A novel method for synthesizing polynucleotides using a diverse library of oligonucleotides

Abstract

A method for synthesizing a target double-stranded (ds) polynucleotide having a predetermined sequence, comprising: a) providing an oligonucleotide library comprising a diversity of oligonucleotide library members in an array device, wherein each library member has a different nucleotide sequence and is contained in separate library containers in an aqueous solution, the diversity comprising single-stranded oligonucleotides (ss oligonucleotides) and double-stranded oligonucleotides (ds oligonucleotides) having at least one overhang and covering at least 10,000 pairs of matched oligonucleotides, b) in a first step, transferring at least a first pair of matched oligonucleotides from the library into a first reaction container using a liquid handler, and assembling the matched oligonucleotides to obtain a plurality of oligonucleotides comprising at least 10,000 pairs of matched oligonucleotides; c) in a second and other step, using a liquid handler to transfer at least second and other pairs of matched oligonucleotides from the library to second and other reaction containers, respectively, and assemble the matched oligonucleotides, thereby obtaining second and other reaction products, each of which contains at least one overhang, respectively, d) assembling the first, second and other reaction products according to a predetermined workflow to produce the target ds polynucleotide with an overhang, optionally followed by a final step to prepare a blunt end, wherein the matched oligonucleotide pairs and assembly workflow used in steps b) and c) are determined using an algorithm to produce the target ds polynucleotide.

Description

Technical Field

[0001] The present invention relates to a novel method for synthesizing double-stranded (ds) polynucleotides having a predetermined sequence using a diverse library of oligonucleotides. Background Art

[0002] Currently, artificial synthesis of polynucleotides is achieved by two methods that are not necessarily exclusive:

[0003] The first method for synthesizing polynucleotides is "chemical synthesis". This is the process of building single-stranded DNA (or RNA) molecules by sequentially connecting nucleotides one by one using phosphoramidite chemistry (Beaucage and Caruthers, 1981). This method allows the construction of DNA molecules with specific predetermined template sequences of any complexity. Chemical methods are popular because of their cheap nature, are easy to parallelize, and in some embodiments allow high-throughput production of DNA or RNA in chips (LeProust et al., 2010). The main and biggest disadvantage of these methods is that the yield of the reaction drops sharply with the length of the synthetic template, thus generally limiting the size of the molecule to about 200 base pairs (bp, or bps).

[0004] The second class of methods for DNA synthesis is the "assembly method", which involves biochemically connecting oligonucleotides and polynucleotides of different sizes and sequences in a specific way to obtain larger molecules with the desired target sequence. The source of these oligonucleotides is usually chemical synthesis, but can also be enzymatic digestion products of naturally occurring DNA. These assembly methods are usually commercialized under the product name "Gene Synthesis", which is a synonym for the synthesis of large polynucleotide chains (1K-5K bp), but not necessarily gene size length. Several methods for assembling smaller polynucleotides into target sequences have been reported in the literature (Stemmer et al., 1995; Smith et al., 2003; Engler et al., 2008; Gibson et al., 2009; Horspool 2010).

[0005] Over the past few years, "Gibson assembly" (Gibson et al., 2009) has become a popular method for connecting several linear ds DNA fragments (ranging in size from about 30 bp to several Kbp). The method involves connecting many ds DNA fragments with pairwise overlapping sequence homology. The overlapping homology regions between the fragments can range from about 15 to 80 bp. Overhangs are not necessarily required, as the enzymatic mechanism of the method is responsible for generating overhangs, filling gaps, and properly connecting the fragments. The enzymatic mechanism utilizes three enzymes: T5 exonuclease, Phusion DNA polymerase, and Taq DNA ligase, all of which are used in an isothermal reaction. The method is simple and versatile and can produce both linear and circular ds DNA products. The disadvantage of this method is that its limitations in automation make it unsuitable for large-scale commercial use.

[0006] A common theme in the construction of DNA molecules of tens of thousands of base pairs is to chemically synthesize small fragments of up to a few hundred nt or bp and then join them together by cloning, ligation, PCA or Gibson assembly.

[0007] Some approaches suggest that it may be possible to pre-construct, by chemical synthesis, oligonucleotide libraries covering the possible genetic space or a desired subset thereof.

[0008] Chari and Church proposed using synthetic oligonucleotides (200 bases) to generate short DNA fragments and assemble them into large DNA segments using in vivo homologous recombination in yeast and Escherichia coli (Chari and Church, 2017).

[0009] WO 2009 / 138954 A2 discloses a method for synthesizing larger polynucleotides by solid phase assembly, wherein defined subunits required for assembling the larger polynucleotide are chemically synthesized as needed.

[0010] Pedersen et al. (US2016 / 0215316A1) proposed using a library containing the space of all possible hexamers (N=4,096 oligomers). Oligonucleotides six base pairs long are then assembled using oligonucleotide linkers to form polynucleotides. There are certain limitations regarding the connection of oligonucleotides and large-scale DNA synthesis. This method is time-consuming due to the use of appropriately designed libraries and manual protocols (e.g., for cloning, using a large number of reagents). These in turn add a lot of cost to the price of synthesis, the cost per bp of which increases with the increase in the length of the target sequence.

[0011] WO2002 / 081490 discloses a method for utilizing the results of genomic sequence information by computer-guided polynucleotide assembly based on information available in a database (e.g., a human genome database). Specifically, it discloses a method for producing a target polynucleotide, wherein the target polynucleotide is parsed into a series of continuous oligonucleotides by a computer program, and the target polynucleotide is produced in a unidirectional or bidirectional manner by sequentially adding de novo synthesized oligonucleotides to the starting oligonucleotides.

[0012] WO2004 / 033619 also discloses a method for utilizing genome sequence information results of computer-guided polynucleotide assembly.

[0013] Although great strides have been made in recent years in the technology of synthesizing DNA, there are still severe limitations in terms of DNA volume, yield and, especially, length. Summary of the invention

[0014] The object of the present invention is to provide an improved method for synthesizing double-stranded (ds) polynucleotides, aiming to shorten the time for synthesizing a target ds polynucleotide.

[0015] This object is achieved by the subject matter of the present invention.

[0016] According to the present invention, there is provided a method for synthesizing a target ds polynucleotide having a predetermined sequence, comprising:

[0017] a) providing an oligonucleotide library within an array device, the oligonucleotide library comprising a plurality of oligonucleotide library members, wherein each library member has a different nucleotide sequence and is contained in separate library containers in an aqueous solution, the diversity comprising single-stranded oligonucleotides (ss oligonucleotides) and double-stranded oligonucleotides (ds oligonucleotides) having at least one overhang and covering at least 10,000 pairs of matched oligonucleotides,

[0018] b) in a first step, transferring at least a first pair of matched oligonucleotides from the library into a first reaction vessel using a liquid handler and assembling the matched oligonucleotides to obtain a first reaction product comprising at least one overhang,

[0019] c) in a second and further step, transferring at least second and further pairs of matched oligonucleotides from the library into second and further reaction vessels, respectively, using a liquid handler, and assembling the matched oligonucleotides, thereby obtaining second and further reaction products, each comprising at least one overhang, respectively,

[0020] d) assembling said first, second and further reaction products in a predetermined workflow to produce said target ds polynucleotide with overhangs, optionally followed by a final step to prepare blunt ends,

[0021] An algorithm is used to determine the matching oligonucleotide pairs and an assembly workflow to generate the target ds polynucleotide.

[0022] Specifically, a series of different target ds polynucleotides are synthesized using the same oligonucleotide library. Specifically, the different target ds polynucleotides have different sequences and are not fragments of each other.

[0023] In particular, the different target ds polynucleotides have a sequence identity of less than 50%, preferably less than 30%. In particular, the different target ds polynucleotides have a sequence identity of less than 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22% or 21%. Even more preferably, the different target ds polynucleotides have less than 20% or 10% sequence identity with each other, in particular they have less than 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 9%, 8%, 7%, 6% or 5% sequence identity.

[0024] In particular, the target ds polynucleotide is a DNA molecule.

[0025] Specifically, one or more amplification steps are performed, for example, by performing PCR, preferably 25 cycles. Specifically, the PCR uses HiFi thermostable DNA polymerase (Phusion or Q5) and two oligonucleotides complementary to each overhang of the assembly fragment, and the complementary oligonucleotides include a cleavage site for a type IIS restriction enzyme (BfuAI). Specifically, the amplified product is contacted with a type IIS restriction enzyme, and the original overhang is introduced into the amplified fragment. Specifically, the amplification step is performed after any one or more of the first, second, third or other assembly steps, wherein the first, second, third or other reaction products are amplified respectively. Specifically, the amplification step is performed after the target ds polynucleotide is assembled, wherein the target ds polynucleotide is amplified.

[0026] Specifically, the predetermined workflow (also called "assembly workflow") is a hierarchical one, with the following specific features:

[0027] Hierarchical workflow refers to parallel or separate production of polynucleotide matching pairs of intermediate assembly produced as intermediates, each intermediate is assembled in a separate reaction compartment, and these intermediates are further assembled to obtain target polynucleotides or a part thereof. According to a specific example, in the first step, the matching pairs of oligonucleotides are combined in parallel and in an independent reaction compartment, thereby producing polynucleotides with a combined size of reagent oligonucleotides and the same overhang length as reagent oligonucleotides in each compartment. In the second step and subsequent steps, the process is repeated iteratively by using previous products or other oligonucleotides as reagents, thereby producing polynucleotides with a combined size of reagent polynucleotides in each layer, and the polynucleotides can keep the same overhang size. If the step before the last step has three compartments, then only two compartments carrying matching pairs are reacted first, and then the further reaction step carried out between the product and the last compartment will produce target polynucleotides. Alternatively, if three compartments contain polynucleotides that can only form two matching pairs in total, then three compartments are combined to produce target polynucleotides.

[0028] In particular, the assembly workflow is automated. In particular, the automated workflow employs a microfluidic processor capable of transferring all or part of the contents of one or several compartments, either serially or in parallel, to other pre-designated compartments that may or may not be empty.

[0029] Specifically, the assembly workflow is sequence-dependent, meaning that the specific order is determined by the template sequence so that when the matching pairs are combined at any step in the workflow, they will produce most of the target ds polynucleotide or ultimately produce the target ds polynucleotide. Specifically, the workflow is determined according to the sequence of the template sequence or the target ds polynucleotide.

[0030] In particular, polynucleotides up to 1,000, 5,000, 10,000 or 100,000 base pairs (bp) in length, or even longer, can be produced at low cost and high speed by the methods described herein.

[0031] The methods described herein specifically include the following components: A) a pre-constructed library of oligonucleotides, which can be designed to cover the entire gene sequence space and organize oligonucleotides in space for efficient access by liquid handlers or microfluidic devices. If the spatial organization of the library reduces the time required to access the necessary oligonucleotides, the access is considered to be effective. Specifically, if the access reduces or shortens the total processing time of the library, the access is considered to be effective, wherein the total processing time is the time spent processing the library members during the synthesis of the target ds polynucleotide. Specifically, compared with other organizations, especially compared with randomly placed oligonucleotides or lexicographic sorting in space, if the access reduces the operating costs or reduces the amount of necessary consumables related to accessing oligonucleotides, the access is further considered to be effective. Specifically, compared with the total processing time of a library organized randomly or lexicographically, if the total processing time of the library is reduced by at least 5%, 10%, 15%, 20%, 25% or 50%, the access is considered to be effective.

[0032] B) Algorithm-determined sequence-specific hierarchical assembly workflow, generating long polynucleotides without mismatches.

[0033] The libraries described herein specifically comprise single-stranded (ss) and double-stranded (ds) oligonucleotides (oligonucleotides), also referred to as library members. These library members are pre-constructed, provided in a storage-stable solution, and located at defined locations within an array device. Oligonucleotides of the library are synthesized and stored in the array device until needed.

[0034] Specifically, an oligonucleotide is a linear polymer of nucleotide monomers and contains "A" representing deoxyadenosine, "T" representing deoxythymidine, "G" representing deoxyguanosine, and "C" representing deoxycytidine, or in addition to conventional bases (A, G, C, T), may contain nucleotide analogs such as inosine and 2'-deoxyinosine and their derivatives (e.g., 7'-deaza-2'-deoxyinosine, 2'-deaza-2'-deoxyinosine), azole- (e.g., benzimidazole, indole, 5-fluoroindole) or nitroazole analogs (e.g., 3-nitropyrrole, 5-nitroindole, 5-nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole) and their derivatives, acyclic sugar analogs (e.g., Such as those derived from hypoxanthine- or indazole derivatives, 3-nitroimidazole or imidazole-4,5-dicarboxamide), 5'-triphosphates of universal base analogs (e.g., derived from indole derivatives), isoquinolones and their derivatives (e.g., methylisoquinolone, 7-propynylisoquinolone), hydrogen-bonded universal base analogs (e.g., pyrrolopyrimidines), and other chemically modified bases (e.g., diaminopurine, 5-methylcytosine, isoguanine, 5-methyl-isocytosine, K-2'-deoxyribose, P-2'-deoxyribose) or other modified bases that may have different base pairing preferences and may pair with more than one natural nucleobase with similar stringency / probability. The monomers are linked by phosphodiester bonds or in some cases by peptidyl bonds or by phosphorothioate bonds or by any other type of nucleotide bond.

[0035] Specifically, the single-stranded DNA oligonucleotide library members (referred to herein as ss oligonucleotides) are or comprise natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine); nucleoside analogs (e.g., inosine or 5-methylisocytosine or 3-nitropyrrole, 5-nitroindole, pyrrolidine, 4-nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 4-aminobenzimidazole, 5-nitroindazole, 3-nitroimidazole, 5-aminoindole, benzimidazole, 5-fluoroindole, indole, methylisoquinolone, pyrrolopyrimidine 7-propynylisoquinolone, 2-aminoadenosine, 2-thiothymidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-amino-adenosine, 7-deaza-adenosine, 7-deaza-guanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine and 2-thiocytidine); chemically or biologically modified bases (including methylated bases); intercalated bases; modified sugars (e.g., ribose, 2'-deoxyribose, arabinose and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite bonds).

[0036] Specifically, the double-stranded DNA oligonucleotide library members (abbreviated herein as ds oligonucleotides) are or comprise natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine); nucleoside analogs (e.g., inosine or 5-methylisocytosine or 3-nitropyrrole, 5-nitroindole, pyrrolidine, 4-nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 4-aminobenzimidazole, 5-nitroindazole, 3-nitroimidazole, 5-aminoindole, benzimidazole, 5-fluoroindole, indole, methylisoquinolone, pyrrolopyrimidine 7-propynylisoquinolone, 2-aminoadenosine, 2-thiothymidine, 3-methyladenosine, 5- methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-amino-adenosine, 7-deaza-adenosine, 7-deaza-guanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine and 2-thiocytidine); chemically or biologically modified bases (including methylated bases); intercalated bases; modified sugars (e.g., ribose, 2'-deoxyribose, arabinose and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite bonds), and are formed by annealing fully or partially complementary single-stranded oligonucleotides.

[0037] Specifically, oligonucleotide library members can be generated by any chemical polynucleotide (oligonucleotide) synthesis method including H-phosphonate, phosphodiester, phosphotriester or phosphite triester synthesis, or any massively parallel oligonucleotide synthesis method such as microarray or microfluidics-based oligonucleotide synthesis (e.g., as described in references (Gao et al. 2001)(LeProust et al. 2010)(Bonde et al. 2014a)).

[0038] Specifically, oligonucleotide library members can be produced by any enzymatic polynucleotide (oligonucleotide) synthesis method, including ssDNA synthesis by DNA polymerase protein or by reverse transcriptase protein, which produces hybrid RNA-ssDNA molecules. Specifically, the enzymatic polynucleotide synthesis reaction can occur in vivo or in vitro.

[0039] Specifically, oligonucleotide library members are generated by synthesizing oligonucleotide sequences from nucleotide building blocks using any polynucleotide synthesis method, wherein the building block consists of "A" representing deoxyadenosine, "T" representing deoxythymidine, "G" representing deoxyguanosine, or "C" representing deoxycytidine, or other natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine), nucleotide analogs, such as inosine and 2'-deoxyinosine and their derivatives (e.g., 7'-deaza-2'-deoxyinosine, 2'-deaza-2'-deoxyinosine), azoles (e.g., benzimidazole, indole, 5-fluoroindole) or nitroazole analogs (e.g., 3-nitropyrrole, 5-nitroindole, , 5-nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole) and their derivatives, acyclic sugar analogs (e.g., those derived from hypoxanthine or indazole derivatives, 3-nitroimidazole or imidazole-4,5-dicarboxamide), 5'-triphosphates of universal base analogs (e.g., derived from indole derivatives), isoquinolone and its derivatives (e.g., methylisoquinolone, 7-propynylisoquinolone), hydrogen-bonded universal base analogs (e.g., pyrrolopyrimidine) or any other chemically modified bases (e.g., diaminopurine, 5-methylcytosine, isoguanine, 5-methyl-isocytosine, K-2'-deoxyribose, P-2'-deoxyribose). The building blocks are linked by phosphodiester bonds or peptidyl bonds or by phosphorothioate bonds or by any other type of nucleotide bonds.

[0040] In a specific embodiment of the present invention, the ss oligonucleotide has a length of 6 to 26 nucleotides. Preferably, the ss oligonucleotide has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides. Preferably, the ss oligonucleotide has a length of up to 26, 25, 24, 23, 22, 21, 20, 19, 18, 17 or 16 nucleotides. In another specific embodiment, the ss oligonucleotide has a length of greater than 26 nucleotides. Preferably, the ss oligonucleotide has a length of less than 100, 90, 80, 70, 60 or 50 nucleotides.

[0041] Specifically, the ds oligonucleotide library members have at least one overhang. The overhang is specifically characterized by being reactive (i.e., capable of hybridizing to another ss oligonucleotide or overhang) as part of a ds oligonucleotide or polynucleotide and / or extending one or more nucleotides of the ds oligonucleotide or polynucleotide.

