A method for biosynthesizing an oligo pool and its application

Abstract

The present invention discloses a biosynthesis method and application of an oligo pool, including the steps of 1) fixing the probe, combining a solid phase carrier with a primer chain; 2) distributing the solid phase carrier as needed, distributing the solid phase carrier or the deprotected product as needed into M tubes according to the volume, where M is selected from any natural number of 1, 2, 3, and 4; 3) chain extension, adding a reaction solution to an enzymatic DNA synthesis system; 4) closing the tubes; 5) deprotection; 6) repeating the above steps 2)-step 5), cumulatively performing step 5) N times, where N is selected from any non-zero natural number; 7) sampling, quality control, and application. Based on the technology of DNA enzymatic synthesis, microspheres are used as solid phase carriers to regulate the types of bases synthesized in each round to control the sequence diversity of the final oligo pool.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA synthesis. Specifically, the present invention relates to a method for biosynthesizing an oligo pool and its application. Background Art

[0002] In recent years, synthetic biology has developed rapidly, and its position in the field of biotechnology has become increasingly prominent. DNA synthesis is one of the most important supporting technologies in the field of synthetic biology. Gene synthesis can not only replicate genes that already exist in nature, but also greatly expand our ability to manipulate genes and proteins through codon optimization and customized modification of genes. On the other hand, the technology for preparing an oligo pool, that is, a technology for a mixture of oligonucleotides containing a large number of specific sequences, enables the exploration or manipulation of a large amount of genetic information in a single experiment in genomics, proteomics, metabolomics, and cell biology research, and has become an indispensable tool in fields such as modern molecular biology, genetic engineering, precision medicine, and synthetic biology.

[0003] As a key fundamental technology in synthetic biology, DNA synthesis is as important as the support of sequencing technology for genomics. Currently, the mainstream technology for DNA synthesis is chemical synthesis. This technology began in the 1950s and 1960s. Subsequently, from the 1960s to the 1970s, the chemical synthesis method of oligonucleotides was continuously improved. In the 1980s, Beaucage and Caruthers developed the phosphoramidite-based DNA synthesis method, which gradually developed into the column synthesis method. This method uses more than a dozen chemical reagents and gradually synthesizes DNA through four steps: deprotection, coupling, capping, and oxidation. Due to the limitations of the chemical synthesis method, obtaining a DNA library with a controllable ratio requires synthesizing a large number of specific DNA sequences and mixing them according to requirements, and it is impossible to precisely adjust the ratio of ATGC in each round through a one-step method.

[0004] Breaking through the traditional chemical organic-phase synthesis system, the enzyme-catalyzed DNA synthesis technology based on a mild aqueous-phase system and independent of templates can achieve the in-situ synthesis of oligo pools. With its advantages in length, precision, flexibility, cost-efficiency, environmental friendliness, and automation, the enzyme-catalyzed DNA synthesis technology is gradually becoming a frontier trend in the field of DNA synthesis, enabling its rapid development in fields such as molecular diagnostics, high-throughput screening, protein engineering, mutant library construction, and single-cell sequencing. In molecular diagnostic systems, the biosynthesis method of oligo pools enables the rapid detection of pathogens such as viruses and bacteria by providing highly specific oligonucleotides. These oligonucleotides can serve as primers for PCR or other amplification techniques to achieve the rapid identification and quantification of pathogen DNA. In high-throughput screening systems, for example, the application of oligo pools has greatly promoted the development of gene editing and CRISPR-Cas9 technologies. By synthesizing sgRNA libraries through high-throughput methods, researchers can efficiently conduct gene function studies and explore disease mechanisms, accelerating the development of new therapeutic methods. In the construction process of protein engineering, the oligo pool technology provides an efficient means for constructing mutant libraries by synthesizing oligonucleotide pools containing specific mutations, enabling researchers to quickly test multiple protein variants and thus optimize protein function and stability. In the process of mutant library construction, the biosynthesis method of oligo pools provides strong technical support for studying gene function and screening beneficial mutations by synthesizing oligonucleotide pools containing different mutations. This method not only improves the construction efficiency of mutant libraries but also provides a new perspective for understanding the relationship between protein structure and function. Single-cell sequencing can sequence hundreds or thousands of cells simultaneously. Oligo pools can efficiently synthesize a large number of high-purity DNA fragments, which are ligated to microbeads or magnetic beads, solving many problems such as high production costs, difficult preparation, low preparation success rates, and unstable effects in different batches, accelerating the wide application of this technology. In summary, the enzyme-catalyzed DNA synthesis technology not only improves the efficiency and accuracy of biosynthesis but also provides strong technical support for fields such as molecular diagnostics, high-throughput screening, protein engineering, and mutant library construction, with broad application prospects and commercial value. Through this method, we can achieve faster and more precise gene manipulation and analysis in biomedical research and clinical diagnosis. Summary of the Invention

[0005] The first aspect protected by the present invention is a biosynthesis method of oligo pools, comprising the following steps: immobilization of probes, cyclic synthesis to regulate nucleotide sequences: on-demand distribution of solid-phase carriers, strand extension, tube combination, and deprotection.

