to generate electrochemical acid via anodic oxidation to affect deprotection. Other EGA reagents include hydrazine; triphenylmethane derivatives; phenylenediamine; ascorbic acid; derivatives of hydroquinone benzoquinone, and substituted benzoquinones, including, but not limited to tetramethylbenzoquinone, methylhydroquinone, tertbutylhydroquinone, and ditertbutylhydroquinone; cathechols such as 3-methylcathechol and 4-methykcathechol; 1,3-dihydroxynaphthalene; tetracyanoquinodimethane; antrachinone; tetracyanoquinodimethane; iodine; N-halosuccinimides, such as N-chlorosuccinimide; alkyldisulfanes; and aromatic systems with nitrogen such as flavines. EGA reagents at a sufficient concentration to allow deprotection of the protecting group, such as a DMT protecting group, may be prepared and administered to the chip prior to the application of current to affect deprotection. For example, EGA reagents may be provided at a concentration of about 0.1 M to about 1 M, from about 0.5 M to about 5 M, from about 0.5 M to about 2 M, such as about 1 M, wherein the upper limit may depend on the solubility of the used reagents. In determination of the optimal parameters, it will generally be desirable to avoid base damage caused by depurination from over-exposure of DNA to acid.

[00261] Likewise, in certain embodiments, localized chemical reactions may occur through the production of photogenerated acid at a sufficient concentration to allow deprotection of the protecting group, such as a DMT protecting group. One aspect of the invention is that the pH of a solution may be changed by photo-generation of acids in a controlled fashion, as disclosed herein.

[00262] As used herein, the term photogenerated acid (PGA) refers to an acid that is produced from a PGA precursor reagent (PGAPR) after irradiation or illumination by photons having a certain wavelength. The wavelengths of the photons may be in any appropriate region of the electromagnetic spectrum, including, for example, infrared, visible, ultraviolet, or x-ray wavelengths. In certain embodiments, the wavelength may range from about 390 nm to about 700 nm, such as from about 400 nm to about 600nm. In certain other embodiments, the wavelength may range from about 200 nm to about 400 nm, such as from about 250 nm to about 350 nm.

[00263] The PGAPR may be any chemical compound that produces PGA upon irradiation, for example irradiation with visible and / or ultraviolet light. Non-limiting examples of PGAPRs may include those disclosed in U.S. Patent No. 6,426,184. For example, PGAPRs that may be used in accordance with various embodiments disclosed herein may include diazonium salts, perhalomethyl triazines, halobisphenyl A, sulfonates, imidylsulfonyl esters, diaryliodonium salts, sulfonium salts, diazosulfonate, and diarylsulfones.

[00264] In certain embodiments, the PGAPR may be present in a solution with at least one solvent. The at least one solvent can be any conventional solvent traditionally used in the chemical reaction, such as, for example, CH2Cl2, CH3CN, toluene, hexane, CH3OH, H2O, and / or an aqueous solution comprising at least one solute, such as NaCl, MgCl2, and phosphate salts. A solution comprising the PGAPR and the at least one solvent may then be irradiated or illuminated by photons having a certain wavelength, resulting in the generation of PGA.

[00265] In certain embodiments disclosed herein, the solution comprising the PGAPR and the at least one solvent may further comprise at least one of buffers, neutralizers, photo- sensitizers, stabilizers, and viscosity additives.

[00266] As used herein, photo-sensitizers may be defined as chemical compounds having a lower excitation energy than the PGAPR used in the solution. The photo-sensitizer may be excited by irradiation, and the excited photo-sensitizer thereby causes the PGAPR to generate the PGA. Thus, the photo-sensitizer may act to lower the required excitation wavelength for generating PGA. Non-limiting examples of suitable photo-sensitizers that may be used in embodiments disclosed herein include, for example, those disclosed in U.S. Patent No. 6,426,184, such as anthracene and derivatives thereof, dicyanoanthracene, thioxanthone, chlorothioxanthenes, pyrene, benzophenone, acetophenone, benzoinyl C1-C12 alkyl ethers, benzoyl triphenylphosphine oxide, Ru2+ complexes, and their derivatives.

[00267] According to certain exemplary embodiments disclosed herein, nucleotide linker molecules may be attached to a solid support, such as a bead, wherein each nucleotide may be protected at the 5'-OH end by an acid-labile protecting group. According to other exemplary embodiments disclosed herein, a universal linker molecule may be attached to the solid support, such as a bead. In certain embodiments, the acid-labile protecting group may be chosen, for example, from dimethoxytrityl (DMT) or methoxytrityl (MMT). In certain embodiments, the nucleotide is attached to a bead, and the bead is located in a well, such as, for example, the well of a multiwell plate.

[00268] The wells of the multiwell plate may then be contacted with the PGAPR solution and light, which acts to generate PGA. The PGA then subsequently deprotects the 5'-OH from the acid-labile protecting group. The deprotected 5'-OH groups may then react with a monomer, such as, for example, an additional nucleotide that is also protected at its 5'-OH end with an acid-labile protecting group. The process may then be repeated to grow the nucleic acid chain to the desired length.

[00269] Generally, no PGA will be generated in wells that are not exposed to light. Accordingly, a predetermined light pattern may be projected onto the wells of the multiwell plate such that only the beads in designated wells will be exposed to PGA and deprotection. The amount of light applied to each well and its duration will vary with parameters such as the amount of reagent to be generated and the size of the well. The light may define a series of pulses of varying frequency or a gradual increase / decrease in frequency. The frequency and duration of the pulse can be adjusted for the optimum generation of reagent. As an example, the light applied to a well may be adjusted for a specified period of time to generate a specified quantity of PGA. The amount of PGA intended for generation will typically be at least enough sufficient to fully catalyze deprotection of the nucleic acid molecules present.

[00270] In embodiments disclosed herein, the multiwell plate may, for example, be a microchip, such as a glass chip with wells comprising a photoresist polymer, such as, for example, SU-8 SUEX (available from by DJ DevCorp). In certain embodiments, the cover of the multiwell plate disclosed herein may be glass, and light may come from an optical semiconductor device, such as a digital micromirror device (DMD). In other exemplary embodiments disclosed herein, the multiwell plate may be a 454 sequencing chip available from Roche. For example, the multiwell plate could be a 454 sequencing chip comprising fiber optic bundles that can be used to direct light through fiber optics to the individual wells or set of wells on the sequencing chip. The microchip may have high density wells, which can capture beads of a size ranging, for example, from about 30 µm to about 40 µm. As one skilled in the art would appreciate, a 454 sequencing chip may be modified as necessary to suit embodiments of the invention disclosed herein.

[00271] The wavelength of light necessary for the generation of PGA may be produced by any optical system known in the art. An exemplary optical system may comprise, for example, a light source, at least one filter, at least one condenser lens, a reflector, a DMD, and a projection lens. In certain embodiments, the optical system may be chosen from fiber optic arrays, liquid- crystal displays (LCD), liquid crystal light valves, acousto-optic scanning light modulators (SLMs), Galvanometric laser scanners, and the like. In certain embodiments, use may be made of photomasks, for example photomasks having a computer-controlled spatial optical modulator as disclosed in U.S. Patent No. 6,426,184.

[00272] The use of beads in combination with the deprotection of nucleotides by PGA may have advantages over the use of two-dimensional surfaces in nucleic acid molecule synthesis. For example, up to 1,000 times greater nucleic acid molecule concentration may be available with the use of a porous bead over a two-dimensional surface. Moreover, the use of beads may allow for the ability to separate (and subsequently release and pool together) only the beads belonging to a single desired fragment. The separation can be done by any feasible means, for example, by the methods disclosed herein; by optical forces, such as optical tweezers; generation of gas bubbles, generated for example through laser heating; micropipetting; acoustic force; etc. Further non-limiting exemplary methods for separating beads may be found in U.S. Published Patent Application No. 2010 / 0216648. Electrowetting

[00273] In some aspects of the invention, "electrowetting" may be employed. Two aspect of the invention where electrowetting may be particularly useful is for the mixing of reagents for (1) nucleic acid synthesis and pooling (Modules 1 and 2) and (2) assembly (Module 3).

[00274] In brief, electrowetting involves modifying the surface tension of liquids on a solid surface using a voltage. Application of an electric field (e.g., alternating or direct), the contact angle between the fluid and surfaces can be modified. For example, by applying a voltage, the wetting properties of a hydrophobic surface can become increasingly hydrophilic and therefore wettable. Electrowetting principle is based on manipulating droplets on a surface comprising an array of electrodes and using voltage to change the interfacial tension. In some embodiments, the array of electrode is not in direct contact with the fluid. In additional embodiments, the array of electrode may be configured such as the support has a hydrophilic side and a hydrophobic side. The droplets subjected to the voltage will move towards the hydrophilic side. In some embodiments, the array or pattern of electrodes may be a high density pattern. When used in conjunction with the phosphoramidite chemistry (as well as other reagents), the array of electrodes should be able to move droplets volumes ranging from 1 pL (and less) to 10 pL. Accordingly, aspects of the invention relate to high voltage complementary semi-conductor microfluidic controller. In some embodiments, the high voltage complementary semi-conductor device (HV-CMOS) has an integrated circuit with high density electrode pattern and high voltage electronics. In some embodiments, the voltage applied is between 15V and 30V. Electrowetting methods are set out in U.S. Patent Publication No. 2012 / 0220497 A1.

[00275] Electrowetting works by using an electric voltage to alter the shape of a liquid drop. In some instances, electrowetting involves a sessile drop positioned on a dielectric-coated electrode. When current is applied, the drop flattens and flows out to the sides, thereby wetting additional surface. When current is removed, the drop returns to its original shape and retracts from the areas covered upon current application. Electrowetting methods are set out in the paper at the following URL: http: / / www.ll.mit.edu / publications / journal / pdf / vol17 no2 / 17 2 4Berry.pdf

[00276] In some embodiments of the invention, nucleic acid synthesis site may have adjacent to is a series of reagents that flow into and recede from the synthesis site when current is applied to the correct reagent location. Thus, the invention includes methods for the synthesis of nucleic acid molecules by the addition and removal of reagents from a synthesis site induced by the addition and removal of current from adjacent reagents. In some instances, the number of reagents adjacent to a nucleic acid synthesis site may be from about 2 to about 10, from about 3 to about 10, from about 4 to about 10, from about 5 to about 10, from about 6 to about 10, etc.

[00277] Electrowetting methods may also be used for fragment assembly and error correction (Module 3). Thus, the invention includes methods for mixing reagents using electrowetting for the assembly and error correction of nucleic acid molecules. Reagents that may be contacted with nucleic acid molecules in these aspects of the invention include exonucleases, mist-match repair endonucleases (MMEs), ligases, buffers, EDTA solutions, etc.