[0042] The library specifically comprises ds oligonucleotides having an overhang and a blunt end. Blunt ends are specifically characterized by a ds terminal extension of one or more base pairs as part of the ds oligonucleotide or polynucleotide.

[0043] Specifically, the library may contain ds oligonucleotides having overhangs at both ends and no blunt ends.

[0044] In some embodiments, the ds oligonucleotide has a length of 6 to 26 base pairs, and the overhang is no more than half the length of each ds oligonucleotide. Specifically, the ds oligonucleotide has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 base pairs. Specifically, the ds oligonucleotide has a length of maximum 26, 25, 24, 23, 22, 21, 20, 19, 18, 17 or 16 base pairs. Specifically, if the ds oligonucleotide is 6 base pairs long, the overhang length is no more than 3 nucleotides. Specifically, if the ds oligonucleotide is 24 base pairs long, the overhang length is no more than 12 nucleotides. In another specific embodiment, the ds oligonucleotide has a length greater than 26 base pairs. Preferably, the ds oligonucleotide has a length less than 100, 90, 80, 70, 60 or 50 base pairs.

[0045] The libraries described herein are specifically composed of physical oligonucleotides and synthesized under standardized conditions. The oligonucleotides are purified, may contain modifications, and are ideally stored in appropriate buffers and / or excipients at standard concentrations and volumes so that they are ready for use.

[0046] Specifically, the oligonucleotide can be kept in solution using any of the following buffers and / or excipients: Tris buffer, TE buffer (Tris-EDTA buffer) or nuclease-free water. Specifically, library members can be stored in Tris buffer, wherein the concentration of the Tris buffer provided is about 10mM (+ / -1mM or 2mM). Specifically, library members can be stored in TE buffer. Specifically, the TE buffer is at least composed of Tris with a concentration of about 10mM (+ / -1mM or 2mM) and a concentration of 0,1,0,2,0,3,0,4,0,5,0,6,0,7,0,8,0,9 or 1,0mM any EDTA composition. Specifically, nuclease-free water is water that has been deionized, filtered and autoclaved and is substantially free of contamination of non-specific endonucleases, exonucleases and RNase activity.

[0047] In particular, all library members are stored in separate array devices using in each case the same or different buffers and / or excipients.

[0048] Libraries described herein can comprise thousands of oligonucleotides. Specifically, libraries described herein comprise diversity oligonucleotide library members, wherein each library member has a different nucleotide sequence, and the diversity covers at least 10,000 pairs of matched oligonucleotides. Specifically, the library comprises at least 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or 100,000 pairs of matched oligonucleotides. Specifically, the library comprises enough pairs of matched oligonucleotides to cover the entire sequence space.

[0049] The oligonucleotide pairs of matching as described herein refer to single-stranded oligonucleotides comprising partial or complete complementary sequences. In a separate compartment, the oligonucleotide pairs of the matching may be present in the library as ss oligonucleotides, or two or more complementary ss oligonucleotides may be included in one compartment, wherein they may anneal to form ds oligonucleotides. The nucleotide sequences of a pair of matching ss oligonucleotides may be complementary by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides, such that the matching pair may form new ds polynucleotide molecules by hybridization of ss oligonucleotide sequences (preferably wherein the ss oligonucleotides partially hybridize), thereby obtaining ds polynucleotides with overhangs.

[0050] The ss-oligonucleotide may in particular be part of a matched pair consisting of two or three hybridization partners. In particular, the ss-oligonucleotide may be used as a first hybridization partner capable of hybridizing with a second hybridization partner, which is another ss-oligonucleotide or a ds-oligonucleotide having complementary overhangs.

[0051] In particular, the ss oligonucleotide can be used as a first hybridization partner capable of hybridizing with two different ss and / or ds oligonucleotides or two different ds polynucleotides used as second and third hybridization partners. In particular, the first hybridization partner is a matched ss oligonucleotide, wherein the first part of the ss oligonucleotide hybridizes with the second hybridization partner and the second part of the ss oligonucleotide hybridizes with the third hybridization partner, thereby obtaining a gapless ds polynucleotide consisting of three hybridization partners.

[0052] A pair of matched ds oligonucleotides is specifically characterized by complementary sequences in each overhang of the ds oligonucleotides, for example wherein each overhang is complementary by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides, such that the matched pair can form a new ds polynucleotide molecule by hybridization of the overhang sequences.

[0053] The libraries described herein may specifically comprise a diversity of double-stranded oligonucleotide library members, wherein each ds oligonucleotide library member has a different nucleotide sequence. Specifically, the diversity encompasses at least 100, 500, 1.000, 2,000, 3,000, 4,000, 5,000, 10,000, 20,000, 40,000, 60,000, 80,000, 100,000, 120,000, 140,000, 160,000, 180,000, or 200,000 different ds oligonucleotides.

[0054] The libraries described herein may specifically comprise diverse single-stranded oligonucleotide library members, wherein each ss oligonucleotide library member has a different nucleotide sequence. Specifically, the diversity encompasses at least 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 20,000, 40,000, 60,000, 80,000, 100,000, 120,000, 140,000, 160,000, 180,000 or 200,000 different ss oligonucleotides. Specifically, the ss oligonucleotides can be used as linkers, particularly in the assembly of ds polynucleotides.

[0055] Specifically, the diversity refers to that different library members are different in at least one base or base pair. A library member may actually include multiple copies of ss or ds oligonucleotides of the same sequence. These multiple copies of library members are specifically contained in only one library container.

[0056] In a specific embodiment of the present invention, the diversity covers phosphorylated ss oligonucleotides and / or ds oligonucleotides. Specific embodiments refer to ss oligonucleotides or ds oligonucleotides modified by any one or more of phosphorylation, methylation, biotinylation or connection with a fluorophore or quencher. Therefore, the library described herein comprises any or all library members that can be unmodified ss oligonucleotides, phosphorylated ss oligonucleotides, methylated ss oligonucleotides, biotinylated ss oligonucleotides, phosphorylated, biotinylated and methylated ss oligonucleotides, unmodified ds oligonucleotides, phosphorylated ds oligonucleotides, methylated ds oligonucleotides, biotinylated ds oligonucleotides and phosphorylated, biotinylated and methylated ds oligonucleotides. Preferably, the library member comprises 5' phosphorylation. Specifically, the library described herein further comprises ss oligonucleotides containing a fluorophore or quencher and ds oligonucleotides containing a fluorophore or quencher.

[0057] In a specific embodiment of the present invention, an oligonucleotide library is provided in an array device, and the library members are contained in separate library containers, each of which is in an aqueous solution. Specifically, the array device is any of a microtiter plate, a microfluidic microculture plate, a capillary set, a microarray or a biochip (preferably a DNA and / or RNA biochip). The array device may include only one, all or any number of the above containers.

[0058] In another specific embodiment of the present invention, more than one different library member can be contained in only one library container. Specifically, the different library members contained in one library container are ss oligonucleotides whose sequences are such that they cannot anneal to each other. Specifically, the different library members contained in one library container are ds oligonucleotides whose sequences are such that they cannot be connected to other ds oligonucleotides contained therein. Specifically, the different library members contained in one library container are ss oligonucleotides and ds oligonucleotides whose sequences are such that they cannot anneal to each other.

[0059] In a specific embodiment, the independent library container is spatially arranged in three-dimensional order, and wherein each compartment is located in the device with the defined coordinates in x, y and z axis. Specifically, the three-dimensional order comprises at least any one of two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, fifty, sixty or even more stacked library containers that are at least partially or completely stacked. Preferably, the library containers are placed in different layers, and these library containers are placed one above another in different layers. Specifically, the layer is placed in a predetermined position in the three-dimensional order. Preferably, each of the library containers in one layer comprises a series of library members spatially arranged in two-dimensional order at a predefined position.

[0060] Specifically, three-dimensional order is predefined by the parameter that is mainly used to shorten the synthesis time.Preferably, the parameter is the frequency of use, and those oligonucleotides that often form matching pairs in DNA sequence are close to each other, such as naturally occurring or the oligonucleotides that are usually used in target ds polynucleotide or its fragment.Due to the need of a large amount of oligonucleotides for building any given sequence, due to the time required for scanning library and retrieval of required oligonucleotides, the spatial distribution of most of oligonucleotides in the library can cause time and resource waste.But, by using the specific distribution of oligonucleotides, the minimum movement occurs in automatic equipment so that the oligonucleotides of matching are transferred in the reaction vessel.For example, oligonucleotides can be stored in microplates, and wherein the first plate comprises the most common oligonucleotide matching pairs, and other plates are arranged in descending order until the last plate comprises the least commonly used oligonucleotides.

[0061] In the methods described herein, a liquid handler is used to transfer oligonucleotides from a library described herein to a reaction vessel. Specifically, the liquid handler can be a droplet handler. Specifically, the liquid handler is automated. A liquid handler can be used to transfer a suitable volume of at least any one of, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 nL, for example, such that 10 of a library member (e.g., a single-stranded oligonucleotide, a matched pair of a single-stranded oligonucleotide and a double-stranded oligonucleotide) is transferred to a reaction vessel. 9 , 10 10 , 10 11 or 10 12 Preferably, at least about 10 copies of a particular oligonucleotide are placed in one reaction vessel. 11 copies (e.g. 6.06×10 11 Preferably, the volume of the oligonucleotide transferred by the liquid handler is 10 to 1000 nL, more preferably it is 10 to 500 nL, even more preferably it is 50 to 250 nL.

[0062] Specifically, the reaction vessel is a compartment unit, such as a hole in any one of a microtiter plate, a microfluidic microculture plate, a capillary set, a microarray or a biochip (preferably a DNA and / or RNA biochip). Specifically, the reaction vessel is characterized by an environment in which a nucleic acid chain is linked to a second nucleic acid chain by complementary chain interactions and hydrogen bonds to produce a double-stranded oligonucleotide. Such conditions include chemical components of an aqueous or organic solution containing nucleic acids and their concentrations (e.g., salts, chelating agents, formamide) and the temperature of the mixture. Other well-known factors (e.g., culture duration or reaction chamber size) may have an impact on the environment.

[0063] According to the method provided herein, the oligonucleotides are transferred from the library to a reaction vessel and assembled to obtain a reaction product. Specifically, the assembly is carried out by any method and / or ligation reaction that hybridizes the ss nucleotide sequence, and the ligation reaction is an enzymatic and / or chemical reaction. Specifically, the ligation reaction is an enzymatic ligation reaction using a ligase or a ribozyme capable of ligation. Preferably, T4 DNA ligase, T7 DNA ligase, T3 DNA ligase, Taq DNA ligase, DNA polymerase or engineered enzyme is used in the ligation reaction. Preferably, the following ligation reaction is used: T4 DNA ligase, at a concentration of 10 sticky end units / μL, and supplemented with 1mM ATP (Sambrook and Russel, 2014, Chapter 1, Scheme 17).

[0064] In particular, the assembly is performed directly by hybridizing matching overhangs or indirectly by hybridizing a suitable ss oligonucleotide adapter, which is a ss oligonucleotide contained in the library, which is selected from the library and transferred to assemble any of the first, second or other reaction products.

[0065] Oligonucleotides are specifically assembled according to the defined workflow. This workflow is specially designed to avoid mispairing or avoid generating a reaction product that cannot be used for assembly to produce a target ds polynucleotide. If there is a partial construct that can be annealed in an alternative manner, then out of control, i.e., uncontrolled polymerization, may occur. In order to avoid the oligonucleotide pairs that will cause the matching of an unwanted construct or out-of-control reaction to be combined together, the oligonucleotide pairs that are matched are assembled with a predetermined assembly step sequence (i.e., specific workflow). Preferably, the specific workflow is not linear but hierarchical, i.e., before the intermediate reaction product is further assembled into other intermediate reaction products or assembled into a target ds polynucleotide sequence, the algorithm for providing this intermediate reaction product is followed, and the intermediate reaction product is limited to the non-continuous portion of the target ds polynucleotide that can be easily produced, thereby avoiding undesirable reaction products as much as possible.

[0066] In a linear workflow, the polynucleotide is assembled in a linear fashion starting from the 3' end of the leading strand, and the next oligonucleotide is added to connect the 3' end of the leading strand to the 5' end of the next oligonucleotide. For example, oligonucleotide B is connected to oligonucleotide A, oligonucleotide C is connected to oligonucleotide B, oligonucleotide D is connected to oligonucleotide C, and so on. This assembly can be achieved simultaneously by adding all oligonucleotides to the reaction vessel at the same time, or the polynucleotide can be extended stepwise by adding oligonucleotides A, B, C, D, etc. to the reaction vessel in sequence.

[0067] For example, when oligonucleotide D can be connected not only to oligonucleotide C but also to oligonucleotide A due to complementary sequences or overhangs, a hierarchical workflow may be necessary. A linear workflow as described above will produce unwanted polynucleotides ADBCD in addition to the desired polynucleotide ABCD. Therefore, it is preferred to assemble the polynucleotides in a hierarchical workflow. Therefore, in two separate reaction vessels, oligonucleotides A and B and oligonucleotides C and D are connected, respectively. The ligation reaction will produce reaction products AB and CD, which can then be transferred to a third reaction vessel, where the desired polynucleotide ABCD is formed after ligation.

[0068] Specifically, the algorithm is used to determine the workflow. Specifically, the algorithm selects matching oligonucleotide pairs and ss oligonucleotide adapters (if necessary), and determines the assembly workflow by determining the best or near-optimal way to assemble the target ds polynucleotides, rather than just by sequence division, so as to avoid mismatches or undesirable reaction products as much as possible. The matching oligonucleotide pairs and assembly workflow are specifically selected to avoid undesirable (wrong) reactions or reaction products, such as palindromic sequences, runaway reactions, and clear assemblies. If there are wrong reaction products in addition to the correct reaction products, these wrong reaction products are appropriately separated from the correct reaction products, such as as follows: using gel electrophoresis to detect oligonucleotides or polynucleotides of a certain size, and cutting and purifying the gel bands corresponding to the size of the desired reaction products. Specifically, the correct reaction product can be detected by incorporating a tag or label into the sequence. Specifically, the oligonucleotide can be captured using a biotinylated oligonucleotide adapter that can hybridize with the overhang of the oligonucleotide, wherein the adapter is fixed on a substrate and coated with streptavidin. The uncaptured wrong products are eliminated by washing, and then the correct product is released from the adapter by increasing the temperature. In particular, other separation methods known in the art may be applied. In particular, such methods may involve chromatography or affinity separation.

[0069] In a specific embodiment of the present invention, the target ds polynucleotide has a length of at least 48 nucleotides. Specifically, the target ds polynucleotide has a length of at least any one of 100, 200, 300, 400, 500, 1,000, 10,000, 100,000, 200,000 or 500,000 nucleotides.

[0070] Typically, a template is used as a model for synthesizing a target ds polynucleotide. Specifically, the nucleotide sequence of the target ds polynucleotide is identical to the nucleotide sequence of the template.

[0071] In a specific embodiment, the target sequence (sequence of interest) (SOI) is provided as a single-stranded template and / or translated into two single-stranded template sequences, based on which the target ds polynucleotide is synthesized. In certain embodiments, the first template comprises the sequence of SOI and the second template comprises the reverse complement sequence of SOI.

[0072] In another embodiment of the present invention, the target ds polynucleotide is a proxy ds polynucleotide having the same sequence as the template, which is further modified to obtain a polynucleotide having a sequence of interest (SOI) different from the sequence of the target ds polynucleotide. Typically, the proxy ds polynucleotide is produced as an intermediate product from which a ds polynucleotide characterized by SOI can be produced by one or more additional mutagenesis steps.

[0073] In particular, the template sequence from which the proxy ds polynucleotide is synthesized is not identical to the SOI. In particular, the template sequence has an identity with the SOI of less than any one of 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92% or 91%, and / or an identity with the SOI of at least any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 or 99%.

[0074] In a specific embodiment of the method provided herein, before being divided into shorter sequences, the terminal nucleotides at the 3' or 5' end or both ends of the sequence of one or each chain in the ds chain are removed. Specifically, they are removed by calculation. Therefore, a template different from SOI is generated. Specifically, any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 terminal nucleotides at the 3' end or 5' end of the sequence is removed to generate a template. Specifically, the nucleotides are removed to generate overhangs and / or to prepare for completion of synthesis by generating flat ends at each end of the target ds polynucleotide.

[0075] Specifically, the template is composed of a single-stranded or double-stranded sequence. Preferably, the template is single-stranded. Specifically, the two single-stranded template sequences are compared to generate a double-stranded template. Specifically, the sequence of the template is divided into shorter sequences (subsequences comprising oligonucleotide library members), and the positions of the library members in the library are digitally annotated. Specifically, the division into subsequences depends on the hierarchical workflow and the library members present in the library.

[0076] Specifically, the target ds polynucleotide has blunt ends at both ends.

[0077] Specifically, the methods provided herein include a completing step.

[0078] Specifically, the finishing step is used to add one or more nucleotides corresponding to those previously removed from the 3' end and the 5' end, respectively, to prepare a template, with the aim of producing a flat end. Specifically, oligonucleotides complementary to the nucleotides at the 3' end and the 5' end, respectively (i.e. complementary to the sticky ends of the polynucleotide) are selected from the library. Specifically, these oligonucleotides are used as primers in a PCR reaction, which is prepared to amplify the final product and add the remaining nucleotides to each chain to synthesize a complete target polynucleotide with a flat end.

[0079] Specifically, the finishing step includes a step of purifying the PCR product using a standard kit (e.g., Monarch PCR & DNA Cleanup Kit (Product No. T1030) from New England Biolabs) to eliminate residual oligonucleotides, enzymes and reagents, thereby leaving the target ds polynucleotide as a DNA product, ready for downstream applications.