[0006] Preferably,

[0007] 1) Probe fixation: The solid phase carrier is combined with the primer chain, and the primer chain characteristics include: the 5' end is connected to the solid carrier and the 3' end is free;

[0008] 2) The primer chain can be RNA or DNA; the primer chain can be single-stranded or double-stranded;

[0009] 3) Solid phase carrier is distributed on demand: solid phase carrier or deprotected product is distributed on demand according to volume, which is M tubes, where M is selected from any natural number of 1, 2, 3, 4;

[0010] 4) Chain extension: Add reaction solution to an enzymatic DNA synthesis system, and add a single or mixed modified nucleotide to each tube. The enzymatic DNA synthesis system contains the key enzyme terminal deoxynucleotidyl transferase or other DNA polymerase, and the 3' end of the modified nucleotide contains a reversible blocking group;

[0011] 5) Combine tubes: Vortex the solution obtained in step 4), resuspend it, put it into the same tube, mix well and remove the liquid;

[0012] 6) Deprotection: Add deprotection solution to the tube in step 5), shake and wash to obtain the deprotection product, so as to remove the reversible blocking group at the 3' end and restore the modification group at the 3' end to -OH;

[0013] 7) Cycle step 3), step 4), step 5), step 6), and execute step 6) N times cumulatively, where N is selected from any non-zero natural number; sampling, quality control, and application; if necessary, the order of step 6) and step 5) can be reversed, that is, deprotection is completed in a single tube and then the tubes are closed.

[0014] As a preferred solution, the M value in the N-times step 3) and the N-1-times step 3) is equal or different.

[0015] As a preferred solution, the solid phase carrier or the deprotected product in the above step 3) is equally divided by volume or distributed according to a ratio.

[0016] As a preferred solution, the removal of the solution in step 5) includes at least one of centrifugation, filtration, adsorption and the like.

[0017] As a preferred embodiment, the above-mentioned solid phase carrier includes a product carrier synthesized in situ on a silicon-based material, glass, resin, or metal carrier; preferably, the product synthesized in situ on a silicon-based material comprises a silicon wafer or nanowire carrier; preferably, the product synthesized in situ on glass comprises a microporous plate carrier; preferably, the product synthesized in situ on a resin comprises insoluble granular polystyrene or polyacrylate; preferably, the product synthesized in situ on a metal carrier comprises precious metals gold or silver.

[0018] Preferably, the above product includes microspheres, and the microspheres include polymer microspheres, preferably polyvinyl alcohol (PVA) or poly(lactic-co-glycolic acid) (PLGA); it also includes magnetic microspheres, preferably streptavidin magnetic beads, NHS magnetic beads, silica magnetic beads, polymer magnetic beads, agarose magnetic beads, carboxyl magnetic beads, etc.; it also includes inorganic microspheres, preferably silica microspheres or Fe 3 O 4 microspheres or TiO 2 microspheres or ZnO microspheres; it also includes natural polymer microspheres, preferably gelatin microspheres, starch microspheres or chitosan microspheres.

[0019] Preferably, the surface of the above microspheres is modified with any one of functional groups such as hydroxyl, amino, carboxyl, aldehyde, epoxy, etc.

[0020] Preferably, the particle size of the above microspheres is 300 nm - 100 μm, and preferably, the particle size of the microspheres includes at least one of 300 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm.

[0021] Preferably, in the above step 1), the primer strand and the solid support are combined by any one of amide bond formation, ester bond formation, carbon-carbon bond coupling, Schiff base reaction, biotin-streptavidin coupling, click chemical reaction, thiol-gold interaction, and through a linker molecule.

[0022] Preferably, in the above step 7), sampling: after performing step 6) N times, the deprotected products synthesized in the Nth and several previous times are resuspended in a buffer with the same volume as the solid support.

[0023] Preferably, the above modified nucleotides contain 3'-O-modified nucleotides, including nucleoside triphosphates with a 3'-O-methyl or 3'-azide or 3'-O-azidomethyl or 3'-O-amino or 3'-aminooxy or 3'-O-allyl group carried at the 3' end.

[0024] Preferably, the above 3'-O-modified nucleotides contain any nucleoside triphosphate with an ester, ether, nitrile, phosphate, carbonate, carbamate, hydroxylamine, borate, nitrate, sugar, phosphoramide, phosphoramidate, benzenesulfonate, sulfate, sulfone, or amino acid carried at the 3' end.