[00278] One problem with electrowetting methods is "splash over" which may occur between mixing areas and also because, in many instances, planar or semi-planar surfaces are used. Thus, unless microfluidic drainage channels, or the like, are employed, there is a possibility of splash over contamination of mixing areas during reagent changes.

[00279] Two means for minimizing this mixing is through the use of microfluidic channels and barriers. Barrier may be placed (e.g., physical barriers such as raised areas) to prevent reagents from moving from one mixing area to another. After a desired reaction is finished, the barrier may be removed. Different reactions may be performed sequentially at different and / or overlapping subsets of mixing areas.

[00280] As mentioned above, the methods of nucleic acid synthesis may be implemented and controlled in a system according to various embodiments described herein by a processor or computing system, such as the exemplary computing system depicted in FIG. 15. For example, applying current (pulse or continuous wave) to selected wells to generate a specific quantity of EGA to fully catalyze deprotection may be controlled by a computing system executing processor executable instructions according to various embodiments of the present teachings. Likewise, applying a light source to selected wells to generate a specific quantity of PGA may also be controlled by a computer system executing processor executable instructions.

[00281] Deblocking may also occur through the use of redox systems. Examples of such system systems include hydroquinone / anthraquinone; pH buffer such as 2,6-lutidine to reduce proton cross talk between active wells and inactive neighboring wells.

[00282] Efficient production of nucleic acid molecules may require that nucleic acid synthesis steps be tailored to the molecules being constructed. Consider the example of the construction of nucleic acid molecules designed for construction of viral genome with a CG / AT ratio of 60 / 40. Nucleic acid molecule building blocks of such a genome will invariable have more Cs and Gs than As and Ts. In such an instance, it may be desirable to have more reactions which add Cs and Gs than As and Ts. As an example, the sequence of base addition may be a repetition of A T C G C A T G C G (SEQ ID NO: 1). Thus, the invention further includes chemical synthesis processes which are tailored for efficient production of specified nucleic acid molecules. In one aspect, this entails adding bases to nucleic acid molecules during chemical synthesis in manner which reflects or closely approximates the prevalence of the bases in those molecules.

[00283] The invention includes, for example, methods which result in high fidelity, microscale production of nucleic acid molecules. Thus, the invention includes methods by which nucleic acid molecules are produced with the following parameters: between <semantics>1×105<annotation encoding="application / x-tex">1 \times 10^{5}< / annotation>< / semantics> and <semantics>5<annotation encoding="application / x-tex">5< / annotation>< / semantics> x 1012 copies of a nucleic acid molecule are generated with an average number of base mis- incorporations of between 1 base in 100 and 1 base in 4,000. The invention includes similar methods with the parameters set out in Table 7.

[00284] In some instances, all of the copies of a nucleic acid molecule will be produced on a single support (e.g., a single bead). In other instances, copies of a nucleic acid molecule will be produced on more than one single support (e.g., from about two to about 20, from about three to about 20, from about four to about 20, from about five to about 20, from about six to about 20, from about three to about 10, etc.). For purposes of the number of copies of a nucleic acid molecule and the number of base mis-incorporations, these numbers may be expressed as a function of copies of a nucleic acid molecule produced on a single support or as a function of all of the copies of a nucleic acid molecule produced in a synthesis run. For example, if one support is used to produce all of the copies of a nucleic acid molecule, then 1.0 x1011 molecules may be produced with an average error rate of 1 base in 310. As a second example, if two supports are used to produce the copies of the same nucleic acid molecule, then 1.0 x1011 molecules may be produced with an average error rate of 1 base in 300 on one support and <semantics>1.0×1011<annotation encoding="application / x-tex">1.0 \times 10^{11}< / annotation>< / semantics> molecules may be produced with an average error rate of 1 base in 320 on the second support. In such an instance, <semantics>2.0×1011<annotation encoding="application / x-tex">2.0 \times 10^{11}< / annotation>< / semantics> molecules may be produced with an average error rate of 1 base in 310. [Image disponible dans le document PDF, Image available in the PDF document]

[00285] Nucleic acid molecules prepared and used in accordance with the invention may contain modified nucleic acid molecules including locked nucleic acids (LNA), peptide nucleic acids (PNA), and the like. A PNA is a polyamide type of DNA analog, and the monomeric units for A, G, T, U, and C are available commercially. Furthermore, nucleic acid molecules of the invention may comprise one or more modified bases selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 8- azaguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid, wybutoxosine, pseudouracil, queosine, inosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6- diaminopurine. The latter modified base can form three hydrogen bonds when base-paired with dT and can increase the Tm of short nucleic acid molecules by as much as 1-2°C per insertion. This effect, however, is complex and is dependent on sequence context.

[00286] 2-Aminopurine can substitute for dA in a nucleic acid molecule. It is a naturally fluorescent base that is sensitive to the local environment making it a useful probe for monitoring the structure and dynamics of DNA hairpins and for detecting the base stacking state of a duplex. 2-aminopurine can be destabilizing and slightly lower the Tm. 5-Bromo-deoxyuridine is a photoreactive halogenated base that can be incorporated into nucleic acid molecules to crosslink them to DNA, RNA or proteins with exposure to UV light. Other modified bases such as inverted dT may be incorporated at the 3'-end of a nucleic acid molecule, leading to a 3'-3' linkage which inhibits both degradation by 3' exonucleases and extension by DNA polymerases. In another embodiment of the invention an inverted dideoxy-T may be placed at the 5' end of a nucleic acid molecule to prevent unwanted 5' ligations. A dideoxy-C (ddC) 3' chain terminator may be used to prevent 3' extension by DNA polymerases. 5-Methyl deoxy-C when substituted for dC will increase the Tm by as much as 0.5°C per insertion. In one embodiment the naturally occurring base deoxy-Inosine may be used which is less destabilizing than mismatches involving the four standard bases. Thus, the invention provides, in part, compositions and methods relating to the synthesis of modified nucleic acid molecules with novel properties and / or functions.

[00287] One modification of the plate format, shown in FIGs. 2 and 2B, is to use a "liquid cover" to the wells. One way this could be performed is for the wells to contain a bilayer. For example, the bottom portion of the well containing the solid support could contain an EGA. Above this could be a lower density, optionally non-miscible, fluid. The lower density fluid layer will prevent or retard the diffusion of acid out of the desired well and over to an undesired well. Further, the lower density fluid can be positioned to make conductive contact with an upper electrode. One example of a commercially available "liquid coverslip" is sold by Ventana Medical Systems, Inc (cat. no. 650-010). This product is a solution used as a barrier between the aqueous reagents and the air, which prevents evaporation, and is designed to provide a stable aqueous environment for applications such as immunohistochemistry and in situ hybridization reactions.

[00288] One exemplary protocol for practicing methods of the invention is as follows. Porous silane-coated magnetic beads (MyOne Beads, Dynal) with a uniform diameter of 1 micron are added to the chip surface by controlled or pulsed flow to ensure uniform distribution of the beads across the microwells (about 1.3 µm diameter) on the chip and to ensure that a maximum number of wells are loaded with one bead. Wells not containing a bead are identified by a pre-synthesis current check that delineate the resistance difference among empty wells and well that contain a conductive magnetic bead.

[00289] A variety of chemistries are possible in the preparation of the bead surface. For example, a number of layers of silane can be produced to impart greater functional surface area to the beads. The silane coating(s) is / are prepared so that there is stable attachment of the hydroxyl functional group; typically through a trimethoxy or triethoxy silane linker, of the silane core to the naked silica bead surface to expose a primary hydroxyl group through with the initial amidite synthetic step is coupled. The fundamental chemistry, developed for a planar array surface electrode, for initiation and coupling in DNA synthesis can be found in Maurer et al., Electrochemically Generated Acid and Its Containment to 100 Micron Reaction Areas for the Production of DNA Microarrays, PLoS ONE 1(1): e34. doi:10.1371 / journal.pone.0000034 (2006).

[00290] Fabrication of the chip: Electrode materials such, as iridium metal up to 50 nm thick are produced on oxidized high-resistivity silicon selected for high conductivity and chemical stability under synthesis, reagent addition and deblocking conditions. Electrodes are connected by ultrasonic bonding to a printed circuit board to provide digitally controlled analogue integrated switch circuits activating electrodes chosen for deblocking a given well. Printed circuit boards are carefully aligned and bonded to the regular microwell structure to generate the synthesis chip. A cover plate providing and sealing the interior volume for reagents and a general complementary circuit electrode is bonded at the perimeter and over the upper surface of the microwell structure to complete the closed synthesis chip.

[00291] Conventional semiconductor or polymer material may be used for forming wells 200. For example, CMOS technology can be used to form wells of desired shape or size in the semiconductor material such as SiO or SiO2. Depending on the desired application, electrodes 202 can be fabricated with wells 200 or separately.

[00292] Administration of nucleic acid synthesis (e.g., DNA synthesis) reagents to the chip can be performed by any number of means. For example, once the beads are loaded into the chip, a computing system controls a series of reagent additions and washings may be carried out to affect phosphoramidite DNA synthesis on the surface of the beads residing in the microwells of the chip. Processor-executable instructions may be employed which determine, for any given population of DNA sequences, the optimal order of DNA synthesis reagent additions and sequence of reagent additions and washing steps relative to volume / cost of reagents and time of a synthesis run. Furthermore, as mentioned above, controller or processor-controlled current to specific wells on the chip determine in which wells electrochemically generated acid may be produced and deprotection to activate the growing nucleic acid molecule on the bead in the well may be chemically prepared to couple the next amidite base added into the reaction vessel. A number of specific configurations of apparatus and components for administration of synthesis reagents and to ensure precise and controlled fluid administration are possible through an optimized development process. Phosphoramidite DNA synthesis steps, conditions and reagents using EGA to affect deprotection can be found in, for examples, Maurer et al., Electrochemically Generated Acid and Its Containment to 100 Micron Reaction Areas for the Production of DNA Microarrays, PLoS ONE 1(1): e34. doi:10.1371 / journal.pone.0000034 (2006) and Egeland and Southern, Electrochemically directed synthesis of oligonucleotides for DNA microarray fabrication, Nucleic Acids Research, 33(14):e125 (2005).

[00293] While in many instances oligonucleotides may be produced using phosphoramidite synthesis chemistry, as well as variations thereof, other methods may also be used to produce oligonucleotides, including PCR, restriction enzyme digest, exonuclease treatment, or template- independent synthesis using a nucleotidyl transferase enzyme. Exemplary methods of template- independent synthesis using a nucleotidyl transferase enzyme are set out in U.S. Patent No. 8,808,989. The nucleotidyl transferase enzyme (e.g., terminal deoxynucleotidyl transferase) is used to incorporate nucleotide analogs having an unmodified 3' hydroxyl and a cleavable protecting group. Because of the protecting group, synthesis pauses with the addition of each new base, whereupon the protecting group is cleaved, leaving a polynucleotide that is essentially identical to a naturally occurring nucleotide (i.e., is recognized by the enzyme as a substrate for further nucleotide addition). Thus, in certain embodiments, the invention includes methods in which oligonucleotides are produced by enzymatic reaction.

