Biosynthesis method and application of oligo pool

Abstract

The invention discloses a biosynthesis method and application of oligo pool. The biosynthesis method comprises the following steps: 1) fixing a probe, and combining a solid-phase carrier with a primer chain; (2) the solid-phase carrier is distributed as required, the solid-phase carrier or a deprotection product is distributed into M tubes as required according to the volume, and M is any natural number selected from 1, 2, 3 and 4; the method comprises the following steps of (1) adding a reaction solution into an enzymatic DNA synthesis system, (2) adding a reaction solution into the enzymatic DNA synthesis system, (3) carrying out chain extension and adding a reaction solution into the enzymatic DNA synthesis system, (4) carrying out tube combination, (5) carrying out deprotection, (6) repeating the steps (2)-(5) and accumulatively executing the step (5) for N times, and (7) carrying out sampling, quality control and application, and on the basis of a DNA enzymatic synthesis technology, regulating and controlling the synthesis types of basic groups in each round by using microspheres as a solid-phase carrier to control the sequence diversity of the oligo pool.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA synthesis, and in particular to a biosynthesis method and application of an oligo pool. Background Art

[0002] In recent years, synthetic biology has developed rapidly, and its position in the field of biotechnology has become increasingly prominent, and DNA synthesis is one of the most important supporting technologies in the field of synthetic biology. Gene synthesis can not only replicate existing genes in nature, but also greatly expand our ability to manipulate genes and proteins through codon optimization and customized gene modification. On the other hand, the preparation technology of oligo pool, a technology that contains a large number of oligonucleotide mixtures with specific sequences, can simultaneously explore or manipulate 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 modern molecular biology, genetic engineering, precision medicine and synthetic biology.

[0003] As a key basic technology in synthetic biology, DNA synthesis is as important as sequencing technology supports genomics. The current mainstream technology for DNA synthesis is chemical synthesis, which began in the 1950s and 1960s. In the 1960s and 1970s, the chemical synthesis methods of oligonucleotides were continuously improved. In the 1980s, Beaucage and Caruthers developed a phosphoramidite-based DNA synthesis method, which gradually developed into a column synthesis method. This method uses more than ten chemical reagents to gradually synthesize DNA through four steps of deprotection, coupling, capping and oxidation. Due to the limitations of chemical synthesis methods, obtaining a DNA library with controllable proportions requires synthesizing a large number of specific DNA sequences and mixing them according to demand, and it is impossible to accurately adjust the ratio of ATGC in each round through a one-step method.

[0004] Breaking through the traditional chemical organic phase synthesis system, the enzymatic DNA synthesis technology based on a mild aqueous phase system and independent of the template can realize the in situ synthesis of the oligo pool. Enzymatic DNA synthesis technology is gradually becoming a cutting-edge trend in the field of DNA synthesis with its advantages in length, accuracy, flexibility, cost efficiency, environmental protection and automation, making it rapidly develop in the fields of molecular diagnosis, high-throughput screening, protein engineering, mutant library construction, single-cell sequencing, etc. In molecular diagnostic systems, the biosynthesis method of the oligo pool makes it possible to quickly detect pathogens such as viruses and bacteria by providing highly specific oligonucleotides. These oligonucleotides can be used as primers for PCR or other amplification techniques to achieve rapid identification and quantification of pathogen DNA. In high-throughput screening systems, the application of oligo pools has greatly promoted the development of gene editing and CRISPR-Cas9 technology. Through the high-throughput synthesis of sgRNA libraries, researchers can efficiently conduct gene function research and disease mechanism exploration, accelerating the development of new treatments. In the process of protein engineering, oligo pool technology provides an efficient means for constructing mutant libraries by synthesizing oligonucleotide pools containing specific mutations, allowing researchers to quickly test multiple protein variants to optimize protein function and stability. In the process of mutant library construction, the biosynthesis method of oligo pool 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 efficiency of mutant library construction, but also provides a new perspective for understanding the relationship between protein structure and function. Single-cell sequencing can sequence hundreds of thousands of cells at the same time. Oligo pool can efficiently synthesize a large number of high-purity DNA fragments and connect them to microbeads or magnetic beads, solving many problems such as high production cost, difficult preparation, low preparation success rate, and unstable effects of different batches, accelerating the widespread application of this technology. In summary, enzymatic DNA synthesis technology not only improves the efficiency and accuracy of biosynthesis, but also provides strong technical support for molecular diagnosis, high-throughput screening, protein engineering and mutant library construction, and has broad application prospects and commercial value. With this approach, we can achieve faster and more precise genetic manipulation and analysis in biomedical research and clinical diagnostics. Summary of the invention

[0005] The first aspect of the present invention is a method for biosynthesis of an oligo pool, comprising the following steps: fixation of probes, cyclic synthesis to regulate nucleotide sequences: distribution of solid phase carriers on demand, chain extension, closure and deprotection.

[0006] As a preferred solution, 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 chain can be RNA or DNA; the primer chain can be single-stranded or double-stranded; 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; 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; 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; 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.

[0007] As a preferred solution, the M value in the N-th step 3) and the N-1-th step 3) is equal or different.

[0008] 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.

[0009] As a preferred solution, the removal of the solution in step 5) includes at least one of centrifugation, filtration, adsorption and the like.

[0010] 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.

[0011] As a preferred embodiment, the above-mentioned product includes microspheres, which include polymer microspheres, preferably polyvinyl alcohol (PVA) or polylactic acid-glycolic acid copolymer; also include magnetic microspheres, preferably streptavidin magnetic beads, NHS magnetic beads, silica magnetic beads, polymer magnetic beads, agarose magnetic beads, carboxyl magnetic beads, etc.; also include inorganic microspheres, preferably silica microspheres or Fe3O4 microspheres or TiO2 microspheres or ZnO microspheres; also include natural polymer microspheres, preferably gelatin microspheres or starch microspheres or chitosan microspheres.