[0025] The second aspect protected by the present invention is to provide a molecular diagnostic system, which includes: a. using the oligo pool biosynthesis method described in any one of the first aspects to design and synthesize molecular markers for diagnosis; b. using the molecular markers selected in a for the molecular diagnosis of diseases.

[0026] The third aspect protected by the present invention is a high-throughput screening system, which includes: a. using the oligo pool biosynthesis method described in any one of the first aspects to design a library required for high-throughput screening; b. using the library in a for high-throughput screening of target molecules or compounds.

[0027] The fourth aspect protected by the present invention is a construction process of protein engineering, which includes: a. using the oligo pool biosynthesis method described in any one of the first aspects to design and construct protein variants; b. using the protein variants in a for functional analysis or optimization.

[0028] The fifth aspect protected by the present invention is that the process includes: a. using the oligo pool biosynthesis method described in any one of the first aspects to design mutations in the mutant library; b. using the mutant library in a for screening and analysis.

[0029] The fifth aspect protected by the present invention is a single-cell or spatial omics sequencing system, which uses the oligo pool biosynthesis method described in any one of the first aspects to synthesize oligonucleotides for single-cell multi-omics sequencing, targeted sequencing or spatial omics sequencing.

[0030] Compared with the prior art, the advantages of the present invention are as follows:

[0031] 1. Compared with the prior art method of synthesizing oligo pool using chemical synthesis technology, the biosynthesis method for sequencing oligo pool provided by the present invention takes into account downstream applications and is a highly novel and innovative method. That is, using enzymatic DNA synthesis technology, the key enzyme is DNA polymerase, such as terminal deoxynucleotidyl transferase (TdT), which includes the following steps: 1. Binding of microspheres to primer strands; 2. Regulating the synthesized nucleotide sequence, mainly through different base reaction systems containing blocking groups, and using the biological enzyme TdT to add blocking group-containing nucleotides at the 3' end respectively. Each round can add 1-4 modified nucleotides; 3. Mixing 1-4 tubes, removing the blocking group, restoring the 3' end modification group to -OH, and then dividing into 1-4 tubes; 4. Repeating steps 2-3. It has high accuracy and good market prospects.

[0032] 2. The biosynthesis technology provided by the present invention synthesizes a 16 nt oligo pool, and only needs to be synthesized once to obtain 4 16different sequences. In traditional chemical gene synthesis, to synthesize 4 16 = 4,294,967,296 different sequences, it is necessary to synthesize 4 16 = 4,294,967,296 times.

[0033] 3. The present invention can perform in-situ synthesis on different solid-phase carriers (silicon-based materials, glass, resin, metal) according to downstream applications. Microspheres are one of them, including polymer microspheres (such as polyvinyl alcohol PVA), magnetic microspheres (such as SA magnetic beads), inorganic microspheres (such as silica microspheres), natural polymer microspheres (such as gum beads), etc. Different solid-phase carriers can be adapted according to different application directions, greatly meeting the needs of the customer market. The probe and the solid-phase carrier can be combined through amide bond formation, ester bond formation, carbon-carbon bond coupling, Schiff base reaction, biotin-streptavidin coupling, click chemical reaction, thiol-gold interaction, through linker molecules, etc. Since the microspheres can exist in a state of solid-liquid mixing, and each microsphere has a very small volume, compared with the single carrier in the chemical synthesis method that can only synthesize 1 sequence, the microspheres can synthesize four nucleotide sequences in one cycle by the form of equal-volume averaging and mixing, greatly reducing the time, manpower and material resources required for synthesis. Therefore, the biosynthetic method provided by the present invention can not only greatly reduce the synthesis cost, but also take into account downstream applications, omitting the steps with cumbersome operations and low yields. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the enzymatic DNA quadruplex synthesis process in Example 1 of the present invention;

[0035] Figure 2 is the PAGE gel electrophoresis result of the SA magnetic bead synthesized sequence in Example 1 of the present invention;

[0036] Figure 3 is the base distribution ratio of the 16 N sample in Example 1 of the present invention;

[0037] Figure 4 is the PAGE gel electrophoresis result of the SA magnetic bead synthesized sequence in Example 2 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Unless otherwise specified, all terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0039] As used herein, the term "oligonucleotide" refers to a polymeric form of nucleotides, ribonucleotides or deoxyribonucleotides, containing natural or non-natural nucleotides, with a length of at least 2, typically from about 5 to about 200, or more commonly up to about 100. Thus, the term encompasses double-stranded and single-stranded DNA and RNA. In addition, oligonucleotides can be nuclease-resistant, including but not limited to 2'-O-methyl ribonucleotides, phosphorothioate nucleotides, dithioate nucleotides, phosphoramidate nucleotides, and methylphosphonate nucleotides.