[00294] Nucleotide triphosphates (e.g., deoxynucleotide triphosphates) (NTPs) suitable for use with enzymatic oligonucleotide synthesis methods will have protecting groups that do not prevent the NTPs from being used by a nucleotidyl transferase as a substrate and can be efficiently removed to allow for addition to an oligonucleotide chain. Thus, in certain embodiments, the invention includes methods where nucleotide addition occurs via enzymatic reaction. In many instances, EGA will be generated as part of the deprotection process. Further, in certain instances, all or part of the oligonucleotide synthesis reaction may be performed in aqueous solutions.

[00295] One aspect of the present invention is the ability to the control the pH of the reaction environment. In certain embodiments, however, DNA degradation may occur due to the presence of strong acids, such as the EGA or PGA. It is known that the acid strength (which may be represented by the pKa value) has an effect on the depurination rate in that the lower the pKa (more acidic), the more significant the degradation of DNA by depurination. This may become obvious when comparing, for example, trichloroacetic acid (TCA) and dichloroacetic acid (DCA). Both TCA and DCA are used for DNA synthesis, but the depurination rate for 3% TCA (pKa = <semantics>0.7<annotation encoding="application / x-tex">0.7< / annotation>< / semantics>) is about four times higher than the depurination rate for <semantics>3%<annotation encoding="application / x-tex">3\%< / annotation>< / semantics> DCA (pKa = <semantics>1.5<annotation encoding="application / x-tex">1.5< / annotation>< / semantics>).

[00296] As disclosed herein, addition of a suitable molecule to act as a proton carrier may reduce the effect of DNA degradation by accepting protons from the EGA or PGA, thereby raising the pKa value of the solution. Any acceptable proton carrier may be used, including every electrochemically stable compound having a pKa ranging from about 0 to about 3. According to certain embodiments, the proton carrier may be at least one compound chosen from 2-chloro-6-methyl pyridine, diphenylamine, 2,2',2'-nitrilotriacetonitrile, pyridazine, urea, and malachite green. At the reaction site, the proton is released from the proton carrier with an optimized acid strength (pKa). This results in a reduction of DNA depurination, and consequently an increase in the quantity and / or quality of nucleic acid molecule synthesis.

[00297] Since a very weak acid may lead to long deprotection times, according to certain embodiments disclosed herein, the proton carrier may have a pKa of about 1. For example, 2- chloro-6-methylpyridine has a pKa value of about 0.72, and diphenylamine has a pKa value of about 0.78. By way of further examples, 2,2',2'-nitrilotriacetonitrile has a pKa value of about 1.1, pyridazine has a pKa value of about 2.1, urea has a pKa value of about 0.2, and malachite green has a pKa value of about 1Ø

[00298] In certain embodiments, the addition of a proton carrier, such as 2-chloro-6- methylpyridine, for example, may reduce the DNA degradation by at least about 15%, at least about 20%, or at least about 25%.

[00299] Using the above-disclosed proton carriers may result in a cost-efficient and simple method to enhance oligonucleotide quality and quantity for EGA-based and / or PGA-based methods.

[00300] In certain embodiments of the invention the nucleic acid molecule or a portion thereof may be subject to a sequence optimization process prior to synthesis. Different computational approaches for sequence modification are known in the art and may be employed to optimize a given nucleotide sequence in terms of 1) efficient assembly and / or 2) improved performance in a given host. To design a nucleotide sequence for optimal assembly, a full- length sequence may be broken down into a defined number of smaller fragments with optimal hybridization properties by means of an algorithm taking into account parameters such as melting temperature, overlap regions, self-hybridization, absence or presence of cloning sites and the like. In certain aspects of the invention, at least part of the desired nucleic acid sequence may encode a polypeptide or protein. In such cases, it may be desirable to optimize the open reading frame for improved performance in a given homologous or heterologous host, such as expression yield or solubility. An increase in gene expression may be achieved, for example, by replacing non-preferred or less preferred codons by preferred codons or by increasing the number of CpG dinucleotides in the open reading frame as described, for example, in U.S. Patent Nos. 5,786,464 and 6,114,148 and U.S. Patent Publication No. 2009 / 0324546 AA.

[00301] In one specific embodiment, an optimized open reading frame may be combined with an algorithm to encrypt a secret message into the open reading frame as described in U.S. Patent Publication No. 2011 / 0119778 AA. Such message may allow the identification or tracking of certain synthetic nucleic acid molecules. In certain aspects of the invention, it may be desired to use an optimization strategy that takes into account multiple different parameters simultaneously including assembly- as well as expression-related sequence properties. One example of a comprehensive multiparameter approach that may be used in the current invention for optimized sequence design is the GENEOPTIMIZER® technology described in U.S. Patent Publication No. 2007 / 0141557 AA. Thus, the invention provides in part aspects of optimal sequence design for downstream applications including assembly and expression strategies. Module 2

[00302] After completion of a synthesis run on Module 1, support-associated (e.g., bead- associated) nucleic acid molecules may be subject to post-processing in Module 2. Processes performed in Module 2 may be performed manually or by computer directed automation controlling such steps as picking and pooling of a bead (e.g., a magnetic bead) from the synthesis microwell array and vapor-phase cleavage and deprotection to prepare the nucleic acid molecules for subsequent assembly steps, as appropriate.

[00303] To expose a microwell array of bead-attached nucleic acid molecules, the cover of the synthesis well, when present, may be removed. In one embodiment, the cover is removed by automatic means in a computer-controlled manner.

[00304] Depending on the application and the number of nucleic acid molecules to be assembled, all of the beads of the microwell array may be pooled or only a subset of the beads. When only a subset of the beads are pooled or when the total number of beads is limited, the number of beads pooled may vary widely and include from about 10 to about 50, from about 50 to about 100, from about 100 to about 1000, from about 50 to about 10,000, from about 100 to about 10,000, or from about 500 to about 10,000 individual beads. These beads may be deposited in any suitable container. One example of a container is the well of a microwell plate (e.g., a well of a 1536 microwell plate). Α. Magnetic Pooling Mechanisms

[00305] In instances where magnetic beads are used, a bead picking instrument comprising, for example a precision-controlled electro-micromagnet can be programmed and controlled to extract and pool individual beads harboring synthesized nucleic acid molecules.

[00306] Automation suitable use with the invention includes a precision-controlled electro- micromagnet picks up the first bead and deposits it into a pooling well (i.e., a well which contains multiple beads for collection of nucleic acid molecules sought to be used in combination). Alternatively, a precision-controlled electro-micromagnet can be used which picks up the first bead and then moves in the X-Y direction to the next position, lowers down in the Z direction to pick up the second bead, back up in the Z direction to get out of the magnetic field range, moves to the third well in the X-Y direction, etc. Thus, the magnet is left "on" and the set of beads (e.g., from about two to about fifty, from about ten to about fifty, from about two to about one hundred, from about ten to about one hundred, from about twenty to about eighty, etc.) is picked up and carried as a string of beads. As a set of beads is collected, this set is then deposited in simultaneously deposited into a pooling well. Of course, multiple sets of beads may be collected and deposited in a single pooling well.

[00307] In some instances, beads may be extracted and pooled using systems as described, for example, in U.S. Patent Publication Nos. 2008 / 0281466 AA or 2008 / 0113361 AA or in U.S. Patent Nos. 6,887,431; 7,347,975 or 7,384,606. In other embodiments of the invention a bead picking instrument with at least one integrated precision-controlled electro-micromagnet may be used. Such a picking instrument may be controlled by a control unit which can be programmed to control the movement of the micromagnet to align with specific microwells. In a further embodiment, the control unit may provide means to control the adjustment of the distance between the micromagnet and the microwell. In a specific embodiment, the micromagnet may be controlled and activated by electric means to allow extraction of single magnetic beads carrying a specific nucleic acid sequence.

[00308] Electro-micromagnets used in aspects of the invention where magnetic beads are used may be hollow magnets or needle shaped and will often be of a size and dimension to focus the magnetic field at its tip to allow for specific targeting of individual beads. In a specific embodiment, the micromagnet may be composed of an electro-magnet and a permanent magnet wherein the activity of the permanent magnet can be controlled by the electro-magnet. Electro- micromagnet used in conjunction with the invention may be in any number or format and may, for example, comprise a single magnet or be arranged together with other micromagnets in a row.

[00309] In certain embodiments of the invention, an electro-micromagnet may be used to extract and pool all magnetic beads contained in the microwells of a single arrays. For this purpose, the electro-micromagnet may be allocated to each microwell to extract the bead- attached nucleic acid molecules in a step-wise manner in a pre-defined or random order. In one embodiment, all nucleic acid molecules required for the assembly of a full-length construct may be synthesized on a single array. According to the amount of nucleic acid molecules required to build a full-length construct, arrays of different sizes and dimensions can be used.

[00310] In another embodiment, the electro-micromagnet may be programmed to target only a portion of the microwells of a specific array to extract and pool a predefined selection of bead- attached nucleic acid molecules. The electro-micromagnet can be programmed to extract and pool beads from the microwells of two or more different plates. The picking may combine full extraction of all beads of a first plate with selective extraction of a portion of beads obtained from a second plate. The first and the second plate may vary in size and dimension.

[00311] Each magnetic bead extracted by the micromagnet may then be transferred to a pooling station by moveable means of the picking instrument. In one embodiment the pooling station may contain a chamber with a microwell plate. In one embodiment the microwell plate may be a 1536 microwell plate. However, microwell plates of other sizes and dimensions (e.g., standard 96 well plates) are known in the art and can be used in the current invention. Defined fractions of nucleic acid molecules can be pooled in individual wells of a microwell plate wherein one pooled fraction contains all nucleic acid molecules required to assemble at least a defined fragment of a full-length construct. In one embodiment, an individual nucleic acid molecule pool may contain all nucleic acid molecules required to assemble a full-length construct. Different nucleic acid molecule pools allocated to each well can be further identified using a machine readable identifier disposed on the microwell plates. B. Non-Magnetic Pooling Mechanisms

[00312] Electrostatic forces may also be used to remove beads and other substrates from synthesis platforms. Using FIGs. 2A and 2B for purposes of illustration, oligonucleotide synthesis substrates (beads in this instance) may have an electrostatic charged and separated from association with a surface or well using an opposite charge. For example, if one or more beads shown in FIGs. 2A and 2B have a positive charge then the lower electrode may be used to generate a positive charge to repel the bead and force it from the well. Magnetic charges can also be used to achieve the same purpose. Residual magnetism may also be employed. In essence, residual magnetism is magnetism that remains in a material after being exposed to magnetic force. In many instances, magnetic substrates will be of small size. Thus, attraction of such substrates will typically not require strong magnetic fields. Residual magnetism may be present in the substrate a selection probe used to bind to the substrate or both. Further, charges may be used to selectively remove a subset of synthesis substrates from a synthesis platform.