[0080] Alternatively, one or both blunt ends of the target ds polynucleotide are generated by selecting a matching ds oligonucleotide having a blunt end or selecting an ss oligonucleotide complementary to the overhang and hybridizing without generating any other overhangs, thereby generating blunt ends.

[0081] In particular, the nucleotide sequence of the target ds polynucleotide SOI or template may be of natural or artificial origin.

[0082] In order to generate a ds polynucleotide with a complex SOI in a simpler and therefore faster assembly workflow, a proxy ds polynucleotide having a target sequence that is less than 100% identical to the SOI can be generated. The proxy ds polynucleotide generated by the assembly method described herein can then be further modified to generate a ds polynucleotide having a nucleotide sequence that is 100% identical to the nucleotide sequence of the SOI. Specifically, the proxy ds polynucleotide is further modified by any of directed mutagenesis, endonucleases, or exonucleases to obtain a nucleotide sequence identical to the nucleotide sequence of the template.

[0083] In another embodiment of the present invention, the target ds polynucleotide is further modified to produce a derivative thereof, which is any of a ds DNA, ss DNA or RNA molecule.

[0084] Specifically, the target ds polynucleotide is modified by site-directed mutagenesis to introduce one or more point mutations, wherein the point mutation is any of nucleotide insertion, deletion or substitution.

[0085] Specifically, the target ds polynucleotide is modified by enzymatic modification using any one or more of methyltransferases, kinases, CRISPR / Cas9, multiple automated genome engineering (MAGE) using λ-red recombination, conjugated assembly genome engineering (CAGE), Argonaute protein family (Ago) or its derivatives, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), large-range nucleases, tyrosine / serine site-specific recombinases (Tyr / Ser SSRs), hybrid molecules, sulfurylases, recombinases, nucleases, DNA polymerases, RNA polymerases or TNases.

[0086] In a specific embodiment of the present invention, the target ds polynucleotide is sequenced to verify the degree of identity with the sequence of the template or SOI. Any suitable sequencing method can be used, such as any of the SNP genotyping methods, including hybridization-based methods (e.g., molecular beacons, SNP microarrays, restriction fragment length polymorphisms, PCR-based methods, including allele-specific PCR, primer extension, 5'-nuclease or oligonucleotide ligation assays, single-stranded conformation polymorphisms, temperature gradient gel electrophoresis, denaturing high-performance liquid chromatography, high-resolution melting of the entire amplicon (HRM), SNPlex and surveyor nuclease assays; sequencing-based mutation analysis, including capillary sequencing or high-throughput sequencing (amplicon sequencing) of the entire PCR amplicon of the PTR. Such high-throughput (HT) amplicon sequencing methods include, but are not limited to, polony sequencing, pyrophosphate sequencing, Illumina (Solexa) sequencing, SOLiD sequencing, semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing, nanopore DNA sequencing, tunnel current DNA sequencing, hybridization sequencing, mass spectrometry sequencing, microfluidics Sanger sequencing, microscopy-based sequencing, RNAP sequencing.

[0087] According to the present invention provided herein, an oligonucleotide library comprising diverse library members is provided within an array device, wherein the library members are single-stranded oligonucleotides (ss oligonucleotides) and double-stranded oligonucleotides (ds oligonucleotides) having at least one overhang, wherein each library member has a different nucleotide sequence and is contained in separate library containers in an aqueous solution, which are spatially arranged in a three-dimensional order, and the diversity covers at least 10,000 pairs of matching oligonucleotides.

[0088] Specifically, the library containers are preferably spatially arranged in a three-dimensional order according to the frequency of use, and wherein the three-dimensional order includes at least any one of two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, fifty, sixty or even more (at least partially or completely stacked) stacked library containers.

[0089] Furthermore, the present invention provides the use of the oligonucleotide library described herein in synthesizing a series of different target double-stranded (ds) polynucleotides having a predetermined sequence, wherein the different target double-stranded (ds) polynucleotides have a sequence identity of less than 50%, preferably less than 30%. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1A Source sequences used to construct the library (only 100 bp fragments are shown), corresponding to four haplotypes of the hypervariable region II of human mitochondria (Anderson et al., 1981: Gene Bank accession number: J01415);

[0091] Figure 1 B Design scaffold of oligonucleotides required to construct any possible combination of haplotypes (assuming full heterozygosity at each polymorphic site). Two chains are shown. Each Z-shaped block is an oligonucleotide pair, where N represents any of the four bases A, T, G, or C. The numbers above and below each oligonucleotide sequence scaffold for each oligonucleotide indicate the length of the oligonucleotide, and in parentheses are the number of oligonucleotides that will be present in the library to cover all possible haplotypes at the variable site. Each ss oligonucleotide should be stored separately in a compartment of the library, except for those that are underlined and stored as annealing pairs;

[0092] Figure 2 A nucleotide sequence of SOI, called DISCOVER (SEQ ID NO: 1);

[0093] Figure 2 B Nucleotide sequences of the 16 oligonucleotides that constitute SOI DISCOVER;

[0094] Figure 2 C forms the dimer structure of the oligonucleotide. D+ and D- are described in the text, but the same structure applies to all other dimers;

[0095] Figure 3 The position of the oligonucleotides in the well plate;

[0096] Figure 3 After A annealing, the contents of columns 1 and 3 are transferred to columns 2 and 4, respectively;

[0097] Figure 3 B After incubating the first ligation reaction, transfer the contents of column 2 to column 4;

[0098] Figure 3 C After the second ligation reaction is incubated, the contents of A4 are transferred to well B4 and incubated for the third and final ligation reaction. Well B4 contains 128 bp of the target ds polynucleotide;

[0099] Figure 4 Acrylamide gel (10%) showing the contents of the method described in Example 2. Lane 1: Reaction D+I ( Figure 2Lane 2: Negative control with 64 bp dsDNA (for ligation). Lane 3: Positive control with 64 bp dsDNA (for ligation). Lanes 4 and 5: Two dilutions of the reaction DI+SC ( Figure 2 Well A4 in C). Lanes 6 and 7: target ds polynucleotide. Lane 8: 50 bp ladder (NEB);

[0100] Figure 5 Part A SOI and its reverse complement (positions 65-100; otherwise Figure 1 A). Elements in italics, bold, and regular font represent different dimers. The underlined portion emphasizes self-complementary overhangs that must be avoided. The upper sequence is SEQ ID NO: 18; the lower sequence is SEQ ID NO: 19;

[0101] Figure 5 B Partial sequence of the template used to generate the proxy ds polynucleotide (positions 65-100). The base pairs with black background indicate the sites that were changed, now making the dimer non-self-complementary. (The resulting modified oligonucleotides are consistent with O- and V+ of Example 2.) Upper sequence SEQ ID NO: 20; Lower sequence SEQ ID NO: 21;

[0102] Figure 5 C. Mutagenic primers used to modify the proxy ds polynucleotide to generate a ds polynucleotide having an SOI. The underlined letters indicate the mutagenic bases. The upper sequence is SEQ ID NO: 22; the lower sequence is SEQ ID NO: 23;

[0103] Figure 6 Arrangement of oligonucleotides transferred from the library of Example 1 on a 96-well plate in preparation for annealing and hierarchical synthesis;

[0104] Figure 7 Agarose gel electrophoresis (2%) showing the results of the hierarchical synthesis method. The top band is the band containing the 608 product. The left lane is the 50 bp ladder;

[0105] Figure 8 Sequence of Example 4. DETAILED DESCRIPTION

[0106] Certain terms used throughout the specification have the following meanings.

[0107] As used herein, the terms "a", "an" and "the" are used herein to mean one or more than one, ie, at least one.

[0108] The term "target sequence" or "SOI" refers to the desired nucleotide or base pair sequence of a ds polynucleotide to be produced by the methods provided herein.

[0109] The term "target double-stranded (ds) polynucleotide" refers to a polynucleotide having a predetermined sequence, which is produced by the synthesis method provided herein. Specifically, the target double-stranded polynucleotide is characterized by a sequence identical and / or corresponding to SOI. If the target ds polynucleotide sequence has a sequence that is less than 100% identical to SOI, the target ds polynucleotide is understood to be a proxy ds polynucleotide that is further modified to generate a sequence identical and / or corresponding to SOI.

[0110] The term "surrogate double-stranded (ds) polynucleotide" refers to a target double-stranded (ds) polynucleotide whose sequence is less than 100% identical to the nucleotide sequence of SOI, and at least 90%, preferably 95%. In order to produce a ds polynucleotide having a sequence identical and / or corresponding to SOI and difficult to synthesize because its sequence can be easily subjected to a clear assembly or runaway reaction, a surrogate double-stranded (ds) polynucleotide can be first synthesized. The sequence of the surrogate double-stranded polynucleotide is designed to avoid palindromic sequences, runaway reactions, and clear assembly and / or promote hierarchical assembly. Specifically, the sequence can be designed by calculation. The synthesized surrogate ds polynucleotide can then be further modified to produce a ds polynucleotide having a nucleotide sequence identical to the nucleotide sequence of SOI. Specifically, the proxy ds polynucleotide is further modified by any of directed mutagenesis, endonucleases or exonucleases, and / or enzymatic modification to obtain a ds polynucleotide having a sequence identical and / or corresponding to the SOI using any of methyltransferases, kinases, CRISPR / Cas9, multiple automated genome engineering (MAGE) using λ-red recombination, conjugated assembly genome engineering (CAGE), Argonaute protein (Ago) or its derivatives, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), large-range nucleases, tyrosine / serine site-specific recombinases (Tyr / Ser SSRs), hybrid molecules, sulfurylases, recombinases, nucleases, DNA polymerases, RNA polymerases or TNases.

[0111] The term "template" refers to a polynucleotide characterized by a specific sequence or polynucleotide sequence that can be used to synthesize and produce a target ds polynucleotide. If a template is used in the synthesis method provided herein, the target ds polynucleotide thus produced has a sequence that is 100% identical to the template.

[0112] Specifically, the template is single-stranded or double-stranded. Such a template can be a natural nucleotide sequence containing the desired product or an artificial nucleotide sequence designed by calculation. Such a template can be identical to the SOI or have less than 100% identity with the SOI, preferably less than 95% identity, but at least 80% identity.

[0113] Preferably, the template is generated by calculation and comprises the sequence of the leading strand of the target ds polynucleotide and the reverse complementary sequence of the target polynucleotide, respectively. Typically, two templates are used in the synthetic methods described herein, one for each strand of the target ds polynucleotide. When the template sequence is designed by calculation, it is preferably compatible with the experimental strategy used for assembly.

[0114] The term "single-stranded DNA oligonucleotide", also referred to as "ssDNA oligonucleotide" or simply "ss oligonucleotide (oligonucleotide)" or "ss oligonucleotide (oligo)", refers to an oligonucleotide that is a linear polymer of nucleotide monomers. The monomers that make up the oligonucleotide are able to specifically bind to natural polynucleotides through regular patterns of monomer-monomer interactions (e.g., Watson-Crick type base pairing, base stacking, Hoogsteen or reverse Hoogsteen type base pairing, wobble base pairing, etc.). The size of the ssDNA oligonucleotides described herein is generally between 6 and 26 nucleotides, but can be longer. The size of the ssDNA oligonucleotides described herein can be between 6 and 220 nucleotides, for example, between 27 and 200 nucleotides. Whenever an oligonucleotide is represented by a letter sequence (uppercase or lowercase) such as "ATGC", it is understood that the nucleotides are in 5'→3' order from left to right, and "A" represents deoxyadenosine, T represents deoxythymidine, "G" represents deoxyguanosine, and "C" represents deoxycytidine. In addition to conventional nucleotides (A, G, C, T), modified nucleotides such as K-2'-deoxyribose, P-2'-deoxyribose, 2'-deoxyinosine, 2'-deoxyxanthosine or nucleotides with nucleobase analogs, such as inosine or 5-methylisocytosine, or 3-nitropyrrole, 5-nitroindole, pyrrolidine, 4-nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 4-aminobenzimidazole, 5-nitroindazole, 3-nitroimidazole, 5-aminoindole, benzimidazole, 5-fluoroindole, indole, methylisoquinolone, pyrrolopyrimidine, 7-propynylisoquinolone can be used. The terminology and atom numbering conventions follow those disclosed in Strachan and Read's Human Molecular Genetics 2 (Wiley-Liss, New York, 1999). Oligonucleotides typically comprise four natural nucleosides (e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose counterparts for RNA) linked by phosphodiester, by peptidyl bonds or by phosphorothioate bonds; however, they may also comprise non-natural nucleotide analogs, e.g., including modified bases, sugars or internucleoside linkages.

[0115] In some embodiments, a single-stranded oligonucleotide library is generated using chemical synthesis, for example, by synthesizing oligonucleotide sequences from monomer-phosphoramidites, dimer-phosphoramidites (Neuner, Cortese and Monaci 1998), or trimer-phosphoramidites (Sondek and Shortle 1992), mixtures of monomer-phosphoramidites, mixtures of dimer-phosphoramidites, mixtures of trimer-phosphoramidites, or combinations thereof.

[0116] In some embodiments, oligonucleotides are produced and purified from naturally occurring sources, or are synthesized in vivo in cells subjected to in vivo mutagenesis using any of a variety of well-known enzymatic methods, such as described in Farzadfard et al. (2014). Specifically, enzymes for synthesizing soft randomized oligonucleotide pools include, but are not limited to, low-fidelity DNA polymerase proteins or low-fidelity reverse transcriptase proteins that incorporate mismatched nucleotides at a high frequency during synthesis. Alternatively, mismatched nucleotides are incorporated into oligonucleotides at a higher frequency by DNA polymerases or reverse transcriptases due to the presence of chemical substances, which is well known to those skilled in the art.

[0117] The term "base pair" or "bp" (used as an abbreviation, singular or plural), also called "bps" (plural), refers to any pair of nucleotides that connects complementary strands of a DNA or RNA molecule and consists of a purine joined to a pyrimidine by hydrogen bonds. The nucleotide pair is adenine and thymine in DNA, adenine and uracil in RNA, and guanine and cytosine in DNA and RNA.

[0118] The term "matched oligonucleotide pair" refers to two or more complementary oligonucleotides, "complementary" means that the nucleotide sequences of similar regions of two single-stranded nucleic acids or overhangs of one or more ds nucleic acids have a nucleotide base composition that allows the single-stranded regions to anneal together in a stable double-stranded hydrogen-bonded region under stringent annealing or amplification conditions, such annealing is also called "hybridization". When a continuous sequence of nucleotides in one single-stranded region is able to form a series of "canonical" hydrogen-bonded base pairs with a similar sequence of nucleotides in another single-stranded region, A pairs with U or T, C pairs with G, the nucleotide sequences are 100% complementary. In addition to the conventional bases (A, G, C, T), analogs such as inosine and 2'-deoxyinosine and their derivatives (e.g. 7'-deaza-2'-deoxyinosine, 2'-deaza-2'-deoxyinosine), azole- (e.g. benzimidazole, indole, 5-fluoroindole) or nitroazole analogs (e.g. 3-nitropyrrole, 5-nitroindole, 5-nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole) and their derivatives, acyclic sugar analogs (e.g. those derived from hypoxanthine or indazole derivatives, 3-nitroimidazole or imidazole-4,5-dicarboxamide), 5'-triphosphates with base analogs (e.g., derived from indole derivatives), isoquinolone and other hydrophobic analogs and any derivatives thereof (e.g., methylisoquinolone, 7-propynylisoquinolone), hydrogen-bonded universal base analogs (e.g., pyrrolopyrimidines), and other chemically modified bases (e.g., diaminopurine, 5-methylcytosine, isoguanine, 5-methyl-isocytosine, K-2'-deoxyribose, P-2'-deoxyribose) may have different base pairing preferences and may pair with more than one natural nucleobase with similar stringency / probability. In some cases, the monomers are linked by phosphodiester or by peptidyl bonds or by phosphorothioate bonds.

[0119] The term "double-stranded DNA oligonucleotide", also referred to as "dsDNA oligonucleotide", or simply "ds oligonucleotide (oligonucleotide)" or "ds oligonucleotide (oligo)", refers to an oligonucleotide that is a linear polymer of nucleotide dimers. The dimer that makes up the oligonucleotide is two complementary nucleotides bound by a regular pattern of monomer-monomer interactions (e.g., Watson-Crick type base pairing, base stacking, Hoogsteen or reverse Hoogsteen type base pairing, wobble base pairing, etc.). The size of the dsDNA oligonucleotides described herein is generally between 6 and 26 base pairs (bp), but can be longer. The size of the dsDNA oligonucleotides described herein can be between 6 and 200 base pairs, for example, between 27 and 200 base pairs. Whenever an oligonucleotide is represented by a sequence of letters (upper or lower case) such as "ATGC", it is understood that the nucleotides are in 5'→3' order from left to right, and that "A" represents deoxyadenosine, "T" represents deoxythymidine, "G" represents deoxyguanosine, and "C" represents deoxycytidine. In addition to conventional nucleotides (A, G, C, T), modified nucleotides such as K-2'-deoxyribose, P-2'-deoxyribose, 2'-deoxyinosine, 2'-deoxyxanthosine or nucleotides with nucleobase analogs, such as inosine or 5-methylisocytosine, or 3-nitropyrrole, 5-nitroindole, pyrrolidine, 4-nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 4-aminobenzimidazole, 5-nitroindazole, 3-nitroimidazole, 5-aminoindole, benzimidazole, 5-fluoroindole, indole, methylisoquinolone, pyrrolopyrimidine, 7-propynylisoquinolone can be used. The terminology and atom numbering conventions follow those disclosed in Strachan and Read's Human Molecular Genetics 2 (Wiley-Liss, New York, 1999). Oligonucleotides typically comprise four natural nucleosides (e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose counterparts for RNA) linked by phosphodiester, by peptidyl bonds or by phosphorothioate bonds; however, they may also comprise non-natural nucleotide analogs, e.g., including modified bases, sugars or internucleoside linkages.