[0040] As used herein, the term "oligo pool", also known as oligonucleotide pool, is a mixture containing thousands of short synthetic oligonucleotides (oligos) that are synthesized simultaneously. After synthesis, these oligonucleotides are removed from the synthesis platform and combined into a single tube. Oligo Pool can be used to construct various types of libraries, such as CRISPR gRNA, mutant libraries, or next-generation sequencing (NGS) target enrichment libraries for high-throughput screening.

[0041] As used herein, the term "primer strand" refers to the starting primer strand used in enzymatic DNA synthesis, which is a short DNA fragment used to initiate the synthesis of a target DNA sequence. The primer strand is a very crucial component in the PCR process. It binds complementarily to the single-stranded template DNA and provides a 3'-OH terminus, enabling DNA polymerase to start synthesizing a new DNA strand from this point. Nucleic acids containing sequences selected to be substantially complementary to each specific sequence to be amplified. The starting primer strand is (T)n, (A)n, (C)n, (G)n, where T represents deoxythymidine nucleotide (abbreviation: deoxythymidine), A represents deoxyadenosine nucleotide (abbreviation: deoxyadenosine), C represents deoxycytidine nucleotide (abbreviation: deoxycytidine), and G represents deoxyguanosine nucleotide (abbreviation: deoxyguanosine).

[0042] The above method of extending one or more natural polynucleotides with a starting primer strand, under TdT reaction conditions, provides one or more natural polynucleotides in the reaction mixture, the natural polynucleotides having a 5'-end linked to a solid support and having a free 3'-hydroxyl group at the 3'-terminal nucleotide; and extending one or more natural polynucleotides with nucleotides of a predetermined sequence by repeated cycles of the following steps: (i) contacting the natural polynucleotide or extended natural polynucleotide having a free 3'-O-hydroxyl group with a 3'-O-blocked nucleoside triphosphate and a TdT variant, such that the natural polynucleotide or extended natural polynucleotide is extended by incorporation of the 3'-O-blocked nucleoside triphosphate to form a 3'-O-blocked extended natural polynucleotide, and (ii) deblocking the extended natural polynucleotide to form an extended natural polynucleotide having a free 3'-hydroxyl group, thereby synthesizing an oligonucleotide of a predetermined sequence on the natural polynucleotide.

[0043] The above polynucleotides include modified nucleotides, such as 3'-O-modified nucleotides, including nucleoside triphosphates carrying a 3'-O-methyl, 3'-azido, 3'-O-azidomethyl, 3'-O-amino, 3'-aminooxy or 3'-O-allyl group at the 3'-terminus. For example, 3'-O-blocked nucleoside triphosphates, including those blocked by a 3'-O-azidomethyl, 3'-aminooxy or 3'-O-allyl group, and the 3'-O-blocking group has a molecular weight of 100 or less. For example, 3'-O-modified nucleotides, including nucleoside triphosphates carrying an ester, ether, nitrile, phosphate, carbonate, carbamate, hydroxylamine, borate, nitrate, sugar, phosphoramide, phosphoramidate, benzenesulfonate, sulfate, sulfone or amino acid at the 3'-terminus.

[0044] The terms "modified" and "blocked" are used interchangeably and are intended to mean a moiety covalently linked to a specific group that prevents chemical change of the group during a chemical or enzymatic process. As long as the specified group is the 3'-hydroxyl of a nucleoside triphosphate, or an extended fragment (or "extended intermediate") in which a 3'-modified / protected / blocked-nucleoside triphosphate is incorporated, the chemical change that is prevented is the further or subsequent extension of the extended fragment (or "extended intermediate") by an enzymatic ligation reaction.

[0045] As used herein, the term "terminal deoxynucleotidyl transferase (TdT)" includes reference to both the purified and recombinant forms of the enzyme. The TdT enzyme can synthesize DNA without relying on a template and can perform DNA synthesis using only single-stranded DNA, so it has the function of de novo creating genomic materials, making it a key biocatalyst for third-generation DNA synthesis. In terms of synthesis strategies, the reaction is mainly terminated by modifying nucleotides and adding reagents with specific chemical groups, or by coupling the TdT enzyme with nucleotides, etc. In the present invention, the TdT enzyme includes, but is not limited to, an enzymatic DNA synthesis kit purchased from Tianjin Zhonghe Gene Technology Co., Ltd., or an expression cassette containing all elements for expressing the TdT enzyme disclosed in Patent CN112746063B and CN110331136B. The bases can be conventional bases (A, G, C, T), unnatural nucleotides such as isocytosine and isoguanine, and their analogs (such as inosine, derivatives of purine and pyrimidine bases, such as N4-methyl deoxyguanosine, azapurines or azapyrimidines, purines or pyrimidines with substituents modified or substituted at any series of chemical positions, such as 2-amino-6-methylamino purine, O6-methyl guanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine and O4-alkyl-pyrimidine, or pyrazolone compounds, such as unsubstituted or 3-substituted pyrazolone[3,4-d]pyrimidine.