[00313] Electrostatic forces for the removal of beads and other substrates from synthesis platforms can be readily calculated. Table 8 below assumes a relative homogeneous electrical field is present and that each bead acts as a single charge point. Nucleic acid molecules carry with them a charge which should be taken into consideration when charge is used to extrude a bead from a well. Further, charge need only be applied to wells that contain substrates with desired nucleic acid molecules (e.g., nucleic acid molecules for assembly into larger nucleic acid molecules. Table 8 [Image disponible dans le document PDF, Image available in the PDF document]

[00314] In another embodiment, a synthesis platform may contain a series of regions that separate from other regions of the synthesis platform. For example, a synthesis platform may contain 100 rows of synthesis areas in a square 10 X 10 arrangement. Further, the synthesis platform may be designed so that it is separable into ten rows of ten synthesis areas. For purposes of illustration, assume that one seeks to produce eight different assembled nucleic acid molecules and these assembled nucleic acid molecules are designed to be formed from the assembly of the following number of oligonucleotides: Table 9 [Image disponible dans le document PDF, Image available in the PDF document]

[00315] Table 9 indicates the numerical designation of the various assembled nucleic acid molecules, the number of oligonucleotides that will be used to assemble the assembled nucleic acid molecules, and the rows in which the oligonucleotides are synthesized in. In this embodiment, rows 1-5 will each have at least one synthesis area in which no oligonucleotides will be produced.

[00316] After synthesis is completed, the separable rows may be separated and the synthesized nucleic acid molecules, collected / processed and assembled, for example, as described elsewhere herein.

[00317] In another aspect, electrolysis can be used to remove beads or other substrates from a synthesis platform. FIG. 18 shows a simplified example of a selective bead removal process from a synthesis chip 1805 using electrolysis according to the present teachings. For example, the synthesis chip 1805 can be a CMOS chip and can contain a plurality of microwells, such as microwell 1810. Each microwell also contains a first electrode 1815 (working electrode) formed at a bottom surface of the microwell and a second electrode 1820 (counter electrode). Each microwell is sized to accommodate a solid substrate, such as a bead 1825 and a volume of working fluid.

[00318] For example, electrode 1815 and 1820 can be similar to the electrodes that are discussed further below in relation to FIG. 9 and Table 11 and can be composed of any number of compounds, including platinum, palladium, copper, gold, aluminum, niobium, niobium oxide, tungsten, titanium, tantalum, molybdenum, nickel, platinum, silver, manganese, neodymium, carbon, and silicon, and an alloy material or a compound material containing one or more of the above-described elements, as well as other elements. A sufficiently high electrical potential difference is provided by a controller (not shown) between the electrode 1815 (working electrode) and electrode 1820 (counter electrode) to cause a current to flow in the fluid in the microwell and to produce an electrochemical reaction, e.g., electrolysis, resulting in the formation of one or more gas bubbles in the microwell.

[00319] For example, if the fluid is water, then about a 0.01V to about 10,000V, a 5V to about a 30V, or about 10V to about a 20V, or about a 15V to about a 30V, voltage can be applied to produce the electrochemical reaction. As the one or more gas bubbles expand and move toward the surface of the microwell 1810, the bead 1825 will also tend to rise. As a result, the volume of the fluid in the microwell 1810 is displaced and the bead 1825 is released from the microwell 1810 into a fluidic channel (not shown), for example in the direction indicated by the arrow in FIG. 18. A current controller, which could be the current controller discussed in relation to FIGs. 2A and 2B, can be used to control the amount of current applied to each electrode, which in turn can control the displacement of the beads from their respective wells. An increase of the current beyond a current threshold can result in potential loss of the bead during the bead removal process and / or heating of the fluid within each well. For example, current limit for one electrode can be multiplied by the number of active electrodes to yield the overall current limit that can be set by the current controller. The generation of the air bubbles generally occurs quickly, usually taking less than a second (although longer times may be used) for the bead to be displaced due to the rising of one or more gas bubbles, and is not dependent on the surface charge or other properties of the bead.

[00320] As noted herein, the generation of gas bubbles in microwell plates can be used as a method to remove beads from the respective well. Gas bubbles can be produced electrochemically in aqueous or non-aqueous buffers (e.g., water, NaCl dissolved in water and more complex non-aqueous buffers like 10 mL MeOH; 35mL ACN; 1 M hydroquinone; 10 mM benzoquinone; 0.25M NEt4pTsO). The inherent properties of the buffer used can have significant implications on the efficiency or performance of the system. For example, the composition of the buffer can influence the surface tension of the bubbles produced. The surface tension is critical for the bubble movement in the well (retention potential). For an efficient removal of beads from a well it is desirable that both, beads and bubbles escape from the well (compared to the bubbles remaining in the well and disturbing the fluidic flow of the system). If the surface tension is too low, the generated bubbles will escape through the gap between well and bead without lifting the bead. If, however, the surface tension is too high, gas bubbles will stick tightly to the walls of the well, which may require additional treatment to remove the gas bubbles such as longer rinsing or rinsing with low surface tension solvents (such as e.g. methanol). Further, high surface tension may also result in beads sticking to gas bubble and not being released into the fluid stream. A favorable surface tension can be achieved by mixing organic solvents (e.g., acetonitrile, isopropanol) and aqueous solutions, preferable 50-90% organic solvents, more preferably, 60-70% organic solvent. The buffer can also be selected to avoid negative effects on the synthesis process. For example, acidic environment has the potential to damage the nascent oligonucleotide chain, and basic condition can promote premature cleavage of the oligonucleotide from the bead. Therefore, a buffered system can be used to avoid the generation of such an undesired condition. Although different buffer systems are available (e.g., HEPES, TRIS, carbonate or ammonium based buffers) a volatile buffer (e.g., ammonium sulfate) is preferred for the application in order to avoid undesired residues which might negatively influence other reaction steps.

[00321] In certain instances a lifting buffer may comprise water, tetraethylammonium-p- toluolsulfonat (NEt4pTsO), acetonitrile and methanol. In an exemplary embodiment a lifting buffer comprising 0.7 M (NEt4pTsO), 50% water, 30% methanol, and 20% acetonitrile can be used. This buffer has a high conductivity required for electrochemical generation of gas bubbles while its surface tension allows for efficient bead lifting and removal of bubbles from wells. Using this buffer, bead lifting can be achieved at a potential of >4.5 V. In certain embodiments a higher potential may be used for optimal bead lifting. For example, 8.5 V may be used without limiting the current. In a typical experiment a current of 20-60 mA may be observed for approximately 3,500 wells.

[00322] In certain embodiments, the displacement of one or more beads from the multiwell plate is controlled and programmed by computer directed automation. For example, when the synthesis chip 1805 is formed as the CMOS chip, each working electrode formed at the bottom of the microwells can be individually addressable, which allows the controller to selectively energize one or more working electrodes at a given time. Thus, one or more beads can be displaced from their respective microwells at the same time or about the same time to be subsequently collected for further processing and analysis.

[00323] For example, the synthesis chip 1805 can have a plurality of microwells, with each microwell having an associated working electrode. As discussed above, the number of wells in the multiwell plate may also vary widely and is limited by factors such as the amount of nucleic acid to be produced and technical factors such as manufacturability and mechanic factors related to use (e.g., the lower size limit of magnetic bead extractors).

[00324] The fluid in the microwell, as discussed further above in relation to the composition and concentration of EGA components, can include an aqueous or non-aqueous buffer solution. For example, the fluid can include relatively simple solutions, such as, water, NaCl dissolved in water, as well as more complex non-aqueous buffers like 10 mL methanol; 35 ml acetonitrile; 1 M hydroquinone; 10 mM benzoquinone; 0.25 M Net4pTsO).

[00325] Once the bead or other substrate is displaced from its corresponding microwell, the bead or other substrate can be collected by a bead collection device that can be programmed and controlled to extract and pool individual beads harboring synthesized nucleic acid molecules. Depending on the application and the number of nucleic acid molecules to be assembled, all of the beads of the microwell array may be pooled or only a subset of the beads.

[00326] By way of example, the bead or other substrate can be collected by a bead collection device 1905, as shown in FIG. 19, and in greater detail in FIG. 20, and placed into a multiwell plate 1910 or other suitable container for further processing and analysis. In this way, the bead collection device can be used to transfer one or more beads or other solid supports from the synthesis chip in an automated fashion to one or more wells of a multiwell collection plate or other suitable container for further processing. In addition, the bead collection device can be used to concentrate the beads or other substrates of similar sizes and dimensions (such as, e.g., vesicles or cells) into a smaller volume.

[00327] The bead collection device 1905 can include a first flow channel 1915 operable to allow the bead 1825 and associated fluid to flow in a first direction, e.g., downward, to be placed in a well of the multiwell plate 1910 or other suitable container. For example, due to gravity, the beads can fall into the selected well of the multiwell plate 1910 and be collected. The bead collection device 1905 can also include a second flow channel 1920 operable to allow fluid, e.g., waste fluid, to flow in a second direction, e.g., upward, opposite the first direction where it can be discarded. A pump 1925, e.g., a syringe pump, and associated tubing 1930 can be used to provide negative pressure, e.g. a vacuum, sufficient to cause the fluid to flow through the second flow channel 1920. The second flow channel 1920 may be equipped with a barrier or pillars to prevent beads from entering the second flow channel. In this way, the bead collection device can be used to collect and pool all of the beads from the synthesis platform having nucleic acid molecules that belong to one fragment. The pooled beads can be transferred to one or more wells of a multiwell plate or other suitable container for further processing, such as cleavage and deprotection to prepare the nucleic acid molecules for subsequent assembly steps as appropriate.

[00328] Each microwell of the multiwell plate 1910 can be sized to accommodate one or more beads. In one example, the multiwell plate 1910 can be supported by a moveable support structure 1930, e.g., a xyz stage, that is operable to be actuated in up to three degrees of freedom. This can allow the bead 1825 collected by the bead collection device to be transferred into a specific microwell on the multiwell plate 1910. Alternately, the bead collection device 1905 can be mounted on a moveable support structure that can allow the bead collection device 1905 to be actuated in up to three degrees of freedom. This process of using the bead collection device to transfer a bead or other substrate into a microwell plate can be performed manually or by computer directed automation.