[0120] The simplest DNA end of a double-stranded molecule is called a flat end. In a flat-ended molecule, both chains terminate at a base pair. Non-flat ends are produced by various overhangs. As used herein, the term "overhang" refers to an extension of unpaired nucleotides at one or both ends of a ds oligonucleotide or polynucleotide molecule. These unpaired nucleotides can form 3' or 5' overhangs in either chain. The simplest case of an overhang is a single nucleotide. The overhang may comprise or be composed of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides or be composed of at least any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides. The overhang is usually no more than half the length of the ds oligonucleotide. For example, if the ds oligonucleotide is 6 nucleotides long, the overhang is no more than 3 nucleotides long, which means that the overhang can also be 1 or 2 nucleotides long. According to another embodiment, if the ds oligonucleotide is 24 nucleotides long, the overhang is no longer than 12 nucleotides long, which means that it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 nucleotides long.

[0121] As used herein, the term "library" refers to a collection of library members (e.g., oligonucleotide libraries) that are nucleic acid fragments and contain at least 10,000 pairs of matched oligonucleotides. Library members can be single-stranded oligonucleotides or double-stranded oligonucleotides. These library members have common features (e.g., given by genomic sequences), but differ in at least one base pair, nucleotide, mutation, and / or phenotype. In addition to those library members with common features, libraries generally also include diversity library members. A specific type of library is a library of random mutants of oligonucleotides, produced by random mutagenesis. Another specific embodiment is a rationally designed (or synthesized) library, such as a library of DNA fragments or oligonucleotides comprising specific engineering. The library described herein comprises library members that are suitably composed of oligonucleotides of different lengths and different sequences, wherein the oligonucleotides can correspond to a certain region of DNA or can even span the entire genetic space. For example, a library can include a variety of oligonucleotides necessary for any and all naturally occurring polynucleotides that may synthesize a human chromosome genome or a mitochondrial genome. In another embodiment, the diversity can cover any and all naturally occurring polynucleotides of eukaryotic species other than humans, such as mice, rats, rabbits, pigs, sheep, plants, fungi or yeast. In yet another embodiment, the diversity can cover any and all naturally occurring polynucleotides of prokaryotes, such as achaeans or bacteria.

[0122] The library provided herein specifically comprises at least 10,000 pairs of matched oligonucleotides, which are single-stranded oligonucleotides, specifically, ss oligonucleotides of different lengths, comprising partially or completely complementary sequences. The matched oligonucleotide pairs can be present in the library as ss oligonucleotides in separate containers, or two or more complementary ss oligonucleotides can be included in one container, in which they can anneal and form ds oligonucleotides. The nucleotide sequences of a pair of matched ss oligonucleotides can be complementary in at least 1, 2 or 3 nucleotides, preferably at least 4 or more nucleotides, so that the matched pairs can form new ds polynucleotide molecules by hybridization of ss oligonucleotide sequences, preferably wherein the ss oligonucleotides partially hybridize, thereby obtaining ds polynucleotides with overhangs.

[0123] The library preferably comprises artificial or chemically synthesized oligonucleotides, or chemically modified oligonucleotides (e.g., including peptidyl nucleic acids or phosphorothioate bonds) synthesized by suitable methods well known in the art. The oligonucleotides contained in the library can also be produced by enzymatic digestion of naturally occurring DNA. The specific characteristics of the members of the oligonucleotide library described herein are that the sequence is different, the mutation or the core base or nucleotide changes, such as the replacement or insertion or deletion of one or more subsequent nucleotides. Generally, the library members are different in at least one or more point mutations. Specifically, in some embodiments, the variation covers each possible naturally occurring core base residue at a certain position. If a mutant is produced by mutagenesis of the parent oligonucleotide, a variety of sequence variations of the parent oligonucleotide are produced.

[0124] The diversity of the libraries described herein may further comprise library members that are phosphorylated, methylated, biotinylated, or linked to a fluorophore or quencher. As described herein, library members may comprise one or more additional phosphoryl groups.

[0125] Methylation of the library members is performed according to suitable DNA methylation methods well known in the art, ie, the addition of methyl groups to DNA molecules, preferably to cysteine ​​or adenine.

[0126] As used herein, biotinylation refers to a process of covalently attaching one or more biotin molecules to a nucleic acid, such as an ss oligonucleotide or a ds oligonucleotide. The library members described herein can be biotinylated by a suitable method well known in the art; preferably, it is a chemical biotinylation method. Oligonucleotides can be readily biotinylated during oligonucleotide synthesis by the phosphoramidite method well known in the art, which employs biotin phosphoramidites.

[0127] The members of the libraries described herein can be conjugated to fluorophores by suitable chemical and enzymatic methods well known in the art. Exemplary methods for fluorescent labeling of nucleic acids can use methods for enzymatic labeling of DNA with fluorescent dyes, for example, using the ARES DNA labeling kit of Thermo Fisher, which uses a two-step method to enzymatically label DNA with fluorescent dyes. Other exemplary methods can use chemical methods to label nucleic acids without enzymatic incorporation of labeled nucleotides, for example, using the ULYSIS nucleic acid labeling kit. Further exemplary methods can use chemical labeling of amine-terminated oligonucleotides to prepare single-labeled fluorescent oligonucleotide conjugates, for example, using the Alexa Fluor oligonucleotide amine labeling kit. Other exemplary methods can use DNA arrays / microarrays and other hybridization techniques.

[0128] The library members can be linked to one or more quenchers (e.g., substances that absorb excitation energy from a fluorophore) by suitable methods well known in the art. Examples of quenchers include, but are not limited to, Dabsyl (dimethylaminoazobenzenesulfonic acid), Black Hole Quenchers, Qxl Quenchers, Iowa black FQ, Iowa black RQ, and IRDye QC-1.

[0129] As used herein, the term "point mutation" or nucleobase change refers to a mutation event that changes a nucleic acid or amino acid sequence at a specific position, for example, by introducing or exchanging a single nucleobase or amino acid or introducing a gap. A point mutation or nucleobase change may include a change in one or more single or adjacent or consecutive nucleobases or amino acid residues in a sequence. In a library comprising a mutant repertoire covering a limited diversity, the frequency of point mutations in a sequence is limited so that the mutant has at least a certain sequence identity with the parental (or reference) sequence, for example, at least any one of 80%, 90%, 95%, 96%, 97%, 98% or 99%.

[0130] "Percent (%) nucleotide sequence identity" is defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in a particular nucleotide sequence, with respect to the nucleotide sequences described herein, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, without considering any conservative substitutions as part of the sequence identity. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0131] As used herein, the term "diversity" refers to the diversity degree of the library provided herein. Specifically, the diversity includes single-stranded and double-stranded oligonucleotides with different lengths and different sequences. For example, the library can include all possible sequence variations of 8-nucleobase-long ss oligonucleotides (referred to as octamers in this article), which are 65,536 different 8-nucleobase-long ss oligonucleotides, and in addition also includes other ss oligonucleotides or ds oligonucleotides of different lengths, which are usually included in the target sequence and therefore more often required to construct any given sequence. The inclusion of commonly used single-stranded or double-stranded oligonucleotides in the diversity of the library reduces the synthesis cost and improves time efficiency.

[0132] In particular, the diversity can cover the entire genome, such as the human genome. In particular, the diversity can cover the entire genetic space. In particular, the diversity can cover the genome or the entire genetic space multiple times in a variety of ways. For example, by covering all possible hexamer, heptamer and / or octamer sequence combinations. For example, the library can also cover all or selected 9-mers and 10-mers or any up to 26-mers.

[0133] According to a specific embodiment, the diversity within the oligonucleotide library described herein is characterized as follows: Diversity can be determined by the number of mutations within the oligonucleotide sequence. For example, in a single oligonucleotide with a length of 16 nucleotides, for four naturally occurring DNA, A, T, G or C nucleotides, the theoretical number of possible single nucleotide changes is 16×3=48. For two single nucleotide changes (double mutants) with four naturally occurring DNA, A, T, G or C nucleotides per oligonucleotide, the possible number of sequences is 6,408. For three single nucleotide changes (triple mutants) per oligonucleotide, this value is 563,904. For quadruple mutations, this value is 36,794,736. These values ​​can be further increased by incorporating non-natural nucleobases into oligonucleotide sequences.

[0134] Exemplary methods for sequencing-based screening of oligonucleotides within a library are as follows: SNP genotyping methods, including hybridization-based methods (e.g., molecular beacons, SNP microarrays, restriction fragment length polymorphisms, PCR-based methods, including allele-specific PCR, primer extension, 5'-nuclease or oligonucleotide ligation assays, single-stranded conformation polymorphisms, temperature gradient gel electrophoresis, denaturing high-performance liquid chromatography, high-resolution melting of the entire amplicon (HRM), SNPlex, and surveyor nuclease assays; sequencing-based mutation analysis, including capillary sequencing or high-throughput sequencing (amplicon sequencing) of the entire PCR amplicon of the PTR. Such high-throughput (HT) amplicon sequencing methods include, but are not limited to, polymerase clone sequencing, pyrophosphate sequencing, Illumina (Solexa) sequencing, SOLiD sequencing, semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing, nanopore DNA sequencing, tunnel current DNA sequencing, hybridization sequencing, mass spectrometry sequencing, microfluidics Sanger sequencing, microscopy-based sequencing, RNAP sequencing.

[0135] Each library member can be individually characterized and labeled with a selectable marker or DNA sequence tag or barcode to facilitate selection of library members in the library or identification of library members in the library. Alternatively, gene mutations can be determined directly by suitable assay methods such as high-throughput sequencing, capillary sequencing, or selection of corresponding oligonucleotides using specific probes that hybridize to predefined sequences.

[0136] It may be necessary to place the library members in separate containers to obtain the oligonucleotide library in the container. According to a specific embodiment, the library is provided in the form of an array such as a DNA biochip, wherein the array comprises a series of spots on a solid support.

[0137] As used herein, the term "mutagenesis" refers to the process of changing the sequence of an oligonucleotide or polynucleotide. Specifically, site-directed mutagenesis refers to a method of producing a specific mutation in a known nucleotide sequence. The mutation is a specific targeted change and may include single or multiple nucleotide insertions, deletions or substitutions. This task may be performed by restriction enzymes, particularly endonucleases and / or exonucleases. Endonucleases cut the phosphodiester bond in the middle of an oligonucleotide or polynucleotide, while exonucleases cut the phosphodiester bond at the 5' or 3' end of an oligonucleotide or polynucleotide.

[0138] As used herein, the term "algorithm" refers to a self-contained sequence of actions to be performed. An algorithm is an efficient method that can be expressed in finite space and time and in a well-defined formal language for computing functions. Starting from an initial state and initial inputs, the instructions describe a computation that, when executed, proceeds through a finite number of well-defined sequential states, ultimately producing an "output" and terminating in a final end state. Transitions from one state to the next are necessarily deterministic.

[0139] The term "workflow" or "assembly workflow" refers to the optimal number of oligonucleotide subsets and their assembly into the sequence of the target ds polynucleotide. In the methods provided herein, the sequence of the template can be divided into subsequences corresponding to the oligonucleotide subsets, avoiding specific nucleotide synthesis problems, such as palindromic sequences, runaway reactions, and clear assembly. In particular, segmentation into shorter oligonucleotides can be very effective in shortening the assembly process and avoiding the requirement to separate unwanted reaction products. Specifically, the connection of the oligonucleotide subsets will produce intermediate reaction products (also referred to as intermediates), and the assembly of the intermediate reaction products will eventually produce the target ds polynucleotide. Preferably, it is best to use the other criteria listed above to select the oligonucleotide subsets. These other criteria include, but are not limited to, minimizing the size of the oligonucleotide subset used in any single connection reaction (e.g., avoiding mismatched connections), thereby minimizing the annealing temperature differences of the oligonucleotide precursor subset members, minimizing the annealing temperature differences of the overhangs of different double-stranded subunits, whether or not a framework conversion adapter or a single-stranded oligonucleotide linker is used, and whether the degree of cross-hybridization between the hybridization-forming parts of the different oligonucleotides constituting the subset is minimized.

[0140] The number of oligonucleotides in a subset may vary. Preferably, the size of the subset is in the range of 1 to 100 or 2 to 100, more preferably in the range of 1 to 50 or 2 to 50, and still more preferably in the range of 1 to 10 or 2 to 10.

[0141] In a subset in which the degree of cross-hybridization has been minimized, a duplex or triplex consisting of a subunit of the set and the complement of any other subunit of the set contains at least one mismatch. In other words, the sequence of the oligonucleotides of such a subset differs from the sequence of each other oligonucleotide of the subset by at least one nucleotide, more preferably by at least two oligonucleotides. The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits of each tag and the length of the subunits.

[0142] A single-stranded oligonucleotide adapter having a sequence complementary to the combined overhang connects adjacent oligonucleotides in the target polynucleotide. The adapter, for example, comprises 6 bases that connect two adjacent oligonucleotides, one at the 3' end and the other at the 5' end, each oligonucleotide having an overhang of 3 bases.

[0143] In a specific embodiment of the invention, the process of determining the assembly workflow is performed by an algorithm. According to the methods provided herein, candidate partitions of the template sequence are systematically examined to find the optimal number of subsets and assembly sequences to partition into for synthesis. Initially, the entire template sequence is considered as a single subset, and thereafter, smaller and smaller subsets are formed with increasing numbers of candidate oligonucleotides of decreasing size until a partition that meets the above-mentioned subset criteria is found.

[0144] The term "assembly" or "assemble" refers to the formation of oligonucleotides or polynucleotides by ligating and / or hybridizing single-stranded and / or double-stranded oligonucleotides. In particular, the assembly is performed by any method of hybridizing ss nucleotide sequences and / or by a ligation reaction that is an enzymatic and / or chemical reaction. Preferably, the assembly is performed by an in vitro ligation method.

[0145] Assembly of the target ds polynucleotides can be performed directly by hybridizing a matching ss oligonucleotide, an overhanging end of a ds oligonucleotide, or indirectly by hybridizing one or more suitable ss oligonucleotide adapters, wherein the ss oligonucleotide adapters are contained in a library and selected and transferred from the library to assemble any of the first, second or other reaction products.

[0146] For direct assembly, oligonucleotide sequences are linked together by their single-stranded oligonucleotide portions or overlaps (i.e., overlaps or overhangs) so that the overlap is only included once in the continuous sequence. When two oligonucleotide sequences are compared with the overlap, a continuous sequence is formed whose length is the length of the two individual oligonucleotides combined minus the length of the overlap. Therefore, a continuous sequence comprising each compared oligonucleotide segment is obtained.

[0147] For indirect assembly, when aligning ss oligonucleotides and connecting them by a single-stranded linker, the target ds polynucleotide or any of the first, second or other reaction products is formed. For example, two (e.g., each 10 bases long) oligonucleotides can be connected by a ss oligonucleotide linker, e.g., 6 bases long, so that the 3 bases at the 3' end of the first oligonucleotide are aligned with the 3 bases at the 5' end of the ss linker, and the 3 bases at the 5' end of the second oligonucleotide are aligned with the 3 bases at the 3' end of the ss linker.

[0148] The term "first, second or other reaction product" refers to the product of the ligation reaction carried out in one or more reaction vessels. In the first step, at least the first pair of matched oligonucleotides are transferred from the library to the first reaction vessel using a liquid handler, and the matched oligonucleotides are assembled in the ligation reaction to form the first reaction product. Specifically, each of the first, second and other reaction products comprises at least one overhang. If the matched oligonucleotides include a first part that hybridizes with the overhang of the reaction product, and also include a second part of another overhang that produces a new reaction product, then this overhang of the reaction product allows further assembly with another matched oligonucleotide in the direction of the overhang, such as to produce a new reaction product with an overhang. Alternatively, if the matched oligonucleotides consist only of a part that hybridizes with the overhang on the total length of the overhang, such as to cover all nucleotides of the overhang, then a flat end can be produced.

[0149] In a specific case, a ds target double-stranded (ds) polynucleotide is generated, which has a blunt end at one or both ends. Preferably, such blunt ends can be obtained by hybridizing any terminal overhang with a matching ss oligonucleotide and / or a ds oligonucleotide (which hybridizes to the entire length of this overhang), but without generating new overhangs, thereby generating blunt ends.

[0150] In the first step, one or more pairs of matched oligonucleotides and one or more ss oligonucleotide adapters are transferred to the first reaction vessel using a liquid handler, and the matched oligonucleotides are assembled to obtain a first reaction product. Preferably, the number of matched pairs transferred to the first reaction vessel is any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25, preferably 4, even more preferably 1, 2 or 3, and the number of ss oligonucleotide adapters transferred is any one of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25, preferably 4, even more preferably 1, 2 or 3.

[0151] In the second step and other steps, at least the second pair and other pairs of matching oligonucleotides are transferred from the library to the second reactor and other reaction vessels, respectively, using a liquid handler, and the matching oligonucleotides are assembled to obtain the second and other reaction products, respectively. In the second step, one or more pairs of matching oligonucleotides and one or more ss oligonucleotide adapters are transferred to the second reaction vessel. Preferably, the number of matching pairs transferred in the second step is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25, any one, preferably 4, even more preferably 1, 2 or 3, and the number of ss oligonucleotide adapters transferred is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25, any one, preferably 4, even more preferably 1, 2 or 3. In the other steps, one or more pairs of matching oligonucleotides and one or more ss oligonucleotide adapters are transferred to the other reaction vessels. Preferably, the number of matching pairs transferred in the further step is any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25, preferably 4, even more preferably 1, 2 or 3, and the number of ss oligonucleotide linkers transferred is any one of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25, preferably 4, even more preferably 1, 2 or 3.