[0046] Cleavage, i.e., deprotection, is required to remove the protecting group (usually dimethoxytrityl, DMT) on the 5'-hydroxyl group before each nucleotide monomer is attached to the growing oligonucleotide chain during the solid-phase phosphoramidite method for DNA synthesis. This protecting group protects the 5'-hydroxyl group in each step of the synthesis to prevent it from reacting with the next monomer to be added. Those skilled in the art will understand that the choice of cleavage agent depends on the type of 3'-nucleotide blocking group used. For example, tris(2-carboxyethyl)phosphine (TCEP) can be used to cleave 3'O-azidomethyl groups, palladium complexes can be used to cleave 3'-O-allyl groups, or sodium nitrite can be used to cleave 3'-O-amino groups. In certain embodiments, the cleavage reaction includes: TCEP, palladium complexes, or sodium nitrite.

[0047] As used herein, the term "solid-phase support" in DNA synthesis is defined as a fixed support for the synthesis reaction, which enables the enzymatic DNA synthesis process to occur on a fixed surface. The starting primer binds to the solid-phase support through any of the following methods: amide bond formation, ester bond formation, carbon-carbon bond coupling, Schiff base reaction, biotin-streptavidin coupling, click chemical reaction, thiol-gold interaction, or through a linker molecule. Nucleotide monomers are attached to the solid-phase support by forming 3'→5' phosphodiester bonds through a coupling reaction, thereby achieving stepwise extension of the DNA strand. In the examples of the present invention, streptavidin magnetic beads (SAbead, Beaver, particle size 300 nm) are used as the solid-phase support, solely for illustrative purposes and not intended to be limiting. Those skilled in the art can make a reasonable choice of the solid-phase support according to actual needs and based on experience. There are still other solid-phase supports that are suitable for the operation of the present invention, including product supports synthesized in situ on silicon-based materials, glass, resins, and metal supports; preferably, the product synthesized in situ on silicon-based materials includes silicon wafers or nanowire supports; preferably, the product synthesized in situ on glass includes microplate supports; preferably, the product synthesized in situ on resins includes insoluble particles of polystyrene or polyacrylate; preferably, the product synthesized in situ on metal supports includes noble metals such as gold or silver. The products include microspheres, which include polymer microspheres, preferably polyvinyl alcohol (PVA) or poly(lactic-co-glycolic acid); also include magnetic microspheres, preferably streptavidin magnetic beads; also include inorganic microspheres, preferably silica microspheres or Fe 3 O 4 microspheres or TiO 2 microspheres or ZnO microspheres; also include natural polymer microspheres, preferably gelatin microspheres, starch microspheres, or chitosan microspheres. The present invention is further illustrated in the following examples, but does not limit the scope of the present invention. Some details of the molecular cloning method may vary depending on the reagent, enzyme, or kit supplier, and should be operated according to the product instructions, which will not be described in detail in the examples.

[0048] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0049] Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0050] Example 1 (16 nt, 4-division method)

[0051] I. Fixation of Probe

[0052] In the present invention, streptavidin magnetic beads (SA beads, Beaver, particle size 300 nm) are used as the solid-phase carrier for the synthesis of 16 N, and then the enzymatic DNA synthesis is carried out through the binding of SA magnetic beads with the initiator, i.e., the primer strand (5'-biotin).

[0053] 1. Take 800 μL of SA magnetic beads into a low-binding EP tube, place the low-binding EP tube on a magnetic rack, and aspirate the supernatant when the magnetic beads are completely adsorbed on the tube wall.

[0054] 2. The concentration of SA magnetic beads is 10 mg / ml, and the final binding concentration is 2 mg / ml. Add 2000 μL of 2* binding buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 M NaCl, 0.01% - 0.1% Tween-20), 80 μL of 20 μM primer strand, and 1920 μL of ddH 2 O.

[0055] 3. Pipette and mix the added liquid with the magnetic beads, place it on an oscillating metal bath at 900 rpm, and bind at 30°C for 30 min.

[0056] 4. After the binding is completed, place the low-binding EP tube on a magnetic rack. After the magnetic beads are completely adsorbed on the tube wall, aspirate the liquid, and add PBS buffer (50 mM KH 2 PO 4 , 100 mM NaCl, 1% Triton, pH 6.8) and pipette and wash 2 times.