[00329] In certain embodiments, the bead collection device 1905 can include an acoustic module and associated power and control circuitry (not shown) that can be operable to vibrate the bead collection device 1905 as a whole or selectively vibrate the first and / or second flow channels 1915, 1920 to facilitate the bead 1825 placement or the fluid removal. The bead collection device 1905 can also include a detector (not shown) operable to detect whether the oligo-bead 1825 has been removed from the bead collection device 1905.

[00330] In one embodiment, the multiwell plate into which the bead is placed includes a fluid-permeable structure to facilitate bead placement into the multiwell plate and the concentration of the bead(s) into a smaller microwell volume. FIG. 21 shows another example multiwell plate 2105, according to the present teachings. The multiwell plate 2105 can include a fluid-permeable structure 2110 (such as, e.g., a micromesh) that can be attached, either permanently or non-permanently, to a top surface of the multiwell plate 2105. The fluid- permeable structure 2110 can be composed of a material that is permeable to fluid, and yet provides sufficient rigidity to hold one or more oligo-beads within a given well. The multiwell plate 2105 can also be composed of two multiwell plates separated by the fluid-permeable structure 2110. In one example, the multiwell plate is a standard 1536 well plate, as known in the art.

[00331] In the example of FIG. 21, a bead collection device, similar to the bead collection device 1905 as shown in FIG. 19, can include a needle or needle-like structure 2115 and associated tubing 2120 to place one or more beads in one or more wells of the multiwell plate 2105 as well to remove excess fluid from the multiwell plate 2105. The needle 2115 can be coupled to a moveable support structure, e.g., a xyz stage, to move the needle 2115 in one or more degrees of freedom to place the beads in the appropriate wells of the multiwell plate 2105. The needle 2115 can also have a needle-in-a-needle or needle-in-a-tube arrangement, such that a first needle is positioned inside a second needle or inside the tubing 2120. Alternatively, the first needle can be positioned adjacent to / parallel to a second needle or tubing 2120. For example, the first needle can be operable to deliver the beads to the multiwell plate 2105 and the second needle or tubing can be operable to remove excess fluid from the multiwell plate 2105. In such setting, the length of the first needle exceeds the length of the second needle or tubing such that in operation only the first needle gets into contact with the fluid-permeable membrane and the liquid volume contained between the fluid-permeable membrane and the bottom of the well, whereas the second needle or tubing gets in contact with the liquid volume contained between the fluid-permeable membrane and the top of the well. Also, the shape of the first and second needle may be the same or may be different. For example, the first needle may have a sharp or pointy end capable of puncturing through the fluid-permeable structure 2110, whereas the second needle or tubing may have a blunt end. Furthermore, the lumen of the first needle may be equal to or smaller than the lumen of the second needle or tubing. The lumen of the first needle should be of a dimension allowing one or more beads of a size as disclosed elsewhere herein and optionally loaded with oligonucleotides to smoothly pass through it into the well, whereas the lumen of the second needle or tubing should be of a dimension allowing excess liquid to be removed by vacuum from the well. Needles are available in a wide variety of outer diameters described by gauge numbers, wherein a smaller gauge number indicates a larger outer diameter. The inner diameter of a needle depends on both gauge and wall thickness. For purposes of illustration, the first needle may for example be a Hamilton® syringe with luer-lock and 25 or 26 gauge, whereas the second needle may be blunt with luer-lock and 17 gauge, 18 gauge or 19 gauge.

[00332] In operation, the needle 2115 can puncture through the fluid-permeable structure 2110 and beads that have been removed from the synthesis chip 1805 can be transferred into the multiwell plate 2105. The beads cannot escape the well of the multiwell plate 2105 because of the fluid-permeable structure 2110, while excess fluid can escape and is removed through the second needle or tubing by vacuum. This arrangement can reduce cross contamination between wells. If needed, the needle 2115 can now be washed and made ready for the next oligo-bead transport. After placing all of the beads into the multiwell plate 2105, a centrifugation step can be performed to ensure that the beads are at the bottom of the wells. Keeping the fluid- permeable structure 2110 on the top of the multiwell plate 2105 can also help with the gaseous deprotection of the nucleic acid molecules attached to the beads. After the deprotection, the multiwell plate 2105 can be opened and the bottom plate with the cleaved / deprotected nucleic acid molecule is ready for gene assembly reactions.

[00333] In other embodiments, rather than using a needle to puncture the fluid-permeable structure, pressure is applied to the top of a selected well in the multiwell collection plate, the pressure being sufficient to rupture the fluid-permeable structure and deliver one or more beads into the selected well of the multiwell collection plate. In certain embodiments, the pressure applied to rupture the fluid-permeable structure is between 0.1-50, 0.1-25, 0.1-20, 0.1-15, 0.1-10, 1-25, 1-20, 1-15, or 1-10 bar.

[00334] In other embodiments, the oligonucleotides synthesized on the microchip can be pooled, concentrated, cleaved and deprotected on a fluid-permeable structure arranged on a top surface of or within a multiwell collection plate. The cleaved oligonucleotides can then be eluted into the well of a multiwell collection plate without having to puncture or otherwise rupture the fluid-permeable structure. FIG. 22 shows an exemplary system by which beads are collected from the synthesis microchip and pooled on a fluid-permeable structure arranged on a top surface of or within a multiwell collection plate. While the beads are retained on the fluid-permeable structure, the oligonucleotides on the beads are cleaved, deprotected, and eluted into a well of the microwell collection plate, in accordance with the present disclosure. Beads 2215 can be removed from the synthesis chip 2205 and collected, pooled, and concentrated, as shown at (1), into an individual well 2225 of the multiwell plate 2220, according to the various mechanisms discussed herein. A vacuum 2230 can be applied to direct the beads 2215 through one end of a fluid conduit 2210 into the well 2225 of the multiwell plate 2220. As discussed above in relation to FIG. 21, the multiwell plate 2220 can include a fluid-permeable structure 2230 either arranged on a top surface of or within the multiwell plate 2220. The fluid conduit 2210 can position the beads 2215 onto a top surface of the fluid-permeable structure 2230. Oligonucleotides can be cleaved from the beads 2215 and deprotected using, for example, an ammonia atmosphere 2240, as shown at (2). Once the oligonucleotides are cleaved and deprotected from the beads 2215, they can be eluted 2245 through the fluid-permeable structure 2230 and collected in the bottom of a well of the multiwell plate 2220, as shown at (3).

[00335] The frame of the multiwell collection plate 2220 can be composed of any suitable material, including, but not limited to, a polystyrene, polyethylene ("PE") or a polypropylene ("PP") material or cyclic olefin copolymer ("COC"), stainless steel, polytetrafluoroethylene ("PTFE") or polycarbonate, and can be solvent compatible. The multiwell collection plate 2220 can be covered on a top surface by a fluid-permeable structure 2230. The fluid-permeable structure 2230 can be composed of any suitable material, including, but not limited to, a polyethylene terephthalate ("PET"), polytetrafluoroethylene ("PTFE"), polypropylene ("PP") or a polyetheretherketone ("PEEK") material, that are fluid and gas permeable, but are able to hold the beads within the well 2225. For example, the fluid-permeable structure may be a polypropylene mesh. The mesh may be placed between two multiwell plates as indicated in FIG. 45B. To avoid lateral flow of liquids (and eluted oligonucleotides) between adjacent wells and / or fix the mesh within the multiwell plate, the mesh may be subject to heat treatment (e.g. on a hot plate) to melt and seal the material along the rim of the well protrusions of the upper and / or lower multiwell plate. The sealing process may be facilitated by applying pressure to the external surface of the upper and / or lower multiwell plate.

[00336] In one embodiment, a bead collection device may comprise a multiwell plate ("filter plate") with a fluid-permeable structure as discussed above and a coaxial needle assembly as shown in FIG. 45A. The needle assembly ensures fast pooling of beads into one or more wells of a filter plate. The needle or needle-like structure may be associated with tubing to place one or more beads in one or more wells of the filter plate. The needle can be coupled to a moveable support structure, e.g., a xyz stage, to move the needle in one or more degrees of freedom to place the beads in the appropriate wells of the filter plate. The needle can also have a needle-in- a-needle or needle-in-a-tube arrangement, such that a first needle is positioned inside a second needle or inside the tubing. Alternatively, the first needle can be positioned adjacent to / parallel to a second needle or tubing. For example, the first needle can be operable to deliver the beads to the filter plate and the second needle or tubing can be operable to provide a washing and / or elution buffer to rinse the beads collected in the filter plate and / or elute the oligonucleotides through the mesh. The needle assembly may be configured to allow for pooling and washing of beads at the same time. The chemical solution that is used for bead unloading by electrolysis preferably contains high concentrations of salt for high conductivity. The salt needs to be removed by washing with a suitable buffer (e.g. 50% acetonitrile, 50% water) before the nucleic acid molecules are cleaved and eluted from the beads. The simultaneous washing can be achieved by using, e.g., a needle-in-needle configuration wherein a first needle is connected with the microfluidic chip and configured to receive and place the beads into a pre-determined well of the microwell plate. The second needle can be connected with one or more reservoirs containing a washing and / or elution reagent and can be configured to transfer liquid from the reservoirs to the well containing pooled beads.

[00337] Once oligonucleotides have been cleaved from the beads and deprotected as described elsewhere herein, they can be eluted from the filter plate through the fluid-permeable mesh into a further multiwell plate arranged below the filter plate as illustrated by FIG. 45C. For this purpose, the filter plate may be moved (e.g., by means of an xyz-stage) from a first washing / purging position to a second elution position. The further multiwell plate may comprise wells of a size and dimension allowing alignment of a well of the filter plate with a well of the further multiwell plate such that oligonucleotides can be eluted simultaneously or subsequently from multiple wells of the filter plate into multiple aligned wells of the further multiwell plate. In certain instances, the protrusions of the lower filter plate may fit into the protrusions of the wells of the further multiwell plate. In other instances the protrusions of both plates may align exactly and be placed in contact with each other. The oligonucleotides may be eluted by centrifugal force or vacuum. For example the filter plate and further microwell plate may be arranged in a fixing device or frame holding both plates together during a centrifugation or vacuum step.

[00338] The multiwell collection plate, filter plate or further multiwell plate can comprise any appropriate number of wells. The most common multiwell plates have 96, 384, or 1536 wells. In certain embodiments, the multiwell collection plate has 1536 wells. The volume of each well in a 1536 well plate is about 12 µl with a working volume of about 3-10 µl. Each well of the multiwell collection plate can accommodate one or more beads from the synthesis chip. For example, in certain embodiments, each well of the multiwell collection plate comprises between 1-1,000, 1-500, 1-350, 1-250, 1-100, 1-50, 100-1,000, 100-500, 100-350, 100-250, 250- 500, 250-350 or about 330 beads transferred from the synthesis chip. Further, the beads may occupy a specific volume of the well. For example, with a 1536 well plate, the beads may occupy up to about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, 0.5%, 0.1%, or 0.05% of the total well volume. In one embodiment, the beads occupy about 0.1% of the total well volume.