[0152] The number of steps and corresponding reaction products is unlimited. To synthesize a large target ds polynucleotide, a series of reaction products need to be generated to assemble into the target polynucleotide, for example, at least 5, 10, 20, 50, 100, 500, 1,000, 5,000 or more may be required.

[0153] As used herein, the terms "hybridize," "hybridization," "hybridizing," "anneal," and "annealing" generally refer to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex can contain two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. The hybridization reaction can constitute a step in a broader process, such as initiating PCR or enzymatic cleavage of a polynucleotide by a ribozyme.

[0154] As used herein, the term "ligation" means a process in which two nucleic acid sequences anneal to each other through an intermolecular chemical bond (eg, hydrogen bond) to form a double strand under appropriate conditions.

[0155] The ligation product, also referred to herein as the reaction product, can be formed by both double-stranded nucleic acid and single-stranded nucleic acid. Double-stranded nucleic acids can be connected by "sticky end" connection or "flat end" connection. In sticky end connection, the staggered ends containing the terminal overhangs can hybridize with the connection partner. In flat end connection, there is no terminal overhang, and successful connection depends on the transient association of the 5' end and the 3' end. Generally, the efficiency of flat end connection is not as good as that of sticky end connection, and various optimization measures (such as adjusting concentration, incubation time and temperature) can be used to improve efficiency. Single-stranded polynucleotides can also be connected.

[0156] The efficiency of the connection between two complementary sequences or fully complementary sequences depends on the operating conditions used, especially stringency. Stringency can be understood as the degree of homology; the higher the stringency, the higher the homology percentage between the sequences. Stringency can be defined in particular by the base composition of the two nucleic acid sequences and / or by the degree of mismatch between the two nucleic acid sequences. By changing conditions (such as salt concentration and temperature), a given nucleic acid sequence can be allowed to be connected only to its exact complementary sequence (high stringency) or to any slightly related sequence (low stringency). Raising the temperature or reducing the salt concentration tends to increase the selectivity of the connection reaction.

[0157] The ligation reaction can be carried out by enzymes, particularly DNA ligases. DNA ligases catalyze the formation of covalent phosphodiester bonds, which permanently connect nucleotides together. In addition, if there is no dsDNA template, T4 DNA ligase can also connect ssDNA, although this is generally a slow reaction. Non-limiting examples of enzymes that can be used for ligation reactions are ATP-dependent double-stranded polynucleotide ligases, NAD+-dependent DNA or RNA ligases, and single-stranded polynucleotide ligases. Non-limiting examples of ligases are Escherichia coli DNA ligase, Thermus filiformis DNA ligase, Thermus thermophilus DNA ligase, Thermus scotoductus DNA ligase (I and II), CircLigase TM (Epicentre; Madison, WI), T3 DNA ligase, T4 DNA ligase, T4 RNA ligase, T7 DNA ligase, Taq ligase, Ampligase ( Technologies Corp.), Van C-type ligase, 9°N DNA ligase, Tsp DNA ligase, DNA ligase I, DNA ligase III, DNA ligase IV, Sso7-T3 DNA ligase, Sso7-T4 DNA ligase, Sso7-T7 DNA ligase, Sso7-Taq DNA ligase, Sso7-E. coli DNA ligase, Sso7-Amp ligase DNA ligase, and thermostable ligase. The ligase can be wild type, mutant isoforms, and genetically engineered variants. The ligation reaction can include a buffer component, a small molecule ligation enhancer, and other reaction components.

[0158] Preferably, T4 DNA ligase is used in the ligation reaction.In the methods provided herein, the ligation reaction is performed under high fidelity conditions that prevent side reactions and minimize mismatches.

[0159] Suitable ligation buffers can be used to assemble intermediate reaction products or target polynucleotides. The ligation buffer is, for example, an aqueous solution, typically in a nuclease-free environment, whose pH value ensures that the selected ligase is active; typically, the pH is about 7-9. Preferably, the pH is maintained by Tris-HCl at a concentration of about 5mM to 50mM. The ligation buffer solution may include one or more nuclease inhibitors, typically calcium ion chelators, such as EDTA. Typically, EDTA is included at a concentration of about 0.1 to 10mM. The ligation buffer includes any cofactor required to make the selected ligase active. Typically, this is a divalent magnesium ion at a concentration of about 0.2mM to 20mM, typically provided as a chloride salt. For T4DNA ligase, ATP is required as a cofactor. The ligase buffer solution may also include a reducing agent, such as dithiothreitol (DTT) or dithioerythritol (DTE), typically at a concentration of about 0.1mM to about 10mM. Optionally, the ligase buffer may include a reagent that reduces the non-specific binding of oligonucleotides and polynucleotides. Exemplary reagents include salmon sperm DNA, herring sperm DNA, serum albumin, Denhardt solution, etc. Preferably, the connection conditions are adjusted so that if the bases in the continuous complementary region of the first and second oligonucleotides form a duplex that matches completely with the target sequence, connection occurs. However, it should be understood that in some embodiments, it is advantageous to allow the non-paired nucleotides on the 5' end of the first oligonucleotide and the 3' end of the second oligonucleotide to assist in detection or reduce the flat end connection. Important parameters in the ligation reaction include temperature, salt concentration, denaturing agents such as the presence or absence of formamide and concentration, the concentration of the first and second oligonucleotides, and the type of ligase used. The method of selecting the hybridization conditions for reaction is well known to those skilled in the art.

[0160] Preferably, connection occurs under stringent hybridization conditions to ensure that only fully matched oligonucleotides hybridize. Typically, stringency is controlled by regulating the temperature at which hybridization occurs while maintaining the salt concentration at a constant value, such as 100 mM NaCl or equivalent. Other factors may be relevant, such as the specific sequence of the first and second oligonucleotides, the length of the first and second oligonucleotides, and the thermal instability of the selected ligase. Preferably, ligation is performed at a temperature close to the melting temperature (melting temperature) of the hybridized oligonucleotide in the connection buffer. More preferably, ligation is performed at a temperature within 10 ° C of the melting temperature of the hybridized oligonucleotide in the connection buffer. Most preferably, ligation is performed at a temperature lower than 0 to 5 ° C of the melting temperature of the hybridized oligonucleotide in the connection buffer.

[0161] One or more amplification reactions can be carried out after the connection. In certain embodiments, before amplification, separation or enrichment of connection products or target polynucleotides can be achieved. Separation can be achieved by various suitable purification methods including affinity purification and gel electrophoresis. For example, connection products or target polynucleotides can be separated by connecting the selective binding agent fixed on the support with the label attached to the capture probe. The support can then be used to separate or separate capture probes and any polynucleotides hybridized with the capture probe from other contents of the sample reaction volume. The separated polynucleotides can then be used for amplification and further sample preparation steps. In certain embodiments, before amplification of the circular target polynucleotides, degradation or selective removal of the capture probe. The amplification of reaction products or target polynucleotides can be achieved by various suitable amplification methods well known to those skilled in the art.

[0162] The term "derivative" refers to an oligonucleotide or polynucleotide that is different from the original oligonucleotide or polynucleotide but retains its essential properties. Derivatives can be produced, for example, using ds polynucleotides (e.g., DNA) as starting material to engineer single-stranded DNA or complementary RNA molecules to introduce one or more point mutations, or to incorporate heterologous moieties or tags by chemical and / or enzymatic methods.

[0163] Generally, derivatives are generally very similar to the original oligonucleotide or polynucleotide, and are identical in many regions. In fact, it is possible to use known computer programs to routinely determine whether any specific nucleic acid molecule or polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence of the present invention. The preferred method for determining the best overall match between the query sequence (sequence of the present invention) and the subject sequence, also referred to as global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of the people such as Brutlag (Comp.App.Blosci.(1990)6:237-245). In the sequence alignment, the query sequence and the subject sequence are both DNA sequences. RNA sequences can be compared by converting U's to T's. The result of the global sequence alignment is the identity percentage. If the subject sequence is shorter than the query sequence due to 5' or 3' deletions rather than internal deletions, the result must be manually corrected. This is because the FASTDB program does not consider the 5' and 3' truncation of the subject sequence when calculating the percentage identity. For example, the subject sequence of 90 bases is compared with the query sequence of 100 bases to determine the identity percentage. The deletion occurs at the 5' end of the subject sequence, so the FASTDB comparison does not show the matching / comparison of the first 10 bases at the 5' end. 10 damaged bases represent 10% of the sequence (the total number of bases in the base number / query sequence at the 5' and 3' ends that are not matched), so 10% is deducted from the identity percentage score calculated by the FASTDB program. If the remaining 90 bases are fully matched, the final identity percentage will be 90%. In another embodiment, the subject sequence of 90 bases is compared with the query sequence of 100 bases. This deletion is an internal deletion, so there is no base that does not match / comparison with the query on the 5' or 3' of the subject sequence. In this case, the percentage identity calculated by FASTDB cannot be manually corrected. Again, only the bases 5' and 3' of the subject sequence that did not match / align with the query sequence were manually corrected.

[0164] The library of the present invention can include thousands of oligonucleotides necessary to cover the entire sequence space. Each oligonucleotide library member can be physically placed in a compartment. All compartments can be conveniently arranged in one or more parts of the device, and these parts are provided together as "array devices". Such array devices can be microtiter plates, microfluid microculture plates, capillary groups, microarrays or biochips (preferably any one or more of DNA and / or RNA biochips). Oligonucleotides can be conveniently transferred from such compartments to other compartments referred to herein as reaction compartments by automated means (e.g., by robots or by using, for example, special fluids of automated liquid handlers), i.e., transferred from one vessel to another vessel. In order to promote the effective assembly of polynucleotides in time, the reaction hierarchy and corresponding vessels corresponding to the frequency of use of oligonucleotide library members can be used. The transfer to new vessels involves the physical movement of the device that picks up one or more oligonucleotide molecules from the corresponding position, or the pneumatic / hydraulic deposition performed by microfluids. Since theoretically constructing any given sequence requires a large amount of oligonucleotides, most of the spatial distributions of library members in the library will cause a waste of time and resources due to the long travel time of scanning libraries and liquid handlers. However, by using a specific distribution of library members, it is possible to ensure that there is minimal movement depending on the target sequence. An example is storage into microplates where the first plate contains the most common oligo pair combinations, proceeding in descending order until the last microplate contains the least commonly used library members.

[0165] Specifically, the independent library container is spatially arranged in a two-dimensional order, wherein each compartment is located in a device at the limited coordinates in the x-axis and y-axis. This order is predetermined by a parameter that is mainly used to shorten the synthesis time. Preferably, the parameter is a frequency of use, and those oligonucleotides are placed close to each other, and those oligonucleotides often form matching pairs in a DNA sequence (e.g., naturally occurring in or generally present in a target ds polynucleotide or its fragment). Even more preferably, the independent library container is spatially arranged in a three-dimensional order, wherein each compartment is located in a device at the limited coordinates in the x-, y-, and z-axis. This order is specifically determined by the frequency of use, and those oligonucleotides are placed close to each other, and those oligonucleotides often form matching pairs in a naturally occurring DNA sequence. Specifically, the spatial arrangement of library members can depend on any one or more of the following parameters: the frequency of use of oligonucleotides, the frequency of occurrence of oligonucleotides in a natural DNA sequence, the frequency of occurrence of oligonucleotides in a set of designed DNA sequences, the processing of microfluidic devices or the access time minimization, the operating cost or the amount of consumables of microfluidic devices.

[0166] In one embodiment, the individual library containers are microplates arranged in stacked plates, optionally barcoded, and accessible to an automated droplet processor. Library members can be conveniently stored in the stacked microplates, wherein the order and stacking are based on decreasing frequency of use.

[0167] As used herein, the term "liquid handler", "automated handler" or "droplet handler" refers to any device used in a method of liquid handling, preferably automated liquid handling, preferably a device used in a sensor-integrated robotic system. As low-volume dispensing becomes more and more common in life sciences, micro syringes with high precision and sealing have emerged. Some manual or electronic holders are designed to precisely control the displacement of the piston to ensure the accuracy of the dispensed volume. In addition to syringes, pipettes are another popular tool for liquid handling. The volume dispensed can be at the microliter or sub-microliter level. It is recommended to use a multichannel pipette to pipette multiple routes at once. There are fixed-volume and adjustable-volume pipettes on the market. The former is more accurate and precise, while the latter has a wider range of applications because the operator can choose different volumes as needed. In addition, high throughput has become crucial in life science research. One of the representative applications is microarray printing. This technology creates an array of biological sample spots, each of which is in the nanoliter range, enabling a large number of experimental analyses while using only a small amount of sample. The process of spotting thousands of biological samples with a handheld dispensing tool is almost an impossible task, which makes automated liquid handling an important aspect.

[0168] Compared with manual liquid handling, robotic workstations have several advantages, as robots can work without fatigue, increase throughput, have stable performance, and ensure accuracy and precision. According to the requirements for platforms with integration and multifunctionality, there are still more complex systems, in which liquid handling tasks are only part of the functionality. The general architecture of liquid handling can be established as follows. First, the control center controls the robot that moves between the dispensing part and the washing station of the robotic workstation. The washing station is used to clean the dispensing head to extend its life and ensure the safety of the sample. The liquid sample is discharged from the dispensing head and deposited on the substrate for further processing. Sensors are included to monitor the status of the dispensing part so that the control center can perform feedback control. Sensors are not always installed on all workstations, but are increasingly used to build feedback loops to provide better performance.

[0169] The term "capillary" refers to any of glass capillaries, microfluidic capillaries, and autonomous microfluidic capillary systems. Capillary microfluidics is an important tool in many different fields. Glass capillary devices have advantages in microfluidic applications due to their axisymmetric fluidity and ability to tolerate organic solvents compared to their photolithographically fabricated polydimethylsiloxane (PDMS) counterparts. In particular, a circular tube is inserted into a square external flow channel, which greatly simplifies the alignment and centering of these devices. These devices can produce small and large droplets ranging in size from 10 to hundreds of μm.

[0170] The term "microtiter plate" refers to any of a well plate, multiwell plate, or microplate. These plates are typically manufactured in a 2:3 rectangular mix with 96, 384, or 1536 wells, but other chamber configurations may also be used. Some other sizes available (less common) are 6, 24, 3456, and 9600 wells. The wells of a microplate typically hold tens of nanoliters to several milliliters of liquid.

[0171] The term "microarray" refers to a support material (e.g., a glass or plastic slide) to which many molecules or fragments, usually of DNA or protein, are attached in a regular pattern. More specifically, it refers to a microscope slide printed with thousands of tiny spots at defined locations, wherein the spots are capable of binding DNA or RNA. Such slides are also commonly referred to as biochips, DNA chips, RNA chips, or gene chips. Such microarrays can bind DNA or RNA in a covalent or non-covalent manner and can therefore be used as array devices that store oligonucleotides at predetermined locations (i.e., spots).

[0172] "Microfluidic devices" are able to manipulate discrete packets of fluid in the form of microdroplets, which offer many benefits for conducting biological and chemical assays. Among these benefits is a significant reduction in the volume of reagents required for the assay, the size of the sample required, and the size of the device itself. This technology also increases the speed of biological and chemical assays by reducing the volume in which processes such as heating, diffusion, and convective mixing occur. Once the droplets are generated, carefully designed droplet manipulation allows large numbers of droplets to be multiplexed, enabling large-scale complex biological and chemical assays.

[0173] The term "microfluidic microplate" refers to the combination of microfluidic technology in the form of microfluidic microplate technology with a 96-well microplate architecture in a standard SBS configuration. Microfluidic microplates allow for improvements in basic workflows, preservation of samples and reagents, improved reaction kinetics, and the ability to increase assay sensitivity through multiple analyte loading (Kai et al., 2012).

[0174] The term "methyltransferase" as used herein may refer to any of a DNA methyltransferase, an RNA methyltransferase, a protein methyltransferase, and a histone methyltransferase. Methyltransferases can be further subdivided into class I methyltransferases (all of which contain a Rossman fold for binding S-adenosylmethionine (SAM)), class II methyltransferases (which contain a SET domain, e.g., a SET domain histone methyltransferase), and class III methyltransferases associated with membranes.

[0175] The term "CRISPR / Cas9" refers to gene editing methods and modifications thereof that are well known to those skilled in the art. Such modifications include, but are not limited to, the fusion of a nuclease-inactivated Cas9 (dCas9) with a cytidine deaminase, which enables site-specific conversion of cytidine to uracil, and mutates the Cas9 protein to generate a form of the Cas9 protein that can only produce single-stranded DNA cuts (nicks).

[0176] The term "multiplex automated genome engineering" or "MAGE" refers to a technique that generally involves introducing multiple nucleic acid sequences into one or more cells to bring the entire cell culture to a state that includes a range of genomic or target region changes. The method can be used to generate a specific configuration of alleles, or can be used to perform combinatorial exploration of designed alleles, optionally including additional random or undesigned changes.

[0177] ssDNA binding protein-mediated recombination, homologous recombination, and MAGE-based methods generally involve introducing multiple oligonucleotides into cells, including the steps of transforming or transfecting the cells using a transformation medium or a transfection medium containing the oligonucleotides, replacing the transformation medium or the transfection medium with a growth medium, incubating the cells in the growth medium, and repeating these steps as necessary or desired until multiple nucleic acid mutations are introduced into the target nucleotide sequence. Increasing the number of mutagenesis cycles generally increases the diversity of mutations introduced.

[0178] MAGE specifically employs the highly efficient lambda phage red recombination system (λRed system), which is a process by which the genome of a cell is reprogrammed to perform a desired function through a form of accelerated directed evolution. The λRed system includes β, γ, and exo genes, whose products are called Beta, Gam, and Exo, respectively. Gam inhibits host RecB, C, D nuclease and SbcC, D nuclease activities so that exogenously added linear DNA is not degraded. The Exo protein is a dsDNA-dependent exonuclease that binds to the end of each chain while degrading the other chain in the 5' to 3' direction. Beta binds to the resulting ssDNA overhangs, ultimately pairing them with complementary chromosomal DNA targets. The λRed system has been widely used for the inactivation of specific genes in Escherichia coli, Salmonella, Citrobacter, and Shigella, and for the introduction of small biotags or single genes into these chromosomes.