[0057] II. Biosynthesis of Oligo

[0058] 1. Divide the well-mixed 800 μL of magnetic beads into 4 tubes evenly, 200 μL per tube, place them on a magnetic rack, and aspirate the liquid after the magnetic beads are completely adsorbed on the tube wall.

[0059] 2. Enzymatic DNA synthesis, and the enzymatic DNA synthesis system is shown in Table 1.

[0060] Table 1: Enzymatic DNA Synthesis System

[0061]

[0062] (1) Add the corresponding reaction solution according to the reaction system, and add four different modified nucleotides to four tubes respectively.

[0063] (2) React with shaking at 40 °C for 1 min, place on a magnetic stand, and aspirate the liquid after the magnetic beads are completely adsorbed on the tube wall.

[0064] (3) Resuspend with 200 µL of PBS buffer, and mix the magnetic beads in the four tubes in the same tube.

[0065] (4) Add the deprotection solution (sodium nitrite), deprotect with shaking at 40 °C for 30 s, place on a magnetic stand, and aspirate the liquid after the magnetic beads are completely adsorbed on the tube wall to remove the nucleotide terminal blocking group and restore the 3'-end modified group to -OH.

[0066] (5) After mixing and pipetting with PBS buffer for cleaning 2 times, aliquot into four tubes evenly.

[0067] (6) Repeat the above steps for a total of 16 times. When synthesizing the 4th, 8th, 12th, and 16th nt, the deprotected products obtained in each step (5) are resuspended with buffer of the same volume as the magnetic beads.

[0068] III. Sample Detection

[0069] 1. Verification by urea-PAGE

[0070] When synthesizing the 4th, 8th, 12th, and 16th nt, resuspend with buffer of the same volume as the magnetic beads. Take 10 µL of the sample for sample preparation and run the gel. Remove the buffer storing the magnetic beads, add 10 µL of 0.1% SDS, heat at 98 °C for 5 min, take the liquid and add it to 10 µL of 2* loading buffer for mixing, and run 20% urea-PAGE. The PAGE gel electrophoresis results of the SA magnetic bead synthesis sequence are as Figure 2 shown.

[0071] 2. Verification by next-generation sequencing

[0072] (1) Add a polyA tail to the sample, referring to the instruction manual of terminal transferase (M0315S, NEB).

[0073] (2) Ligation of index + product enrichment: PCR amplification

[0074] Table 2: PCR amplification system

[0075]

[0076] 1) After adding a polyA tail to the magnetic beads, add the PCR amplification liquid.

[0077] 2) Perform PCR according to the PCR program: 95°C for 3 min; 95°C for 15 s, 50°C for 15 s, 72°C for 15 s, 26 cycles; 72°C for 5 min; 12°C for ∞

[0078] 3) Send the amplified liquid out for library construction and sequencing

[0079] 4) Sequencing result analysis: Sequence the synthesis result, filter the sequencing result through the forward target and the reverse target (polyA), then count the number of bases at each position and calculate the proportion. As Figure 3 shown, it can be seen that the random distribution of bases is relatively uniform. Therefore, our synthesis mode can ensure the diversity and uniformity of the sequence.

[0080] Example 2 (20 nt, the last round of dichotomy, only C / G)

[0081] I. Fixation of the probe

[0082] In the present invention, streptavidin magnetic beads (SA bead, Beaver, particle size of 500 nm) are used as the solid-phase carrier for the synthesis of 20 N, and then the enzymatic DNA synthesis is carried out through the binding of SA magnetic beads with the initiator, that is, the primer strand (5'-biotin).

[0083] 1. Take 800 μL of SA magnetic beads into a low-adsorption EP tube, place the low-adsorption EP tube on a magnetic rack, and aspirate the supernatant when the magnetic beads are completely adsorbed on the tube wall;

[0084] 2. The concentration of SA magnetic beads is 10 mg / ml, and the final binding concentration is 2 mg / ml. Add 2000 μL of 2* binding buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 M NaCl, 0.01% - 0.1% Tween-20), 80 μL of 20 μM primer strand, and 1920 μL of ddH 2 O;

[0085] 3. Pipette and mix the added liquid with the magnetic beads, place it on an oscillating metal bath at 900 rpm, and bind at 30°C for 30 min;

[0086] 4. After the binding is completed, place the low-adsorption EP tube on a magnetic rack, aspirate the liquid after the magnetic beads are completely adsorbed on the tube wall, and add PBS buffer (50 mM KH 2 PO 4 , 100 mM NaCl, 1% Triton, pH 6.8) and pipette and wash 2 times.