[00339] Other methods may also be used to collect and pool nucleic acid synthesis substrates, including (1) "grabbing", for example by the use of tweezers like devices which operate based upon mechanical (e.g., actual grabbing), optical, sonic, magnetic principles, (2) "destroying" structures surrounding nucleic acid synthesis substrates by methods such as chemical dissolution or through the use of lasers, (3) moving nucleic acid synthesis substrates by, for example, the use of thermal, electrostatic, magnetic, fluidic energy, (4) hybrid gripper which combine, for examples, (a) magnetic and fluidic flushing, (b) magnetic and piezoelectric methods, and (c) electrostatic lifting and fluidic flushing, (5) magnetic fixing / collecting using, for example, modulated permanent magnets, external coils, planar coils on synthesis substrates, etc., (6) electrostatic lifting & collecting, and (7) flux direction (e.g., the addition of fluid to the bottom of a well to lift substrates).

[00340] Additionally or alternatively, beads can be removed from the synthesis chip using other non-magnetic techniques, including, but not limited to, gravity-fed, dielectrophoresis, valving, or using a weir structure on the synthesis microchip. For example, if the bead contains a dielectric material, then a non-uniform electric field can be generated using, for example, the electrodes of the synthesis microchip, to produce a force (dielectrophoresis) that can be used to selectively remove beads from their respective wells on the synthesis microchip. By way of yet another example, the synthesis microchip can include a valving, weir, or barrier-like structure that can be used to alter the flow of the removed beads in a particular direction, such as to direct the beads to a particular well of the multiwell collection plate. C. Electrochemically Generated Base Cleavage of Nucleic Acids

[00341] In certain embodiments disclosed herein, the synthesized nucleic acid molecules may be cleaved directly from the beads, or other suitable solid support, in the microchip and eluted into a suitable container, such as, for example a multiwell plate. In certain embodiments, the nucleic acid molecules can be cleaved from the one or more beads using an electrochemically generated base (EGB), as described herein. Nucleic acid molecules that belong to the same fragment can be pooled together at this stage as described herein. After cleavage, a concentration step may be performed, for example, using a reverse phase material or any other suitable resin, as described herein.

[00342] In certain embodiments disclosed herein, an EGB may be used to cleave the synthesized nucleic acid molecule from the bead, or other suitable solid support, directly in the well of the microchip. In a first step, protected nucleic acid molecules are synthesized according to methods disclosed herein and coupled to a bead via a base-cleavable linker, such as, for example, succinate. As used herein, a base-cleavable linker may be defined as any linker capable of being cleaved from a solid support by a base catalyzed reaction. The nucleic acid molecules are synthesized on a bead or other solid support in a microchip comprising at least one electrode, such as a platinum electrode, as disclosed herein. Next, a compound, such as azomethane, may be chemically reduced on the at least one electrode to yield an EGB. Other compounds that may be chemically reduced on the at least one electrode to yield an EBG include, for example, azobenzene, anthraquinone, aromatic halides (where the halide is iodine or bromine), and carbon disulfide. The EGB then acts to cleave the synthesized nucleic acid molecules from the beads or other suitable solid support.

[00343] As the EGB is only generated on activated electrodes, a site-selective cleavage of the desired nucleic acid molecules may be accomplished. The activation of the electrode causes the cleavage of the nucleic acid molecules that are linked to the bead with the base-cleavable linker, and the cleavage can be done in a highly selective manner so that only electrodes that are selected and activated have the nucleic acid molecules cleaved. The cleaved nucleic acid molecules are then free in solution and available for further processing. FIG. 25 shows an exemplary reaction wherein a nucleic acid molecule is bound to a bead via a base-cleavable linker. FIG. 25 shows electrons from the electrode acting to reduce azomethane to the base, methylamine. The methylamine then acts to cleave the nucleic acid molecule from the bead into the solution. In certain exemplary embodiments, an EGB may be generated from 1M dimethyldiazene, via activation of an electrode at about 7.5V for about 5 minutes. D. Photolytic and Reductive Cleavage of Nucleic Acids

[00344] In certain other exemplary embodiments, the synthesized nucleic acid molecule may also be cleaved from the bead or other solid support by other cleavage means known in the art, such as photolytic cleavage and reductive cleavage. In certain embodiments, cleavage conditions may be generated in a site selective manner, such as in selected wells and / or on selected beads. In certain exemplary embodiments, a universal linker such as a UNYLINKERIM or a preloaded base is located between the synthesized nucleic acid molecules and the cleavable linker in order to prevent 3' phosphorylation of the desired nucleic acid molecules.

[00345] For photolytic cleavage, a photocleavage linker may be irradiated with lightwaves, such as UV lightwaves. The lightwaves trigger the cleavage of the nucleic acid molecules from the solid support (e.g., the bead). In certain embodiments, spatial control may be achieved with a light source, such as a mirror device like a digital micro mirror device. In certain exemplary embodiments, the light source illuminates selected synthesis positions (e.g., wells and / or beads), thereby cleaving selected nucleic acid molecules. FIG. 31 illustrates an exemplary embodiment wherein a photocleavable linker is bound to a bead and an oligonucleotide. Nonlimiting examples of photocleavable linkers that may be mentioned include, for example, o-nitrobenzyl, desyl, trans-o-cinnamoyl, m-nitrophenyl, and benzylsulfonyl groups.

[00346] For reductive cleavage, a linker may be used that can be cleaved via an electrochemically reduced compound. In one exemplary embodiment, a disulfide linker may be used, as illustrated in FIG. 32. When a disulfide linker is used, for example and as shown in FIG. 32, 2-hydroxyethyl disulfide may be electrochemically reduced to generate 2- mercaptoethanol in situ on the desired synthesis positions. In certain embodiments, the reducing reagent, such as the 2-hydroxyethyl disulfide, is only reduced on the desired activated electrodes such that cleavage is site specific. The resulting monosulfide acts to cleave the desired oligonucleotide from the disulfide linker. E. Enrichment of Nucleic Acids Using Solid Phase Materials

[00347] In certain embodiments, after the cleaved nucleic acid molecules are released into a solution, it may be desirable to perform a concentration step. For example, in certain embodiments the cleaved nucleic acid molecule volume may be concentrated so that the volume is compatible for use in certain multiwell plates for pooling as discussed herein, including, for example, 20 µL wells or a 1536 microwell plate (10 µL wells). Several exemplary embodiments are feasible for enrichment of the cleaved nucleic acid molecules.

[00348] In certain embodiments disclosed herein, a solid phase material may be used to absorb cleaved nucleic acid molecules, wherein the solid phase material is in a direct flow path following the cleavage step, such that cleaved nucleic acid molecules flow to the solid phase material, and then to an optional washing step, followed by an elution step, in a cycle of cleavage, absorption, optionally washing, and elution. Alternatively, in certain other exemplary embodiments, a solid phase material may be used in multiple positions of a multiwell plate, such that the cleavage, absorption, optional washing, and elution steps may occur one after each other, in parallel. Such parallel cleavage, absorption, and elution may be advantageous over cyclic cleavage, absorption, and elution, as it may in certain embodiments be more time efficient.

[00349] Absorption of the cleaved nucleic acid molecules may be performed by any means known in the art. For example, in certain embodiments, absorption of the cleaved nucleic acid molecule may be by a reverse phase resin material. When a reverse phase resin material is used to concentrate oligonucleotides that have been cleaved by an EGB, the cleavage reaction with the EGB may be carried out in an aqueous solution, or in certain embodiments, an aqueous solution comprising a small amount of at least one organic solvent, such as 20% acetonitrile. Subsequently, the nucleic acid molecule solution having a relatively low concentration of nucleic acid molecules may be pumped through a small amount of a reverse phase material. As used herein, reverse phase material refers to hydrophobic material having an affinity for hydrophobic compounds, thereby allowing hydrophilic compounds to elute in an aqueous solution. Reverse phase materials may include, for example, silica-based reverse phase materials, such as, for example, C18 modified silica or, in certain embodiments, polystyrene reverse phase materials. The protected nucleic acid molecules will remain on the reverse phase material, which may then be washed and dried, for example with nitrogen and / or air.

[00350] Next, the protected nucleic acid molecules may be eluted with an organic solution comprising at least an agent for removing a protecting group from a nucleic acid molecule, such as, for example, methylamine in a water / ethanol mixture. Any additional suitable organic solvents may be used, including, for example, at least one of acetonitrile, methanol, tetrahydrofuran, and isopropyl alcohol. In certain embodiments, the nucleic acid molecules may be eluted with 80% acetonitrile.

[00351] In certain embodiments, absorption of the cleaved nucleic acid molecule may be by a size exclusion material, such as cross-linked dextran gels (e.g., Sephadex®). The cleaved nucleic acid molecules may be retained in the size exclusion material and then eluted via gravity, such as in a centrifuge.

[00352] In certain other embodiments disclosed herein, absorption of the cleaved nucleic acid molecules may be by an anion exchange resin. Exemplary anion exchange resins may include, for example, resins comprising quaternary ammonium groups as the ion exchange groups, for example in styrene based resins such as crosslinked polystyrene or in acrylic resins. In certain embodiments, the absorption may occur at a relatively low anion concentration, such as about 1 mM. In certain exemplary embodiments, 1 mM Cl- ions may be used. When an anion exchange resin is used, elution of the cleaved nucleic acid molecules may be performed with a relatively high anion concentration, such as about 1 M. In certain exemplary embodiments, 1 M Cl- ions may be used.

[00353] In certain other embodiments disclosed herein, absorption of the cleaved nucleic acids may occur on silica beads, such as those available from ThermoScientific and sold as the Silica Bead DNA Gel Extraction Kit. In this embodiment, the cleaved nucleic acids and at least one chaotropic salt are applied to the silica solid phase at a given pH. The presence of at least one chaotropic salt allows the cleaved nucleic acid to aborb to the silica. In certain embodiments, an optional washing step may be performed, followed by elution of the nucleic acids from the silica by an eluent. In certain embodiments, the cleaved nucleic acids may be absorbed to the silica at a given pH, such as an acidic pH, and then eluted from the silica as the pH gradient of the eluent changes, for example, increases to a more basic pH. In certain embodiments, the eluent may be water or a tris-EDTA buffer, such as a tris-acetate EDTA buffer or a tris-borate EDTA buffer.