[0179] The term "conjugative assembly genome engineering" or "CAGE" refers to a precise genome assembly method that uses conjugation to hierarchically combine different genotypes from multiple E. coli strains into a single chimeric genome. CAGE allows the large-scale transfer of specific genomic regions between strains without the limitations of in vitro manipulation. The strains are assembled in pairs by establishing a donor strain that contains the conjugation machinery and a recipient strain that receives DNA from the donor. In the strain pair, the transfer of conjugative origins and the targeted placement of selection markers in the donor and recipient genomes enable the controlled transfer and selection of desired donor-recipient chimeric genomes. By design, the selection markers act as genomic anchor points and are recycled in subsequent rounds of hierarchical genome transfer.

[0180] "Ago" refers to the Argonaute protein that has been shown to provide DNA-based DNA interference, in which a single-stranded DNA guide can direct Ago-based cleavage of a plasmid DNA target. A key advantage is that, unlike CRISPR-Cas9, no protospacer adjacent motif (PAM) is required.

[0181] Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) recognize DNA target sites ranging in size from 25 to 40 bp in a sequence-specific manner through their DNA binding domains and generate staggered double-strand breaks through the action of the Fok1 nuclease domain on opposite DNA strands.

[0182] "Meganucleases" (also called homing endonucleases) recognize specific DNA sequences of 14 to 40 bp, cleave them and induce DSBs. Meganucleases are quite efficient and they require only a single tailored biopolymer for each target site.

[0183] "Tyrosine / serine site-specific recombinases" or "Tyr / Ser SSRs" typically recognize target sequences of 30 to 40 bp in length and are one of the earliest genome engineering tools capable of homology-directed repair (HDR) in mammalian genomes. In short, the target site consists of three parts, flanked by two short DNA sequences with inverted repeats, and recombination can occur between a pair of target sites, where the DNA sequence between the target sites can be deleted, reversed, or replaced. Notably, Tyr SSRs utilize a strand exchange mechanism without generating double-strand breaks, Ser SSRs do generate double-strand breaks, but unlike simpler designed double-strand nucleases, SSRs require coordinated cutting and religation with the presence of donor DNA.

[0184] The foregoing description will be more fully understood with reference to the following examples. However, these examples merely represent methods of implementing one or more embodiments of the present invention and should not be construed as limiting the scope of the present invention.

[0185] Examples

[0186] In the following examples, the production of oligonucleotide libraries, the processing and verification of their content and properties are described. In addition, how to synthesize polynucleotides according to the methods provided herein is described.

[0187] Example 1: Generation of a library

[0188] 1.1 Determining the spatial structure of genetic information

[0189] A. First, all oligonucleotide sequences included in the library must be listed. These sequences are pre-calculated from a set of input sequences covering all potential desired targets. This information can come from a variety of criteria, such as subsets of possible combinations (e.g., all heptamers, all octamers, etc.), predicted results from digesting a genome with a set of restriction enzymes, or any other computational criteria.

[0190] In this example, the human mitochondrial genome (GenBank Accession No. J01415; Anderson et al., 1981) with 16569 base pairs was used as the basis for the library. Ideally, all reported sequences would be as follows and Figure 1 and processed in the same manner as described in .

[0191] The reference genome sequence was divided into oligonucleotide dimers ranging in length from 8 to 26 bp. Similarly, the reverse complement sequence was calculated and divided into oligonucleotides ranging in length from 8 to 26 nt. This resulted in a total of 2,070 oligonucleotides that could form ds structures with 4 nt overhangs (see Figure 1). Next, the same process was repeated by first transferring sequence 1, then 2, then 3, up to 15 nucleotides. As a result, there were 2070×16=33,120 oligonucleotides in the library.

[0192] If we count only those that overlap by 4 bp, there are at least 16,544 matching pairs in this database. In general, variant sequences should be treated in a similar manner, which increases diversity in a nonlinear manner. For example, a window of about 100 bp containing only 16 polymorphic sites adds more than 400 oligonucleotides and nearly 20,000 matching pairs ( Figure 1 ). Combinatorics means that, when more variable sites are considered, the oligonucleotide library is populated in a non-polynomial manner with the number of sequence variants considered in its design.

[0193] Some oligonucleotides are conserved between haplotypes and are distributed in the library as paired elements ( Figure 1 B) Oligonucleotides spanning the variable sites (and depending on the degree of variability) are maintained independently as ssDNA elements.

[0194] B. The two-dimensional arrangement of the library is determined by sorting the library members according to the preferred criteria. Here, first according to the sequence shift, then according to its first appearance order in the sequence and by alternate conjugation to sort the 16-aggressors. When alternative oligonucleotides appear for a given position, they are subclassified according to their frequency of occurrence. Oligonucleotides that are conservative between all input sequences are assigned together with their conjugation at the same position.

[0195] Alternative criteria reflecting individual usage of oligonucleotides and relative usage of their matched pairs may be by lexicographic order, length, contiguity of matched pairs, frequency, or any other arbitrary but known manner.

[0196] C. Next, the first sequence is distributed into a 2-dimensional array corresponding to the positions where the actual oligonucleotides are placed in the 1536 microwells of the microplate.

[0197] D. Add subsequent oligonucleotides until all 1535 remaining wells are occupied with oligonucleotides in an order reflecting the sorting preference of step B.

[0198] E. Then, repeat step C with the next 1536 oligonucleotides, and so on, until all 33,120 or more oligonucleotides are distributed in the microplate wells.

[0199] F. Information is stored digitally to track the location of each oligonucleotide. In subsequent steps, this serves two purposes: first, it is used as a lookup table to facilitate access to the oligonucleotides, and second, it allows the use and access frequency of each oligonucleotide to be monitored in order to track available capacity.

[0200] 1.2. Library synthesis

[0201] Once the sequence is properly structured, the actual synthesis of each oligonucleotide is performed. Physically, the library used to construct the human mitochondrial genome consists of 27 1536 microwell plates (Corning 1536 well plates, Sigma Aldrich ProductNr.CLS3726-50EA), which are made of polypropylene (preferably polypropylene, but any material that minimizes the adsorption of DNA to the surface can be used). Each plate is clearly labeled and / or barcoded for easy access and content bookkeeping.

[0202] As determined above, each generated oligonucleotide is located in its predetermined plate. In this example, the oligonucleotide is phosphorylated at the 5' end. Other applications may require treatment with other modifications at the 3', 5' or both ends, such as di- or triphosphates, biotin, TEG or thiol modifiers, or methylation. Oligonucleotides are stored in aqueous solution (nuclease-free ddH2O or TRIS 10mM pH 8.0 and 1mM EDTA) at a concentration of 200μM in a volume of 10μL / oligonucleotide / microwell (Sambrook and Russell, 2014).

[0203] The actual production of the library can be carried out using standard methods of molecular biology, by digesting the naturally occurring DNA with nucleases, using chemical constructions such as oligomer synthesizers, and then separating and purifying by HPLC, capillary electrophoresis or other techniques. Since the synthesis and modification of oligonucleotides are standard, they can also be outsourced from many services. According to this example, an automatic DNA synthesizer is used to produce the library, which repeatedly carries out the chemical reaction of deoxynucleoside phosphoramidites to make the polynucleotide covalent bonds (Beaucage and Cartuthers, 1981) of the mononucleotides and the solid phase.

[0204] Store the library at -20°C when not in use for a short period of time, or at -80°C for long-term storage.

[0205] 1.3 Use of the library

[0206] A. Thaw the library by placing the plate at 3 °C for at least 60 minutes, then keep the library on ice or on a cooler plate at 3-5 °C.

[0207] B. Vortex each microplate at 2500 rpm for 30 seconds on an orbital mixer and then centrifuge at 900 rpm for 1 minute in a centrifuge.

[0208] C. Using a small volume droplet handler (TPP Lab Tech Mosquito X1), transfer 100 nL (recommended range: 50-250 nL) to a new 384-well microplate (other volumes such as 96 or 1536, or surfaces may also be used) containing 1.8 μL of solution or solution droplets (recommended range is 1-5 μL) where oligonucleotides are bound and / or further reacted.

[0209] D. Annotate the used volumes of each microwell in a digital database to ensure that there is always enough of all required oligonucleotides for the next round of use. Note that some liquid handlers provide accurate and real-time measurement of the used and remaining volumes in each well visited. This feature can facilitate more precise tracking.

[0210] E. Once the library is used, it is returned to -80°C for storage.

[0211] 1.4 Determining the characteristics of the library

[0212] The main characteristics defining the library of the invention are: i) a defined length of the oligonucleotides, ii) single-stranded and / or double-stranded oligonucleotides with at least one overhang, and iii) a certain number of oligonucleotides. The main characteristics of the library used in this example are: i) the oligonucleotide length is 8 to 26 nt, ii) there are single-stranded and double-stranded oligonucleotides with at least one overhang, and iii) at least 33,120 oligonucleotides are included in the library.

[0213] For quality control purposes, it is desirable to be able to verify that these characteristics hold true.

[0214] I. Verify the length of the oligonucleotide.

[0215] Using a droplet handler, take aliquots of 5-10 nL from each microwell and merge into a common solution. Alternatively, take random aliquots and merge into 10 different pooled solutions in a way that each oligonucleotide is only in one of the pools. Mix one or more pools by vortexing. A small aliquot of a few μL from each pooled solution is subjected to capillary electrophoresis (Kemp, 1998). Alternatively, the samples can be analyzed on a 25% acrylamide gel and compared to a standard ladder of ssDNA from 6 to 24 bp.

[0216] II. Verification of the structures of the oligonucleotides present in the library.

[0217] ss oligonucleotides, ds oligonucleotides, and ds oligonucleotides with ss overhangs are distinguished by comparing a denatured but untreated sample of a given oligonucleotide with a sample treated with an exonuclease such as E. coli Exonuclease I (e.g., Thermo Scientific Exonuclease I, Product No. EN0581). This enzyme digests ssDNA into mononucleotides and dinucleotides but leaves dsDNA intact (Lehman and Nussbaum, 1964). Thus, when examined by capillary electrophoresis, the untreated and treated samples give one of the following results:

[0218] - The untreated sample showed a single band in the range of 6-26 nt, while the treated sample showed no band. This means that the original sample consisted of ss DNA.

[0219] - Untreated sample showed a single band in the range of 6-26 nt, while treated samples showed the same band. This means that the original sample consisted of ds DNA (no overhangs).

[0220] - The untreated sample shows two distinct bands, both in the range of 6-26 nt, and the treated sample shows a single band whose length coincides with the smallest band of the untreated sample. This means that the original sample consists of a DNA dimer with one overhang. The length of the overhang is the difference between the sizes of the two bands of the untreated sample, and the length of the ds portion is the length indicated on the treated sample.

[0221] - The untreated sample showed a single band in the range of 6-26 nt, and the treated sample showed a single band whose length was shorter than that of the untreated sample. This means that the original sample consists of a DNA dimer with two overhangs of equal size. The length of the overhang is the difference between the sizes of the treated and untreated samples, and the length of the ds portion is the length indicated by the band of the treated sample.

[0222] - The untreated sample showed two bands, both in the range of 6-26 nt, while the treated sample showed a single band whose length was shorter than that of the two untreated samples. This means that the original sample consists of a DNA dimer with two overhangs of different sizes. The length of the overhangs is determined by the difference in the size of each band relative to the size given by the treated sample, and the length of the ds portion is the length indicated on the treated sample.

[0223] Other analytical techniques, such as HPLC, can also reveal the composition of the untreated sample in its spectrum, directly indicating the presence of a single species of DNA or two of them, directly proving the nature of the oligonucleotide in one well of the library. In addition, circular dichroism can be used to distinguish between single-stranded and double-stranded DNA, even dsDNA with overhangs.

[0224] III. Verify the number of oligonucleotides and the number of matching pairs.

[0225] Combine 50-100 nL samples of the contents of each microwell into a common solution that is annealed by heating at 95°C for 3 minutes and allowed to cool to at least room temperature or down to 16°C. Add the appropriate buffer necessary for ligation, including necessary cofactors such as Mg+, ATP, etc. Add enough ligase (e.g., T4 ligase, NEB, product number M0202) to catalyze the reaction (1 U / μL reaction solution). Incubate the reaction mixture at room temperature for 1 hour or at 16°C overnight.

[0226] By assuming that if there are enough matching pairs, the ligase will covalently link them, thus generating DNA molecules of a certain length range with random sequences. The distribution of lengths is resolved by electrophoresis on TAE with agarose 2-4%. The samples are run with an appropriate ladder (on a separate lane; 50 or 100 bp is recommended) showing a DNA smear along the sample lane without discrete bands. By cutting the gel guided by the ladder, a narrow range of about 100-200 bp is isolated (Sambrook and Russell, 2014; Chapter 5). DNA is isolated from the excised agarose block following a standard protocol for gel extraction (e.g. Zymoclean Gel DNA Recovery Kit, Zymo research, product number D4001T). After purification, the samples are deep sequenced to determine the different sequences in the library (Bentley et al., 2008).

[0227] In order to estimate the number of oligonucleotides and matching pairs, the following analysis was performed. If the starting material of the reaction consists of 6 to 26 nt of DNA, and the sequence is not highly repetitive, it can be inferred that there are at least 2×N×100 / 26 oligonucleotides (N is the number of reported sequences) on average, and at most 2×N×200 / 6 oligonucleotides and almost identical matching pairs. Further bioinformatics analysis was performed as follows to extract the sequences of the oligonucleotides. Take the first 6nt of one of the sequences, search and match the pattern in the complete sequence library, and annotate the number of occurrences. Repeat for 7nt, then 8nt, and so on until 26nt. By using a statistical T test, it can be determined which value is significantly different from the random value. This unique pattern is stored in the list of putative oligonucleotides, and all its occurrences are eliminated from the database. Repeat the process for the remaining sequences until only DNA subsequences of 6 to 26nt remain, which cannot be further divided and are now added to the list of patterns. The number of identified oligonucleotides is called M. Since these oligonucleotides are connected to at least one other oligonucleotide, this means that, together with their partial complementary sequences in the opposite strand, the continuous oligonucleotides are part of the matching pair. Therefore, except for those oligonucleotides at the end, there are at least as many matching pairs as the number of oligonucleotides identified. For example, there are about MN matching pairs. Statistical analysis and bootstrap simulation are performed to determine whether the number identified is a subsample of a larger set of at least 33,120 oligonucleotides.

[0228] Example 2: Synthesis of 128 bp target DNA molecule

[0229] In this example, it is demonstrated how to synthesize a 128 bp sequence by the method proposed herein. Figure 2 A shows the target sequence, which is called DISCOVER and is constructed from 16 matching pairs ( Figure 2 B), the matching pair formed a 4 nt overhang on each strand (see Figure 2 C) and 8 16nt ds oligonucleotides with 8 complementary sites. Each ds oligonucleotide is represented by the letters D, I, S, C, O, V, E, R, and their constituent leading and lagging strands are represented by + and - superscripts, respectively. The oligonucleotides are part of the library generated in Example 1. It has the following characteristics: all oligonucleotides are phosphorylated at the 5' end, they are provided in nuclease-free ddH20 at a concentration of 200 μM, and the oligonucleotides used are single-stranded and pure.

[0230] A. Prepare annealing solution.

[0231] In a reaction tube, prepare 252 μL of ddH2O with TRIS-HCl (50 mM), MgCl2 (10 mM), DTT (10 mM), and ATP (1 mM). Set the pH to 7.5. Some commercial buffers are prepared to mix in H2O, such as New England Biolabs' Ligase Reaction Buffer, Product No. B0202S, and easily contain the ATP required for ligase activity. Mix the solution thoroughly by vortexing. Dispense 28 μL of this solution mixture into 8 microwells in a 4×2 array. Transfer 1 μL of each oligonucleotide to the predetermined microwell of the plate and mix thoroughly by pipetting:

[0232] Pipette D+ and D- into well A1

[0233] Pipette I+ and I- into well A2

[0234] Pipette S+ and S- into well A3

[0235] Pipette C+ and C- into well A4

[0236] Pipette O+ and O- into well B1

[0237] Pipette V+ and V- into well B2

[0238] Pipette E+ and E- into well B3

[0239] Pipette R+ and R- into well B4

[0240] B. Annealing.

[0241] The plate was sealed and incubated in a thermal cycler at 95°C for 5 minutes to allow the matched pairs of ss oligonucleotides to anneal. The temperature was then decreased to 16°C by a ramp function that decreased the temperature by 1°C per minute. Once completed, the double-stranded oligonucleotides were kept at 16°C.

[0242] C. Prepare ligation solution.

[0243] Prepare the ligation solution on ice by mixing 13.3 μL of nuclease-free ddH2O, 2 μL of ligase buffer and 4 μL of ATP in the following order, with a final concentration of 1 mM. The ligation solution is thoroughly mixed by vortexing and centrifugation. Add 0.7 μL of T4 ligase (NEB, product number M0202), add 1 unit per μL of final solution, and mix it thoroughly by gently pipetting. Keep the solution on ice until needed. Transfer 2.5 μL of the ligation solution to each of the 8 microwells containing the ds oligonucleotide of B and mix by pipetting. Afterwards, seal the plate again.

[0244] D. Connection rounds.

[0245] For the first round of ligation, the following wells were combined as follows: D+I, S+C, O+V, E+R. This was done by transferring the contents of one well to another (it is also possible to transfer the contents of both wells to the new well). A scheme was used where the contents of the leftmost well were transferred to the contents of the rightmost well ( Figure 3 A). Incubate the ligation reaction mixture at 16°C for at least one hour. Repeat this process by combining wells DI+SC and OV+ER ( Figure 3 B) and incubate them again for one hour. For the final round of ligation, combine the DISC+OVER wells and incubate them for another hour ( Figure 3 C). The final volume containing the 128 bp product is 140 μL.