[0087] II. Biosynthesis of oligo

[0088] 1. Divide 800 µL of the well-mixed magnetic beads into 4 tubes evenly, with 200 µL in each tube. Place them on a magnetic stand. After the magnetic beads are completely adsorbed to the tube wall, aspirate the liquid.

[0089] 2. Enzymatic DNA synthesis. The enzymatic DNA synthesis system is the same as in Table 1.

[0090] (1) Add the corresponding reaction solutions according to the reaction system. Add four different modified nucleotides to the four tubes respectively.

[0091] (2) React with shaking at 40 °C for 1 min. Place it on a magnetic stand. After the magnetic beads are completely adsorbed to the tube wall, aspirate the liquid.

[0092] (3) Resuspend with 200 µL of PBS buffer. Put the four tubes of magnetic beads into the same tube and mix well.

[0093] (4) Add the deprotection solution (sodium nitrite). Deprotect with shaking at 40 °C for 30 s. Place it on a magnetic stand. After the magnetic beads are completely adsorbed to the tube wall, aspirate the liquid to remove the nucleotide terminal blocking group and restore the 3'-end modified group to -OH.

[0094] (5) After mixing and pipetting and washing 2 times with PBS buffer, divide it evenly into four tubes.

[0095] (6) Repeat the above steps 19 times. When synthesizing the 20th nt, there are only bases C / G, divided into two tubes. When synthesizing the 5th, 10th, 15th, and 20th nt, resuspend with buffer of the same volume as the magnetic beads.

[0096] III. Sample Detection

[0097] Verification by Urea-PAGE

[0098] When synthesizing the 4th, 8th, 12th, and 16th nt, resuspend with buffer of the same volume as the magnetic beads. Take 10 µL of the sample for sample preparation and run the gel. Remove the buffer storing the magnetic beads, add 10 µL of 0.1% SDS, heat at 98 °C for 5 min. Take the liquid and add it to 10 µL of 2* loading buffer and mix well. Run 20% urea-PAGE. The PAGE gel electrophoresis result of the SA magnetic bead synthesized sequence is as Figure 2 shown. It can be seen from the gel picture that the reaction efficiency is very high.

[0099] Example 3 (15 nt, one-division method in the last round)

[0100] I. Fixation of Probe

[0101] The present invention uses streptavidin magnetic beads (SA beads, Beaver, particle size 5 μm) as a solid-phase carrier for the synthesis of 15N, and then carries out enzymatic DNA synthesis through the binding of SA magnetic beads to the initiator, that is, the primer strand (5'-biotin).

[0102] 1. Take 800 μL of SA magnetic beads into a low-binding EP tube, place the low-binding EP tube on a magnetic rack, and aspirate the supernatant when the magnetic beads are completely adsorbed on the tube wall;

[0103] 2. The concentration of SA magnetic beads is 10 mg / ml, and the final binding concentration is 2 mg / ml. Add 2000 μL of 2* binding buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 M NaCl, 0.01% - 0.1% Tween-20), 80 μL of 20 μM primer strand, and 1920 μL of ddH 2 O;

[0104] 3. Pipette and mix the added liquid with the magnetic beads, place it on an oscillating metal bath at 900 rpm, and bind at 30°C for 30 min;

[0105] 4. After the binding is completed, place the low-binding EP tube on a magnetic rack, aspirate the liquid after the magnetic beads are completely adsorbed on the tube wall, and add PBS buffer (50 mM KH 2 PO 4 , 100 mM NaCl, 1% Triton, pH 6.8) and pipette and wash 2 times.

[0106] II. Biosynthesis of oligo

[0107] 1. Divide the well-mixed 800 μL of magnetic beads into 4 tubes, 200 μL per tube, place them on a magnetic rack, and aspirate the liquid after the magnetic beads are completely adsorbed on the tube wall;

[0108] 2. Enzymatic DNA synthesis, and the enzymatic DNA synthesis system is the same as in Table 1;

[0109] (1) Add the corresponding reaction solutions according to the reaction system, and add four different modified nucleotides to the four tubes respectively;

[0110] (2) React with oscillation at 40°C for 1 min, place it on a magnetic rack, and aspirate the liquid after the magnetic beads are completely adsorbed on the tube wall;

[0111] (3) Resuspend with 200 μL of PBS buffer, and mix the four tubes of magnetic beads in the same tube

[0112] (4) Add the deprotection solution (sodium nitrite), shake at 40 °C for deprotection for 30 s, place it on a magnetic stand, and after the magnetic beads are completely adsorbed on the tube wall, suck out the liquid to remove the nucleotide terminal blocking group and restore the 3'-end modification group to -OH;

[0113] (5) After mixing and pipetting with PBS buffer for washing 2 times, divide them into four tubes evenly;

[0114] (6) Repeat the above steps 14 times. When synthesizing the 15th nt, there is only one tube with base C. When synthesizing the 5th, 10th, and 15th nt, resuspend with buffer of the same volume as the magnetic beads.