[00354] In certain embodiments, the volume of the elution solution may be chosen to be low enough to enable dispensing the resulting solution in a multiwell plate, such as a 1536 microwell plate, with a fraction collector. The synthesized nucleic acid molecules may now be deprotected and pooled in the wells of a multiwell plate, preferably in volumes between 0.1 µL and 25 µL, <semantics>0.1μL<annotation encoding="application / x-tex">0.1 \mu L< / annotation>< / semantics> and <semantics>10μL<annotation encoding="application / x-tex">10 \mu L< / annotation>< / semantics>, <semantics>1μL<annotation encoding="application / x-tex">1 \mu L< / annotation>< / semantics> and <semantics>25μL<annotation encoding="application / x-tex">25 \mu L< / annotation>< / semantics>, <semantics>1μL<annotation encoding="application / x-tex">1 \mu L< / annotation>< / semantics> and <semantics>10μL<annotation encoding="application / x-tex">10 \mu L< / annotation>< / semantics>, <semantics>5μL<annotation encoding="application / x-tex">5 \mu L< / annotation>< / semantics> and <semantics>25μL<annotation encoding="application / x-tex">25 \mu L< / annotation>< / semantics>, <semantics>5μL<annotation encoding="application / x-tex">5 \mu L< / annotation>< / semantics> and <semantics>10μL<annotation encoding="application / x-tex">10 \mu L< / annotation>< / semantics>, or about <semantics>10<annotation encoding="application / x-tex">10< / annotation>< / semantics> μL.

[00355] In certain embodiments, the solution comprising the synthesized nucleic acid molecules may then be evaporated, for example in a speedvac, before further processing. Optionally, in certain embodiments, an enzyme mix may be added to the dried nucleic acid molecule mixtures in order to start the gene assembly process or any other desired reaction.

[00356] In alternative embodiments, use may be made of a multiwell plate, such as a 1536 filter plate, that has been loaded with a solid phase resin, such as a reverse phase resin, a silica material, or an anion exchange resin. After the nucleic acid molecules are cleaved from the one or more beads by the EGB, as disclosed herein, the cleaved nucleic acid molecules may be rinsed in an individual well of the filter plate, wherein the nucleic acid molecules will bind to the solid phase resin. Next, the protection group on the nucleic acid molecules may be removed using known techniques, including, for example, gas phase or liquid deprotection. In certain embodiments, a washing step may be used to remove any free protecting groups and / or short truncated nucleic acid molecules.

[00357] Another method disclosed herein for the retrieval of nucleic acid molecules after synthesis is the use of a microfluidic chip for recovering nucleic acid molecules, such as nucleic acid molecules belonging to the same fragment from complex microarrays. The microfluidic chip used for retrieving the nucleic acid molecules may, for example, be similar to the microwell plates disclosed herein or may be similar to the microfluidic chip disclosed in Autebert, J. et al., Hierarchical Hydrodynamic Flow Confinement: Efficient Use and Retrieval of Chemicals for Microscale Chemistry on Surfaces, Langmuir 2014, 30(12) 3640-3645.

[00358] Disclosed herein are methods of retrieving nucleic acid molecules from a multiwell plate, a microfluidic chip or a microarray using micro-elution. It has been shown that hybridized nucleic acid molecules may be recovered by local denaturation from surface treatment with a NaOH solution, such as about 0.5M NaOH. Therefore, one method for retrieving nucleic acid molecules comprises preparation of two complementary sets of nucleic acid molecules on two microfluidic chips, wherein members of the first set of nucleic acid molecules can be hybridized to members of the second set of nucleic acid molecules and vice versa. In certain embodiments, the same type of microfluidic chip or microarray is used for the synthesis of both sets of nucleic acid molecules.

[00359] Synthesis of the complementary sets of nucleic acid molecules could be by any means known in the art, such as, for example, via inkjet or other techniques, including the synthetic techniques disclosed herein. In certain exemplary embodiments, the nucleic acid molecules may be synthesized on a bead, and the microfluidic chip may comprise a plurality of wells in which the beads may be located with each well having an associated working electrode that is individually addressable. The first and / or second sets of nucleic acid molecules may be prepared in one or more wells of a multiwell plate, a microfluidic chip or a microarray and may be prepared in an average amount of from about 1 attomole to about 1 picomole, from about 10 attomole to about 1 picomole, from about 10 attomole to about 100 picomole, from about 10 attomole to about 100 attomole etc.

[00360] As disclosed herein, a first set of nucleic acid molecules is synthesized on a first microfluidic chip, and a second set of nucleic acid molecules that is complementary to the first set is synthesized on a second microfluidic chip. In certain embodiments, the synthesized sets of nucleic acid molecules are synthesized such that nucleic acid molecules required for the assembly of a specific fragment are located in the same region on both of the chips. For example, if both sets of nucleic acid molecules are synthesized on identical microfluidic chips, then a subset of nucleic acid molecules associated with a fragment to be assembled from the first set nucleic acid molecules may be synthesized in a specific x-y location (e.g., region or wells) on the first chip. The same subset of nucleic acid molecules associated with the same fragment to be assembled from the second set of nucleic acid molecules may also be synthesized at the same x-y location on the second chip.

[00361] After synthesis, the nucleic acid molecules from the first chip are cleaved off and deprotected. The side protection groups of the nucleic acid molecules from the second chip are removed, but according to embodiments disclosed herein, the nucleic acid molecules on the second chip remain linked to the surface. To achieve this, for example the first and second sets of nucleic acid molecules may be attached to the surface (e.g. a bead in a well) via first and second types of linkers, respectively, wherein the first type of linker allows the first set of nucleic acid molecules to be cleaved off under deprotection conditions, and wherein the second type of linker allows the second set of nucleic acid molecules to remain linked to the surface under deprotection conditions. For example, the first type of linker may be a chemically-cleavable linker and the second type of linker may be a photocleavable or reductive cleavable linker as discussed elsewhere herein. Next, the second chip is contacted with the deprotected nucleic acid molecules from the first chip under hybridizing conditions such that the deprotected nucleic acid molecules from the first chip hybridize to nucleic acid molecules on the second chip. Optionally, at least one washing step may be performed to wash away non-bound nucleic acid molecules from the second chip.

[00362] Next, the nucleic acid molecules from the first chip that are hybridized to the second chip are denatured with a denaturing solution. In certain embodiments, the bound nucleic acid molecules may be chemically denatured with a basic solution, such as a NaOH solution. In certain embodiments, a 0.5M NaOH solution may be used to denature the nucleic acid molecules. The denatured nucleic acid molecules are then pooled and collected.

[00363] In certain embodiments, a microfluidic device may be used to add solutions (e.g., buffer or denaturing solution) or transfer the nucleic acid molecules cleaved from the first microfluidic chip to specific regions on the second microfluidic chip, such as wells in a multiwell plate. As will be appreciated by one skilled in the art, after cleavage and deprotection, a nucleic acid molecule synthesized in a specific x-y location of the first chip may be combined with the same nucleic acid molecule synthesized in the same x-y location of the second chip for the hybridization step. In this way, the same nucleic acid molecules may be combined physically in the same well on the second chip, without the presence of other nucleic acid molecules.

[00364] The microfluidic device may also be used to remove solutions (e.g., buffer or denaturing solution) and denatured nucleic acid molecules from specific locations, such as wells of a multiwell plate, where they may be collected, for example in a new multiwell plate or to a droplet making device for further processing, such as fragment assembly. In certain embodiments, the microfluidic device may be similar to the microfluidic probe disclosed in Autebert, J. et al., Hierarchical Hydrodynamic Flow Confinement: Efficient Use and Retrieval of Chemicals for Microscale Chemistry on Surfaces, Langmuir 2014, 30(12) 3640-3645.

[00365] FIG. 26 shows an exemplary microfluidic device according to certain embodiments disclosed herein. As shown in a FIG. 26, at least one buffer solution may be added to the second microfluidic chip 2605 via a first inlet channel 2601 in the microfluidic device 2600. Additionally, the denaturing solution and the first microarray's deprotected nucleic acid molecules may be added to the second microfluidic chip 2605 via a second inlet channel 2602 in the microfluidic device 2600. The at least one buffer solution may act to "nest" the denaturing solution and nucleic acid molecules, as shown in FIG. 26. The at least one buffer solution may then proceed out of the microfluidic device 2600 through a first outlet channel 2604, while the denaturing solution and nucleic acid molecules may proceed out of the microfluidic device 2600 through a second outlet channel 2603. The nucleic acid molecules from the second outlet channel 2603 may then be collected for further processing.

[00366] The above-described use of a microfluidic chip and microfluidic device for recovering nucleic acid molecules may have several advantages over using mixtures from multiwell plates. For example, the dilution of the nucleic acid molecules may be very low, as the microfluidic chip may contain only microliter or even nanoliter volumes. Additionally, nucleic acid molecules belonging to the same fragment can be combined physically, without the presence of other nucleic acid molecules. Other methods known in the art that amplify the desired nucleic acid molecules from a mixture by, for example, PCR may be more time consuming and have more contamination. Finally, the hybridization step adds an error correction step, because only nucleic acid molecule sequences with no or minor errors cleaved from the first microfluidic chip will hybridize to the second microfluidic chip. The hybridization step also adds a cleaning step, as very short nucleic acid molecules can be removed by optimizing the hybridization conditions.

[00367] Pooling stations used in the practice of the invention may further contain a microwell handling device which comprises controllable moveable means for moving the microwell plate from a first to at least a second position in X and / or Y and / or Z direction and can be programmed to perform liquid handling steps. Such pooling stations may further be equipped with a pipetting device and a suction apparatus allowing for controlled addition and removal of reagents. Alternatively the removal of liquid can be performed by vacuum means or using containers qualified for acoustic handling. The pipetting device may further be connected to reagent reservoirs and mixing means to mix and add defined amounts of reagents required for purification and subsequent processing and assembly steps. Integrated liquid handling devices combining the respective functions are known by those skilled in the art.

[00368] In a specific embodiment, the pooling station integrates means to allow for further combining of one or more nucleic acid molecule pools from first and second wells into a third well to yield a larger nucleic acid molecule pool. Such step-wise pooling may be required in cases where variants or libraries of full-length constructs are assembled from identical and variable sequence elements.

[00369] Pooling stations used in the practice of the invention may further contain a magnet located beneath the microwell plate. In a specific embodiment such a plate magnet may serve as counterpart to the micromagnet in order to trigger release of the extracted beads into the recipient microwell. Alternatively the electro-micromagnet may be a hollow magnet connected to a capillary that can be flushed with liquid to blow out the bound bead into the recipient well. Other means of bead release may also be employed.

[00370] With respect to pooling of nucleic acid molecules, this may be done any number of ways. For example, synthesis substrates may be collected and placed in a single container. Alternatively, nucleic acid molecules may be released from synthesis substrates and then contacted with each other. Further, nucleic acid molecules may be assembled by hybridization. This means that more than one assembly may occur in the same container. In other words, the invention includes methods by which assembly of more than one (e.g., two, three, four, five, six, etc.) nucleic acid molecule occurs from smaller, chemically synthesized nucleic acid molecules. One application where the assembly of more than one larger nucleic acid molecule (e.g., replicable nucleic acid molecules) may be useful is where the assembled nucleic acid molecules are intended for insertion into the same cell. Thus, one of the assembled nucleic acid molecules could be a chromosome and another could be a plasmid.