[0246] E. Purification.

[0247] Prepare an agarose gel 2% (50mL TAE provided with 5 μL SYBR Safe DNA stain containing 1mg agarose) with 11 wells. 4.5 μL of a 50bp ladder (New England Biolabs product number N3236 or Invitrogen product number 10416014) is added to the first lane, and the 140 μL solution obtained in step D is distributed to the remaining holes. The gel is run for 50 minutes at 85V, 200mA and 12W. After electrophoresis is complete, the gel is placed on a UV transmission illuminator, and the gel band corresponding to the 128bp fragment is cut. The purification of these bands can be carried out by a commercial kit (such as Zymoclean, see previous examples) for the purpose or according to any standard protocol for this purpose.

[0248] F. Amplification.

[0249] To further increase the amount of product, the product obtained in step D was amplified by PCR (Sambrook and Russell, 2014; Chapter 8). The starting 16nt D- and R+ were used as primers for the amplification. After amplification, the enzyme and primers were removed from the construct and divided into two aliquots, one for further use, which was labeled and stored at -20°C, and the other for sequence verification of the construct.

[0250] Figure 4 An acrylamide gel showing the intermediate steps and the final result of the method is depicted. In lanes 6 and 7, the upper band corresponds to the 128 bp target ds polynucleotide. The construct was isolated (from a 2% agarose gel; not shown), purified, amplified and Sanger sequenced for both chains. The resulting sequence was identical to the target and its reverse complement.

[0251] Example 3: Post-processing of target DNA sequences for complex sequence or RNA synthesis.

[0252] 3.1 Design of surrogate ds polynucleotides

[0253] In this example, ds polynucleotides were synthesized, and the workflow generally includes ambiguous steps, such as self-complementary oligonucleotide dimers (e.g., Figure 5 A). Since such self-complementary dimers had to be excluded from the workflow to avoid unwanted runaway reactions, the template sequence was designed by replacing the self-complementary elements with different bases in a way that the resulting assembly workflow was well defined. Based on this template, a proxy ds polynucleotide was synthesized.

[0254] exist Figure 5 In A, the target sequence is described. The underlined parts represent those parts of the sequence that are capable of self-complementarity and self-polymerization. To avoid these sequences, a template sequence containing two base pair modifications spanning three oligonucleotides was designed ( Figure 5 B).

[0255] As shown in Example 2, a proxy ds polynucleotide was synthesized by the methods described herein. The proxy sequence was chosen to be identical to the oligonucleotides O- and V+ of Example 2, and therefore, its synthesis was performed exactly as described above.

[0256] Once the proxy ds polynucleotides are synthesized, ds polynucleotides having the same sequence as the target sequence are generated as follows: The principle of site-directed mutagenesis is applied, i.e., after PCR amplification, the part of the target sequence excluded in the synthesized proxy ds polynucleotide is replaced with the original target sequence.

[0257] After synthesis, the 128 bp proxy ds polynucleotide was purified and prepared for PCR reaction. In this reaction mixture, not only the 3' terminal primer was included, but also a pair of "mutagenous primers" (AttB). These mutagenic primers had ten nucleotides on either side of the mutagenic element (three bases in this example) that completely overlapped with the proxy sequence. According to these provisions, standard PCR was performed to retrieve ds polynucleotides with the same sequence as SOI (Sambrook and Russell, 2014; Chapter 13), by using, in this example, a commercial kit with standardized reaction conditions and reagents (Taq PCR kit, New England Biolabs, product number E5000S).

[0258] 3.2 RNA production

[0259] RNA molecules with a given target sequence must also be generated using a proxy ds polynucleotide. This is done in two steps. First, the reverse complement sequence of the RNA sequence of interest (i.e., the DNA sequence) must be calculated. The DNA sequence is the sequence that will be synthesized. Second, a specific promoter sequence is incorporated into the template DNA sequence so that it can be recognized by a DNA-dependent enzyme that subsequently transcribes the DNA into RNA (Rio, 2011). In this example, we used the T7 RNA polymerase I system.

[0260] The necessary steps are:

[0261] A. Design of DNA template For a given target RNA sequence, its DNA reverse complement sequence was calculated, which included the T7 RNA pol promoter sequence TAATACGACTCACTATAG (SEQ ID NO: 24) at the 5' end of the reverse complement sequence.

[0262] B. Synthesis of Proxy ds Polynucleotides. As described in Example 2, a proxy dsDNA polynucleotide was synthesized according to the DNA template of step 3.2.A (see also Examples 1 and 3.1). After the proxy DNA was synthesized, its ends were modified to generate blunt ends. The ss overhangs were flattened by incubation with 1 unit / μg of E. coli polymerase I Large Klenow fragment at 25°C for 15 minutes in the presence of 33uM of each dNTP, and inactivated by adding ETDA 10mM and heating at 75°C for 20 minutes (purchased from New England Biolabs, product number M0210; Sambrook and Russell, 2014; Chapter 12). Next, the proxy ds polynucleotide was purified, amplified and purified again: the following RNA synthesis reaction requires a minimum of 1μg of DNA.

[0263] C. RNA transcription, post-treatment and purification. Follow the standard protocol for RNA transcription (e.g., HiScribe T7ARCA mRNA kit, New England Biolabs, product number E2060, and several others), which includes synthesizing RNA by surrogate DNA. In order to synthesize RNA by surrogate DNA, the following protocol was applied:

[0264] RNA molecules were generated by dissolving 1-3ug DNA in a solution consisting of 2μL 2X rNTP mix, 2μL T7 RNA polymerase mix, and 18μL nuclease-free water, followed by incubation at 37°C for 30 minutes. The reaction was terminated by adding 2μL DNase and incubating at 37°C for 15 minutes to digest the template DNA, and the resulting RNA was purified using a spin column as described in the previous example.

[0265] Example 4: Synthesis of 608 bp target DNA molecule

[0266] In this example, it is demonstrated how to synthesize a 608 bp target ds polynucleotide (SOI is the sequence "Ribbon_test_608", SEQ ID NO: 26) using the methods provided herein. The oligonucleotide is part of the library generated in Example 1. The oligonucleotide has the same properties as in Example 2.

[0267] Oligonucleotides were prepared in an asymmetric manner in the reaction plate to obtain partial constructs of different sizes at the fourth ligation. A 608 bp sequence was obtained by completing four rounds of ligation to obtain one 128 bp reaction product and three 160 bp reaction products, which were then purified and subjected to two more rounds of ligation to obtain 608 bp per strand of the target ds polynucleotide.

[0268] 4.1 Preparation of annealing solution

[0269] Prepare 864 μL of a master mix of annealing solution consisting of 772 μL ddH2O and 92 μL T4 ligase buffer. Dispense 21.6 μL of this solution mix into 38 microwells. Transfer 0.7 μL of each oligonucleotide (150 μM) to the designated microwells of the plate and mix by pipetting.

[0270] Partially complementary ss oligonucleotides are from the library of Example 1 and are placed in specific wells of a 96-well plate, such as Figure 6 For simplicity, the oligonucleotides are named according to the position on the plate where they are placed for annealing. As in Example 2, the leading and lagging strands are indicated by + and - superscripts, respectively; see the sequences with SEQ ID NOs: 27 to 102 in FASTA format. Note that the wells in rows EG, columns 2-7 are intentionally left empty.

[0271] 4.2 Annealing

[0272] Annealing was performed as in Example 2.

[0273] 4.3 Preparation of ligation solution

[0274] The ligation solution was prepared similarly to Example 2, but the amount was adjusted to 80 μL, sufficient for 38 reaction wells, namely: 7.2 μL nuclease-free ddH2O, 8 μL ligase buffer, 40 μL ATP, after vortexing, 24.8 μL T4 ligase, mixed by pipetting.

[0275] 2 μL of the resulting solution was transferred using a dispenser to each of the 38 reaction wells in B in preparation for ligation, followed by gentle mixing using a multichannel pipette.

[0276] 4.4 Front four wheel connection

[0277] For the first round of ligation, transfer the entire contents from the wells in rows A and C of columns (1-7) to rows B and D, and from wells E1 and G1 to F1 and H1, respectively. Transfer with a multichannel pipette and mix gently. This protocol is equivalent to Example 2: transfer the leftmost contents to the rightmost well. Seal the plate and incubate the reaction mixture in a thermal cycler at 16°C for at least 1 hour. Note that rows E to G of rows 2-7 remain empty.

[0278] For the second round of ligation, open the plate and transfer the entire contents from the wells in row B of columns (1-7) to row D and from well F1 to H1 by pipetting and mix. Seal the plate again and incubate at 16°C for at least one hour.

[0279] For the third round of ligation, the plate was opened and the entire contents were transferred by pipetting from the wells in row D of columns 1-7 to row H and then mixed. The plate was sealed again and incubated at 16°C for at least one hour.

[0280] For the fourth round of ligation, the plate was opened and the entire contents were transferred from wells H2, H4 and H6 to wells H3, H5 and H7 respectively by pipetting and then mixed. Note that well H1 remained unchanged. The plate was sealed again and incubated at 16°C for at least one hour.

[0281] 4.5 Intermediate Purification

[0282] Prepare three kinds of agarose gels as in Example 2 Part E, with 7 lane combs, including 50bp ladder bands. The contents in the H1 hole from Part D are distributed in six lanes of the gel (33 μ L for each lane). Contents H3, H5 and H7 are distributed in three lanes of each of the other two gels (41 μ L for each lane). Run the gel as described in Example 2 Part E, then excise the band as required (128 for the 2-4 lanes of gel 1, and 80bp for the remaining lanes of gel 1 and gel 2). Purify as described in Example 2 Part E, merge in the same purification column sample containing the same synthon. Use 10 μ L ddH2O (as shown in the Zymoclean purification kit) to elute each of the 4 samples, warm at 35 ℃ to improve elution efficiency. The contents are transferred to the PCR reaction tube strips, and are labeled as S1 to S4.

[0283] 0.5 μL samples of S1 and S4 were taken and diluted in 0.5 μL ddH2O. These samples were used to estimate the DNA concentration by spectrophotometry at 260 nm (nanodrop 2000, Thermo Fisher Scientific), yielding 1.52 μg / μL and 1.98 μg / μL, respectively. It was assumed that the molar concentration ranges of samples S2 and S3 were similar.

[0284] 4.6 Preparation of Ligation Solution

[0285] Place samples on ice. Add 0.5 μL ddH2O to samples S1 and S4 (to compensate for the 0.5 μL used for measurement in Part E). Prepare ligation reactions by adding 1.14 μL ligase buffer to each sample. Add 0.3 μL T4 ligase to S1 and S3. Mix the solutions by pipetting.

[0286] 4.7 The last two rounds of connection

[0287] For the fifth round of ligation reaction, transfer the entire contents from tubes 1 and 3 to tubes 2 and 4, respectively, by pipetting and then mix. Close the tubes. Incubate the reactions in a thermal cycler at 16°C for 80 minutes.

[0288] For the last round of ligation, transfer the entire contents from tube 2 to tube 4 separately by pipetting and mix. Close the tubes. Incubate the reactions in a thermal cycler at 16°C for 80 minutes. This completes the hierarchical synthesis process.

[0289] 4.8 Final purification

[0290] Purify through a 2% agarose gel using an 8-lane comb. As in Example 2, Part E, the first lane contains a 50 bp ladder. Mix the complete sample with 10 μL of purple loading mold without SDS and dispense into a single lane. Run the gel at 100 V, 200 mA, 12 W for 45 minutes. Figure 7 The resulting gel is shown. The upper band corresponding to the expected size of 608 bp was excised and purified by Zymo Gel Extraction Kit as in Example 2, Part E, using 20 μL ddH2O water warmed to 35°C. If 0.5 μL of this sample was used, the solution was estimated to contain 10 ng / μL by spectrophotometry.

[0291] 4.9 Sequencing

[0292] The solution was divided into two samples, one of about 10 μL and one of 9.5 μL. Primers ("primer 1" and "primer 2") were added to each solution and sequenced using the Sanger method. The sequencing results in the central reliable region confirmed that the target ds polynucleotide had complete sequence identity with SOI.

[0293] Example 5: Synthesis of 10,000 bp DNA molecules

[0294] In this example, based on the library design of Example 1, the construction of a ds polynucleotide consisting of a 10,000 bp target sequence is demonstrated by using a 26 bp oligonucleotide that forms a ds dimer with 4 nucleotide overhangs.

[0295] 5.1. Sequence processing

[0296] A. Calculate the reverse complement sequence of the leading strand of the target sequence and remove the last 4 nucleotides at the 3' end in both sequences (leading strand and reverse complement sequence). This generates two single-stranded template sequences, one corresponding to the leading strand of SOI and the other corresponding to the reverse complement sequence of SOI, minus the 4 nucleotides at the 3' end.

[0297] B. The sequences of the two ss templates are aligned to obtain the double-stranded template sequence, which is then divided into shorter sequences (called oligonucleotide subsets or subsequences) that appear in the oligonucleotides contained in the library, and their positions in the library are annotated with numbers.

[0298] C. Define a workflow that allows for unambiguous assembly of the subsequences identified in step B.

[0299] 5.2. Reactions

[0300] Unless otherwise stated, all the following steps were performed at 16 °C and all solutions were prepared and kept on ice.

[0301] A. Prepare 700 μL of 2X Ligase Buffer in ddH2O and dispense 1.8 μL of this master mix into each well of a 384-well microplate.

[0302] B. Extract 0.1 μL of each oligonucleotide library member (which corresponds to a subsequence of the ss template sequence (determined in 4.1, step B), which is the leading strand of SOI minus the 4 nucleotides at the 3' end) from the library in the order of appearance in the target sequence, and dispense into the wells of a 348-well microplate, starting with wells A1, B1, ... P1, and then proceeding to subsequent columns A2, B2, etc. until all oligonucleotides are dispensed into the wells.

[0303] C. Extract 0.1 μL of each oligonucleotide library member (which corresponds to a subsequence of the ss template sequence (determined in 4.1 step B), which is the reverse complement of SOI minus 4 nucleotides at the 3' end) in reverse sequence order and dispense into the microplate in step B, starting again from well A1 until all oligonucleotides have been dispensed into the wells. At this point, each microwell contains two oligonucleotides with 22 complementary bp and an overhang consisting of 4 nucleotides. In summary, the wells should now contain matched pairs of oligonucleotide library members.

[0304] D. Seal the microplate and anneal in a thermal cycler, starting at 95°C and decreasing to 16°C at a rate of 1°C / min.

[0305] E. Prepare 800 μL of a ligation reaction solution containing a master mix of T4 ligase in ddH2O at a concentration of 20 viscosity units / μL and dispense 2 μL of this solution into each of the 384 wells of the plate.

[0306] F. Centrifuge the plate by pulse at 1000 g in a centrifuge.

[0307] G. The rows containing the solution are listed using the following formula: 2 t-1 k, where t is the number of layers, t = 1, 2, 3, 4, and k is the index of the row of filled holes, r = k = 1, ..., 16 / 2 t-1 In this way, in the first level, all rows are enumerated, in the second level, only half, and so on.

[0308] H. Use a multichannel micropipette or liquid handler to transfer the contents of the wells in each odd-indexed row to the wells in the rightmost column of even-indexed columns.

[0309] I. Immediately after transferring the contents, gently mix the solution by direct pipetting using a micropipette or processor.

[0310] J. Incubate the reaction for 60 minutes to allow the ligation reaction to complete.

[0311] K. Repeat steps GJ four more times until only the last row (P) of the microplate is filled, leaving a total of 24 filled wells.

[0312] L. Transfer the contents of each of the 24 wells (containing 48 μL) into 24 reaction tubes and prepare for purification in a column according to the Monarch PCR & DNA Cleanup Kit (Product No. T1030) from New England Biolabs to obtain 6 μL of a purified solution containing only intermediate reaction products exceeding 100 bps.

[0313] M. Transfer the purified solution to three fresh strips of 8 PCR tubes and arrange them in 8 rows x 3 columns.

[0314] N. Take 17.5 μL of the solution from step E and add 7.5 μL of ligase buffer (10X) to make a final concentration of 7X and dispense 1 μL of this solution into each test tube.

[0315] O. The reaction was carried out three more times in the same manner as steps HJ to obtain three filled tubes (one in each column of the last row).

[0316] P. The contents of column 1 are transferred to column 2, while column 3 remains unchanged.

[0317] Q. Incubate the reaction for 1 hour.

[0318] R. Transfer the contents of column 2 to column 3.

[0319] S. Incubate the reaction for 1 hour.

[0320] T. Prepare a 0.8% agarose gel and load the samples along with a 10 kbp ladder. Run the gel at 100 V for 45 minutes.

[0321] U. Extract and purify the band corresponding to 10K bp from the gel slice using standard protocols and kits (Zymo clean is recommended in this example, see also Example 1).

[0322] 5.3. Completion and expansion

[0323] A. Two 26 bp long oligonucleotides were selected from the library that are complementary to the last 26 nucleotides at the 3' end of the SOI, i.e. they also include the 4 nucleotides deleted in step A of point 4.1. These two oligonucleotides are used as primers in a PCR reaction that is ready to amplify the final product and add the remaining 4 bps to each strand to complete the 10,000 bp sequence with blunt ends.

[0324] B. Purify the PCR product using a standard kit as described in step L of point 4.2 to remove residual oligonucleotides, enzymes and reagents, leaving the final DNA product, i.e., a ds polynucleotide with the same sequence as SOI, ready for downstream applications.

[0325] References

[0326] Anderson,S.,Bankier,A.T.,Barrell,B.G.et al.(1981)Sequence andorganization of the human mitochondrial genome.Nature,290:457-465.