[0115] III. Sample Detection

[0116] The steps are the same as those in Example 1.

[0117] Example 4 (15 nt, penultimate round of trichotomy)

[0118] The steps are the same as those in Example 3, except that in the step of biosynthesizing oligo, (6) Repeat the above steps 13 times. When synthesizing the 14th nt, there are three tubes with bases A / T / C. When synthesizing the 15th nt, combine the tubes and take the 5th, 10th, and 15th nt to resuspend with buffer of the same volume as the magnetic beads.

[0119] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims.

Claims

1. A method for biosynthesis of an oligo pool, characterized in that: The process includes the following steps: probe fixation, cyclic synthesis to regulate nucleotide sequence; solid phase carrier distribution as required, chain extension, closure and deprotection; 1) Probe fixation: The solid phase carrier is combined with the primer chain, and the primer chain characteristics include: the 5' end is connected to the solid carrier and the 3' end is free; 2) The primer strand is single-stranded deoxyribonucleic acid (DNA); 3) Solid phase carrier is distributed as needed: the solid phase carrier or the deprotected product is distributed as needed into M tubes according to the volume, where M is selected from any natural number of 1, 2, 3, and 4; 4) Chain extension: adding a reaction solution to an enzymatic DNA synthesis system, and adding a single modified nucleotide to each tube, wherein the enzymatic DNA synthesis system contains a key enzyme terminal deoxynucleotidyl transferase, and the 3' end of the modified nucleotide contains a reversible blocking group; 5) Combine tubes: Vortex the solution obtained in step 4), resuspend it, put it into the same tube, mix well and remove the liquid; 6) Deprotection: Add deprotection solution to the tube in step 5), shake and wash to obtain the deprotection product, so as to remove the reversible blocking group at the 3' end and restore the modification group at the 3' end to -OH; 7) Cycle step 3), step 4), step 5), step 6), and execute step 6) N times cumulatively, where N is selected from any non-zero natural number; sampling, quality control, and application; or swap the order of step 6) and step 5), i.e., complete deprotection in a single tube and then close the tubes.

2. The method for biosynthesis of oligo pool according to claim 1, characterized in that: The M value in the N-times step 3) and the N-1-times step 3) is equal or different.

3. The method for biosynthesis of oligo pool according to claim 1, characterized in that: In step 3), the solid phase carrier or the deprotected product is divided equally by volume or distributed according to a ratio.

4. The method for biosynthesis of oligo pool according to claim 1, characterized in that: The removal of the solution in step 5) includes at least one of centrifugation, filtration or adsorption.

5. The method for biosynthesis of oligo pool according to claim 1, characterized in that: The solid phase carrier includes a product carrier synthesized in situ on a silicon-based material, glass, resin or metal carrier.

6. The method for biosynthesis of oligo pool according to claim 5, characterized in that: The product comprises microspheres comprising polymer microspheres.

7. The method for biosynthesis of an oligo pool according to claim 6, characterized in that: The surface of the microsphere is modified with any functional group of hydroxyl, amino, carboxyl, aldehyde or epoxy.

8. The method for biosynthesis of oligo pool according to claim 7, characterized in that: The particle size of the microspheres is 300nm-100μm.

9. The method for biosynthesis of an oligo pool according to claim 1, characterized in that: In the step 1), the primer chain is combined with the solid phase carrier by any of amide bond formation, ester bond formation, carbon-carbon bond coupling, Schiff base reaction, biotin-streptavidin coupling, click chemistry reaction, thiol-gold interaction, and linker molecules.

10. The method for biosynthesis of an oligo pool according to claim 1, characterized in that: Sampling in step 7): perform step 6) N times, and resuspend the deprotection products of the Nth synthesis and several times before the Nth synthesis in a buffer with the same volume as the solid phase carrier.

11. The method for biosynthesis of an oligo pool according to claim 1, characterized in that: The modified nucleotides include 3'-O modified nucleotides, including nucleoside triphosphates carrying 3'-O-methyl or 3'-azido or 3'-O-azidomethyl or 3'-O-amino or 3'-aminooxy or 3'-O-allyl groups at the 3' end.

12. The method for biosynthesis of an oligo pool according to claim 1, characterized in that: The 3'-O modified nucleotides include any nucleoside triphosphate carrying ester, ether, nitrile, phosphate, carbonate, carbamate, hydroxylamine, borate, nitrate, sugar, phosphoramide, phosphoramidate, benzenesulfonate, sulfate, sulfone or amino acid at the 3' end.

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