[00371] Once desired pools of nucleic acid molecules have been generated, bead-attached nucleic acid molecules will often be further processed, for example, to obtain functional nucleic acid molecules for downstream reactions. After chain synthesis the 5'-terminal 5'-hydroxy group is usually protected, for example, with a dimethoxytrityl (DMT) group; the internucleosidic phosphate or phosphorothioate moieties may also be protected, for example, with 2-cyanoethyl groups; and the exocyclic amino groups in all nucleic bases (except for T and U) may be protected, for example, with acyl protecting groups. Usually, the 5'-terminal DMT group is cleaved after the last synthesis cycle on the support before the bead-attached nucleic acid molecules are pooled. However, all protection groups have to be removed in a deprotection step before the nucleic acid molecules can be effectively used in subsequent processes.

[00372] In one embodiment of the invention, deprotection is performed, for example, without releasing the nucleic acid molecule form the bead. This can be carried out by choosing a base-stable, non-cleavable linker. Respective linkers are known by the skilled person.

[00373] In one embodiment, nucleic acid molecules are released from the beads prior to downstream assembly. If cleavage of nucleic acid molecule is required, cleavage and deprotection may be performed in a single step. Release of the nucleic acid molecules may be achieved by cleaving the linker attaching the 3'-end of the nucleic acid molecule to the bead (e.g., a magnetic bead) with a suitable reagent. Suitable reagents and conditions for cleavage depend on the nature of the linkage as described elsewhere herein and are known by those skilled in the art. In certain embodiments, nucleic acid molecules are released from the beads using, for example, an EGB, a photocleavable linker, or a reducing linker, as described herein.

[00374] In one embodiment of the invention, nucleic acid molecules are attached to the solid support (e.g., a magnetic or non-magnetic bead) via succinyl groups. In certain embodiments, a universal linker may be located between the succinyl group and the nucleic acid molecules. The succinyl linker may be cleaved by the use of, for example, concentrated aqueous ammonium hydroxide. The reaction is usually carried out at temperatures between 50°C and 80°C for at least one to about eight hours. In certain embodiments, the succinyl linker may be cleaved by the use of ammonia gas, using increased heat and pressure, such as, for example, a temperature of about 80°C, and a pressure of about 3 bar for a time of about 2 hours. Of course, cleavage conditions may vary depending on the protocol and the protecting groups used. In embodiments wherein aqueous ammonium hydroxide is used, the ammonia solution may then be removed by evaporation, leaving the nucleic acid molecules ready for purification.

[00375] In one embodiment, cleavage may be carried out by vapor-phase processing. In vapor-phase processing, nucleic acid molecules may be cleaved in a closed chamber in a gaseous environment comprising gaseous cleavage / deprotection reagent, such as gaseous ammonia or ammonium hydroxide vapors. Respective methods are set out, for example, in U.S. Patent Nos. 5,514,789 or 5,738,829, the disclosures of which are incorporated herein by reference.

[00376] The above reaction will typically also triggers cleavage of other protecting groups including the cyanoethyl group and the group protecting the heterocyclic primary amine. Thus, a single cleavage reaction may be used, when appropriate, to remove all protecting groups present.

[00377] Linkers used in the practice of the invention may be cleaved using at least two approaches: (a) simultaneously under the same conditions as the deprotection step or (b) subsequently utilizing a different condition or reagent for linker cleavage after the completion of the deprotection step. Various methods to remove universal linkers from a nucleic acid molecule are described in the art such as, for example, U.S. Patent Publication No. 2002 / 0143166 A1, the disclosure of which is incorporated herein by reference.

[00378] For downstream applications, it may be required to purify the pooled and deprotected nucleic acid molecules to remove the cleaved groups, for example, by precipitation. It may further be required to separate the nucleic acid molecule mixture from the magnetic particles or other support. In one embodiment, a plate magnet located beneath the microwell plate can be used to immobilize the beads in the wells while the nucleic acid molecules can be eluted, for example, by suction. Alternatively, in the absence of a plate magnet, the beads may be automatically removed from the wells by magnetic means while the nucleic acid molecules would be retained in the well to obtain femtomoles of individual pools of high quality nucleic acid molecules at picomole concentration ready for further processing or use.

[00379] In some instances, nucleic acid molecules may be separated from solid support while the solid supports remain localized in the same or similar location as to where the nucleic acid molecules were synthesized. In such instances, typically after synthesis completion, oligonucleotide synthesis reagents may be removed from contact with synthesis supports, followed by the addition of one or more reagents for release of the constructed oligonucleotide, also referred to as cleavage reagents. These releasing reagents may be in forms such as liquid or gaseous. Gaseous reagents are referred to above.

[00380] In many instances, the cleavage reagent agent will be volatile (e.g., it can be removed via freeze drying) and non-ionic. The cleaved oligonucleot...

Claims

<pat:ClaimStatement>THE EMBODIMENTS OF THE INVENTION FOR WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:< / pat:ClaimStatement> <pat:Claims com:id="claims"> <pat:Claim com:id="CLM-00001"> <pat:ClaimNumber>1< / pat:ClaimNumber> <pat:ClaimText>1. A system for synthesis of a nucleic acid molecule, the system comprising: a CMOS microchip comprising a plurality of well structures formed thereon, each well of the plurality of well structures sized to accommodate a bead for synthesis of the nucleic acid molecule, wherein each well has formed therein a first electrode at a bottom of the well that is individually controllable by a controller and a second electrode in operable connection with each well; and a lid member arranged on top of the microchip and comprising a fluidic channel formed therein to provide fluid path for the bead, wherein (i) the second electrode is placed at the bottom or along one or more sidewall of each well, or (ii) the lid member comprises the second electrode, wherein the controller is operable to provide a voltage between the first electrode and the second electrode to cause fluid in the well to undergo electrolysis producing one or more bubbles in the fluid to rise to a top of the well along with the bead, a bead-collection device operable to collect the bead that is removed from the well, and a first multiwell collection plate operable to receive the bead that is collected from the bead-collection device, wherein the microchip comprises a top inlet / outlet and a bottom inlet / outlet, and wherein the microchip is arranged in a vertical position such that it is emptied or drained by purging a gas or a solvent in a direction from the top inlet / outlet of the microchip to the bottom inlet / outlet of the microchip to efficiently remove reagents or gas through the bottom inlet / outlet of the microchip. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00002"> <pat:ClaimNumber>2< / pat:ClaimNumber> <pat:ClaimText>2. The system for synthesis of a nucleic acid molecule according to claim 1, wherein the first multiwell collection plate comprises a plurality of well structures and a fluid-permeable structure formed on a top surface of or within the plurality of well structures, optionally wherein the total volume of each well of the first multiwell collection plate is between 1 µl and 200 µl. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00003"> <pat:ClaimNumber>3< / pat:ClaimNumber> <pat:ClaimText>3. The system for synthesis of a nucleic acid molecule according to claim 1 or 2, further comprising a controller that is operable to move the bead collection device in one or more degrees of freedom to deliver the beads that are collected in the bead collection device into the plurality of wells of the first multiwell collection plate. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00004"> <pat:ClaimNumber>4< / pat:ClaimNumber> <pat:ClaimText>4. The system for synthesis of a nucleic acid molecule according to any one of claims 1-3, wherein the microchip is programmed to extract the bead from a specific well of interest in the microchip and deliver the bead via the bead collection device to an addressable well in the plurality of wells in the first multiwell collection plate. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00005"> <pat:ClaimNumber>5< / pat:ClaimNumber> <pat:ClaimText>5. The system for synthesis of a nucleic acid molecule according to any one of claims 1-4, wherein each well of the plurality of well structures of the microchip comprises well material including a high-aspect ratio photoresist material, optionally wherein the photoresist material is epoxy-based resist. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00006"> <pat:ClaimNumber>6< / pat:ClaimNumber> <pat:ClaimText>6. The system for synthesis of a nucleic acid molecule according to any one of claims 1-5, wherein the bead collection device comprises a needle or needle-like structure and associated tubing configured to place one or more beads in one or more wells of the multiwell plate. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00007"> <pat:ClaimNumber>7< / pat:ClaimNumber> <pat:ClaimText>7. The system for synthesis of a nucleic acid molecule according to any one of claims 1-6, wherein the diameter of each well of the plurality of well structures of the microchip is between about 10 μm and about 90 μm. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00008"> <pat:ClaimNumber>8< / pat:ClaimNumber> <pat:ClaimText>8. The system for synthesis of a nucleic acid molecule of any one of claims 1- 7, wherein the microchip comprises between 1,000 and 100,000 wells. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00009"> <pat:ClaimNumber>9< / pat:ClaimNumber> <pat:ClaimText>9. The system for synthesis of a nucleic acid molecule according to any one of claims 1-8, wherein the bead is a monodisperse bead and wherein the diameter of the monodisperse bead is smaller than the diameter of each well by about 5% to about 20%. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00010"> <pat:ClaimNumber>10< / pat:ClaimNumber> <pat:ClaimText>10. The system for synthesis of a nucleic acid molecule according to any one of claims 1-9 wherein the microchip is arranged in a holder, and wherein the system further comprises: reagent reservoirs with reagents for oligonucleotide synthesis, wherein the microchip is in fluid communication with the reagents, a gas reservoir and a waste collection, a first manifold connecting the microchip with the reagent reservoirs and further connecting the microchip and the reagent reservoirs with the waste collection, a second manifold connecting the microchip with the gas reservoir and the waste collection, and wherein in a first configuration valves are set to allow for washing and priming of the first manifold and draining of the microchip and flow paths are connected with the waste collection of the first manifold, and wherein in a second configuration valves are set to allow filling of the emptied microchip with reagents for oligonucleotide synthesis and flow path is connected with the waste collection of the second manifold. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00011"> <pat:ClaimNumber>11< / pat:ClaimNumber> <pat:ClaimText>11. The system for synthesis of a nucleic acid molecule according to claim 1, wherein the solvent is acetonitrile and / or wherein the gas is argon. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00012"> <pat:ClaimNumber>12< / pat:ClaimNumber> <pat:ClaimText>12. The system for synthesis of a nucleic acid molecule according to claim 10 or 11, wherein the system is further equipped with a flow restrictor device to allow controlling of the flow rates of liquid or gas through the fluidic channels. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00013"> <pat:ClaimNumber>13< / pat:ClaimNumber> <pat:ClaimText>13. The system for synthesis of a nucleic acid molecule according to any one of claims 10 to 12, wherein the fluidic transport is pressure-driven and the manifolds comprise zero dead-volume valves. < / pat:ClaimText> < / pat:Claim> < / pat:Claims>