[0327] Beaucage,S.L.and Caruthers,M.H.(1981)Deoxynucleosidephosphoramidites-a new class of key intermediates for deoxypolynucleotidesynthesis.Tetrahedron Letters 22:1859-1862.

[0328] Bentley,D.R.,et al.(65authors)(2008)Accurate Whole Human GenomeSequencing using Reversible Terminator Chemistry.Nature,456:53-59.

[0329] Bonde,M.T.,Kosuri,S.,Genee,H.J.,Sarup-Lytzen,K.,Church,G.M.,Sommer,M.O.A.and Wang H.H.(2014)Direct Mutagenesis of Thousands of Genomic TargetsUsing Microarray-Derived Oligonucleotides.ACS Synthetic Biology 4(1):17-22.

[0330] Chari,R.and Church,G.M.(2017)Beyond editing to writing largegenomes.Nature Reviews Genetics,In Press.

[0331] Engler,C.,Kandzia,R.and Marillonnet,S.(2008)A one pot,one step,precision cloning method with high through put capability.PloS One 3(11):e3647.

[0332] Farzadfard,F.and Timothy,K.L.(2014)Genomically Encoded Analog Memorywith Precise in Vivo DNA Writing in Living Cell Populations.Science 346(6211):1256272.

[0333] Gao,X.,LeProust,E.M.,Zhang,H.,Srivannavit,O.Gulari,E.,Yu,P.,Nishiguchi,C.,Xiang,Q.and Zhou,X.(2001)A Flexible Light-Directed DNA ChipSynthesis Gated by Deprotection Using Solution Photogenerated Acids.NucleicAcids Research 29(22):4744-50.

[0334] Gibson,D.G.,Young,L.,Chuang,R.Y.,Venter,J.C.,Hutchison III,C.A.andSmith,H.O.(2009)Enzymatic assembly of DNA molecules up to several hundredkilobases.Nature Methods,6(5):343-345.

[0335] Horspool,D.R.,Coope,R.J.N.and Holt,R.A.(2010)Efficient assembly ofvery short oligonucleotides using T4 DNA Ligase.BMC Research Notes,3291-299.

[0336] Kai,J.,Puntambekar A.,Santiago N.,Lee S.H.,Sehy D.W.,Moore V.,HanJ.and Ahn C.H.(2012)A novel microfluidic microplate as the next generationassay platform for enzyme linked immunoassays(ELISA).Lab Chip,12(21):4257-62

[0337] Kemp,G.(1998)Capillary electrophoresis:a versatile family ofanalytical techniques.Biotechnology and Applied Biochemistry 27:9-17.

[0338] Lehman,I.R.and Nussbaum,A.L.(1964)The deoxyribonucleases ofEscherichia coli.V.On the specificity of exonuclease I(phosphodiesterase),Journal of Biological Chemistry,239:2628-2636.

[0339] LeProust,E.M.,Peck,B.J.,Spirin,K.,McCuen,H.B.,Moore,B.,Namsaraev,E.,and Caruthers,M.H.(2010)Synthesis of high-quality libraries of long(150mer)oligonucleotides by a novel depurination controlled process.Nucleic AcidsResearch,38(8),2522-2540.

[0340] Neuner,P.,Cortese,R.and Monaci,P.(1998)Codon-Based Mutagenesis UsingDimer-Phosphoramidites.Nucleic Acids Research 26(5):1223-27.

[0341] Rio, D.C. (2011). RNA: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.

[0342] Sambrook, J., and Russell, D.W. (2014). Molecular Cloning. A Laboratory Manual. (3rd ed.). New York: Cold Spring Harbor Laboratory Press.

[0343] Smith H.O., Hutchison III, C.A., Pfannkoch, C. and Venter J.C. (2003) Generating a synthetic genome by whole genome assembly: X174 bacteriophage from synthetic oligonucleotides. Proceedings of the Natural Academy of Sciences of the USA, 100(26): 15440 - 15445.

[0344] Sondek, J., and Shortle, D. (1992). A General Strategy for Random Insertion and Substitution Mutagenesis: Substoichiometric Coupling of Trinucleotide Phosphoramidites. Proceedings of the National Academy of Sciences 89(8): 3581 - 85.

[0345] Stemmer, W.P., Crameri, A., Ha, K.D., Brennan, T.M. and Heyneker, H.L. (1995) Single - step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene, 1614: 49 - 53. Sequence Listing <110> Riben Biolabs LLC <120> A novel method for synthesizing polynucleotides using a diverse library of oligonucleotides <130> RB001P <160> 104 <170> BiSSAP 1.3 <210> 1 <211> 128 <212> DNA <213> Artificial sequence <220> <223> Polynucleotide <400> 1 tttcttcttt cgagtttcta tatccgtcgc tggtctgaac ggaaaaatca tcgcacaatc 60 tatgcctacc gttggctgct catgcgtcct tccgacaatc ccatgttcgt ctcgcatccg 120 tttcctgc 128 <210> 2 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 2 tttcttcttt cgagtt 16 <210> 3 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 3 tagaaactcg aaagaa 16 <210> 4 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 4 tctatatccg tcgctg 16 <210> 5 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 5 agaccagcga cggata 16 <210> 6 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 6 gtctgaacgg aaaaat 16 <210> 7 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 7 gatgattttt ccgttc 16 <210> 8 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 8 catcgcacaa tctatg 16 <210> 9 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 9 taggcataga ttgtgc 16 <210> 10 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 10 cctaccgttg gctgct 16 <210> 11 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 11 catgagcagc caacgg 16 <210> 12 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 12 catgcgtcct tccgac 16 <210> 13 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 13 gattgtcgga aggacg 16 <210> 14 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 14 aatcccatgt tcgtct 16 <210> 15 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 15 tgcgagacga acatgg 16 <210> 16 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 16 cgcatccgtt tcctgc 16 <210> 17 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 17 gcgtgcagga aacgga 16 <210> 18 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Polynucleotide <400> 18 cctaccgttg gctgctaatc cgtccttccg acaatc 36 <210> 19 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Polynucleotide <400> 19 ggcaaccgac gattaggcag gaaggctgtt ag 32 <210> 20 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Polynucleotide <400> 20 cctaccgttg gctgctcatg cgtccttccg acaatc 36 <210> twenty one <211> 32 <212> DNA <213> Artificial sequence <220> <223> Polynucleotide <400> twenty one ggcaaccgac gagtacgcag gaaggctgtt ag 32 <210> twenty two <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Polynucleotide <400> twenty two gttggctgct aatccgtcct tccg 24 <210> twenty three <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Polynucleotide <400> twenty three cggaaggacg catgagcagc caac 24 <210> twenty four <211> 18 <212> DNA <213> Artificial sequence <220> <223> Polynucleotide <400> twenty four taatacgact cactatag 18 <210> 25 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 25 aagaaagctc aaagat 16 <210> 26 <211> 608 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide <400> 26 tttcttcttt cgagtttcta tatccgtcgc tggtctgaac ggaaaaatca tcgcacaatc 60 tatgcctacc gttggctgct catgcgtcct tccgacaatc ccatgttcgt ctcgcatccg 120 tttcctgcac gcaccccccc ctgtactttg gaaagcggcc atcttaacac tctcccaact 180 ttttaaatgc gtcgaagccc tgggcatctg gtttccacta gcctagtcgg gtgttggata 240 cgccgagagt tatggtgtag ctgtgtgcgc gaaccgacgg gtggaaattg ctgaccgatt 300 ttcaaatagt tctcaggaag ccgatggcag ttacggcttg cgactcgggg caccgtgagc 360 ctcttctccc tctagaagtc gaagcaaggg acactatcct aaatgccatg gactagcgcg 420 cgcgaaatcg atgcactcct tattaatgtg atctgcgcaa gttgttcagc catcggtcat 480 tttgcgttga tattcggttc tttgatttgc gtgccatgct tataacagga cacttattgt 540 gccccagctc ttctcatgca agtgcggttt tctctagcta ctgtggtgtg cgctcatcaa 600 tactccag 608 <210> 27 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 27 tttcttcttt cgagtt 16 <210> 28 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 28 tagaaactcg aaagaa 16 <210> 29 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 29 tctatatccg tcgctg 16 <210> 30 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 30 agaccagcga cggata 16 <210> 31 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 31 gtctgaacgg aaaaat 16 <210> 32 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 32 gatgattttt ccgttc 16 <210> 33 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 33 catcgcacaa tctatg 16 <210> 34 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 34 taggcataga ttgtgc 16 <210> 35 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 35 cctaccgttg gctgct 16 <210> 36 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 36 catgagcagc caacgg 16 <210> 37 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 37 catgcgtcct tccgac 16 <210> 38 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 38 gattgtcgga aggacg 16 <210> 39 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 39 aatcccatgt tcgtct 16 <210> 40 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 40 tgcgagacga acatgg 16 <210> 41 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 41 cgcatccgtt tcctgc 16 <210> 42 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 42 gcgtgcagga aacgga 16 <210> 43 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 43 acgcaccccc ccctgt 16 <210> 44 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 44 aagtacaggg gggggt 16 <210> 45 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 45 actttggaaa gcggcc 16 <210> 46 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 46 agatggccgc tttcca 16 <210> 47 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 47 atcttaacac tctccc 16 <210> 48 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 48 agttgggaga gtgtta 16 <210> 49 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 49 aactttttaa atgcgt 16 <210> 50 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 50 ttcgacgcat ttaaaa 16 <210> 51 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 51 cgaagccctg ggcatc 16 <210> 52 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 52 accagatgcc cagggc 16 <210> 53 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 53 tggtttccac tagcct 16 <210> 54 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 54 gactaggcta gtggaa 16 <210> 55 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 55 agtcgggtgt tggata 16 <210> 56 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 56 ggcgtatcca acaccc 16 <210> 57 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 57 cgccgagagt tatggt 16 <210> 58 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 58 ctacaccata actctc 16 <210> 59 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 59 gtagctgtgt gcgcga 16 <210> 60 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 60 cggttcgcgc acacag 16 <210> 61 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 61 accgacgggt ggaaat 16 <210> 62 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 62 agcaatttcc acccgt 16 <210> 63 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 63 tgctgaccga ttttca 16 <210> 64 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 64 tatttgaaaa tcggtc 16 <210> 65 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 65 aatagttctc aggaag 16 <210> 66 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 66 tcggcttcct gagaac 16 <210> 67 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 67 ccgatggcag ttacgg 16 <210> 68 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 68 caagccgtaa ctgcca 16 <210> 69 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 69 cttgcgactc ggggca 16 <210> 70 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 70 acggtgcccc gagtcg 16 <210> 71 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 71 ccgtgagcct cttctc 16 <210> 72 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 72 gagggagaag aggctc 16 <210> 73 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 73 cctctagaag tcgaag 16 <210> 74 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 74 cttgcttcga cttcta 16 <210> 75 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 75 caagggacac tatcct 16 <210> 76 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 76 atttaggata gtgtcc 16 <210> 77 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 77 aaatgccatg gactag 16 <210> 78 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 78 cgcgctagtc catggc 16 <210> 79 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 79 cgcgcgcgaa atcgat 16 <210> 80 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 80 gtgcatcgat ttcgcg 16 <210> 81 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 81 gcactccttattaatg 16 <210> 82 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 82 atcacattaa taagga 16 <210> 83 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 83 tgatctgcgc aagttg 16 <210> 84 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 84 tgaacaactt gcgcag 16 <210> 85 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 85 ttcagccatc ggtcat 16 <210> 86 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 86 caaaatgacc gatggc 16 <210> 87 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 87 tttgcgttga tattcg 16 <210> 88 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 88 gaaccgaata tcaacg 16 <210> 89 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 89 gttctttgat ttgcgt 16 <210> 90 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 90 tggcacgcaa atcaaa 16 <210> 91 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 91 gccatgctta taacag 16 <210> 92 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 92 tgtcctgtta taagca 16 <210> 93 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 93 gacacttatt gtgccc 16 <210> 94 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 94 gctggggcac aataag 16 <210> 95 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 95 cagctcttct catgca 16 <210> 96 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 96 cacttgcatg agaaga 16 <210> 97 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 97 agtgcggttt tctcta 16 <210> 98 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 98 tagctagaga aaaccg 16 <210> 99 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 99 gctactgtgg tgtgcg 16 <210> 100 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 100 tgagcgcaca ccacag 16 <210> 101 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 101 ctcatcaata ctccag 16 <210> 102 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 102 ttatctggag tattga 16 <210> 103 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 103 tttcttcttt cgagtttcta tatccgtcgc tg 32 <210> 104 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 104 ttatctggag tattgatgag cgcac 25

Claims

1. A method for synthesizing a target double-stranded (ds) polynucleotide having a predetermined sequence, comprising: a) providing an oligonucleotide library within an array device, the oligonucleotide library comprising a diversity of oligonucleotide library members, wherein each library member has a different nucleotide sequence and is contained in separate library containers in an aqueous solution, the diversity comprising single-stranded oligonucleotides (ss oligonucleotides) and double-stranded oligonucleotides (ds oligonucleotides) having at least one overhang and covering at least 10,000 pairs of matched oligonucleotides, wherein the ss oligonucleotides are 6 to 26 nt in length, and wherein the ds oligonucleotides are 6 to 26 bp in length, wherein the individual library containers are spatially arranged in a three-dimensional order, wherein each container is located within the device at a defined coordinate within the x-, y- and z-axes, the order being specifically determined by the frequency of use, placing those oligonucleotides in close proximity to one another, those oligonucleotides often forming matching pairs in naturally occurring DNA sequences, said oligonucleotides being oligonucleotides that occur naturally or are commonly used in target ds polynucleotides or fragments thereof, b) in a first step, transferring at least a first pair of matched oligonucleotides from the library into a first reaction vessel using a liquid handler and assembling the matched oligonucleotides to obtain a first reaction product comprising at least one overhang, c) in second and further steps, transferring at least second and further pairs of matched oligonucleotides from the library into second and further reaction vessels, respectively, using a liquid handler, and assembling the matched oligonucleotides, thereby obtaining second and further reaction products, each comprising at least one overhang, respectively, d) assembling the first, second and other reaction products in a predetermined workflow to generate the target ds polynucleotide having overhangs, wherein an algorithm is used to determine the matched oligonucleotide pairs used in steps b) and c) and a hierarchical assembly workflow to generate the target ds polynucleotide, and The same oligonucleotide library is used to synthesize a series of different target ds polynucleotides.

2. The method of claim 1, wherein the sequence identity of the different target ds polynucleotides is less than 50%.

3. The method of claim 1 or 2, wherein the overhangs of the ds oligonucleotides do not exceed half the length of the respective ds oligonucleotides.

4. The method according to claim 1, wherein the diversity comprises ss oligonucleotides and / or ds oligonucleotides modified by any one or more of phosphorylation, methylation, biotinylation or linkage to a fluorophore or a quencher. The method of claim 1 , wherein the target ds polynucleotide is at least 48 bp in length. The method according to claim 1 , wherein the assembling is performed by a ligation reaction, which is an enzymatic and / or chemical reaction. The method according to claim 6 , wherein the ligation reaction is an enzymatic ligation reaction using a ligase or a polymerase or a ribozyme. The method according to claim 7 , wherein the ligase is T3, T4 or T7 DNA ligase.

9. The method of claim 1, wherein the assembly is performed directly by hybridizing matching overhangs or indirectly by hybridizing a suitable ss oligonucleotide adapter, the ss oligonucleotide adapter being an ss oligonucleotide contained in the library, which is selected from the library and transferred to assemble any of the first, second or other reaction products.

10. The method of claim 1, wherein the nucleotide sequence of the target ds polynucleotide is identical to that of the template.

11. The method according to claim 1, wherein the target ds polynucleotide is further modified by any of site-directed mutagenesis, endonuclease or exonuclease to obtain the polynucleotide having a target sequence.

12. The method of claim 1, wherein the target ds polynucleotide is further modified to produce a derivative of the target ds polynucleotide, the derivative being any one of a ds DNA, ss DNA or RNA molecule.

13. The method according to claim 12, wherein the modification method is any enzymatic modification, using any of methyltransferase, kinase, CRISPR / Cas9, multiplex automated genome engineering (MAGE), conjugated assembly genome engineering (CAGE), Argonaute protein (Ago) or its derivatives, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), hybrid molecule, sulfurylase, recombinase, nuclease, DNA polymerase, RNA polymerase or TNase.

14. The method of claim 1, wherein the three-dimensional sequence comprises at least two at least partially stacked library containers.

15. The method of claim 1, wherein the array device is any of a microtiter plate, a microfluidic microplate, a capillary array, a microarray, or a biochip. The method according to claim 15 , wherein the biochip is a DNA or RNA biochip.

17. The method of claim 1, wherein the target ds polynucleotide is sequenced to verify the degree of sequence identity with a template or target sequence.

18. An oligonucleotide library provided in an array device, comprising a diversity of library members covering at least 10,000 pairs of matched oligonucleotides comprising partially complementary sequences, wherein the library members are single-stranded oligonucleotides (ss oligonucleotides) or double-stranded oligonucleotides (ds oligonucleotides) having at least one overhang, wherein each library member has a different nucleotide sequence and is contained in separate library containers in an aqueous solution, wherein the containers are spatially arranged in a three-dimensional order, wherein each container is located in the device at defined coordinates within the x-, y- and z-axes, the order being specifically determined by the frequency of use, placing those oligonucleotides in close proximity to each other, those oligonucleotides often forming matching pairs in naturally occurring DNA sequences, and the oligonucleotides are oligonucleotides that occur naturally or are commonly used in target ds polynucleotides or fragments thereof.

19. The oligonucleotide library according to claim 18, wherein the three-dimensional order comprises at least two at least partially stacked library containers.

20. Use of the oligonucleotide library according to claim 18 or 19 for synthesizing a series of different target double-stranded (ds) polynucleotides having a predefined sequence, wherein the sequence identity of the different target double-stranded (ds) polynucleotides is less than 50%.

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