[0012] As a preferred embodiment, the surface of the microspheres is modified with any functional group such as hydroxyl, amino, carboxyl, aldehyde, epoxy, etc.

[0013] As a preferred embodiment, the particle size of the upper microsphere is 300 nm-100 μm. Preferably, the particle size of the microsphere 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, and 100 μm.

[0014] As a preferred embodiment, in the above step 1), the primer chain and the solid phase carrier are combined 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, or through a linker molecule. As a preferred solution, in the above step 7), sampling is performed: after executing step 6) N times, the deprotection products synthesized N times and several times before the N times are resuspended in a buffer having the same volume as the solid phase carrier.

[0015] As a preferred embodiment, 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.

[0016] As a preferred embodiment, the above-mentioned 3'-O modified nucleotides include any nucleoside triphosphate carrying ester, ether, nitrile, phosphate, carbonate, carbamate, hydroxylamine, borate, nitrate, sugar, phosphoamide, phosphoamidate, benzenesulfonate, sulfate, sulfone, and amino acid at the 3' end.

[0017] The second aspect of the present invention is to provide a molecular diagnostic system, which includes: a. using the oligo pool biosynthesis method as 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 molecular diagnosis of diseases.

[0018] The third aspect protected by the present invention is a high-throughput screening system, which comprises: a. using the oligo pool biosynthesis method as described in any one of the first aspects to design a library required for high-throughput screening; b. using the library in a to perform high-throughput screening of target molecules or compounds.

[0019] The fourth aspect protected by the present invention is a protein engineering construction process, which comprises: a. using the oligo pool biosynthesis method as described in any one of the first aspects to design and construct protein variants; b. using the protein variants in a to perform functional analysis or optimization.

[0020] The fifth aspect protected by the present invention is that the process comprises: a. using the oligo pool biosynthesis method as 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.

[0021] The fifth aspect protected by the present invention is a single-cell or spatial omics sequencing system, which uses the oligo pool biosynthesis method as described in any one of the first aspects to synthesize oligonucleotides for single-cell multi-omics sequencing, targeted sequencing or spatial omics sequencing.

[0022] Compared with the prior art, the advantages of the present invention are: 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 is a highly novel and innovative method that takes into account downstream applications. That is, the enzymatic DNA synthesis technology is used, and the key enzyme is DNA polymerase, such as terminal deoxynucleotidyl transferase (TdT), which includes the following steps: 1. The microsphere is combined with the primer chain; 2. The synthesized nucleotide sequence is regulated, mainly through different base reaction systems containing blocking groups, and the biological enzyme TdT is used to add nucleotides containing blocking groups at the 3' end respectively, and 1-4 modified nucleotides can be added in each round; 3. Mix 1-4 tubes to remove the blocking group, restore the 3' end modification group to -OH, and then divide into 1-4 tubes; 4. Repeat steps 2-3. It has a high accuracy rate and good market prospects.

[0023] 2. The biosynthesis technology provided by the present invention synthesizes a 16 nt oligo pool, and only one synthesis is required to obtain 4 16 In traditional chemical gene synthesis, 416 =4,294,967,296 different sequences, 4 16 =4,294,967,296 times.

[0024] 3. The present invention can be synthesized in situ 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 glue beads), etc. Different solid phase carriers can be adapted according to different application directions, which greatly meets 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 chemistry reaction, thiol-gold interaction, and through linker molecules. Since the microspheres can exist in a state of solid-liquid mixing, and each microsphere is very small, compared with a single carrier of the chemical synthesis method that can only synthesize one sequence, the microspheres can synthesize four nucleotide sequences in one cycle by equal volume equalization and mixing, which greatly reduces the time, manpower and material resources required for synthesis. Therefore, the biosynthesis method provided by the present invention can not only greatly reduce the synthesis cost, but also take into account downstream applications, eliminating the steps of cumbersome operation and low yield. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 This is the enzymatic DNA tetralysis synthesis process in Example 1 of the present invention; Figure 2 The PAGE gel electrophoresis results of the SA magnetic bead synthetic sequence in Example 1 of the present invention; Figure 3 is the base distribution ratio of 16 N samples in Example 1 of the present invention; Figure 4 This is the PAGE gel electrophoresis result of the SA magnetic bead synthetic sequence in Example 2 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Unless otherwise specified, all terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs.

[0026] As used herein, the term "oligonucleotide" refers to a polymeric form of nucleotides, either ribonucleotides or deoxyribonucleic acids, containing natural or non-natural nucleotides, and having a length of at least 2, usually between about 5 and about 200, or more usually about 100. Thus, the term encompasses double-stranded and single-stranded DNA and RNA. In addition, oligonucleotides may be resistant to nucleases, including but not limited to 2'-O-methyl ribonucleotides, thionucleotides, dithionucleotides, phosphoramidite nucleotides, and methylphosphonate nucleotides.

[0027] The term "oligo pool" used here is also called an oligonucleotide pool, which is a mixture of thousands of short synthetic oligonucleotides (oligos) that are synthesized simultaneously. After the synthesis is completed, the oligonucleotides are removed from the synthesis platform and merged into a separate tube. Oligo Pool can be used to construct various types of libraries, such as CRISPR gRNA, mutation libraries, or next-generation sequencing (NGS) target enrichment libraries for high-throughput screening.

[0028] The term "primer chain" used herein refers to the starting primer chain used in enzymatic DNA synthesis, which is a short DNA fragment used to initiate the synthesis of the target DNA sequence. The primer chain is a very critical component in the PCR process. It complementarily binds to the template DNA single strand and provides a 3'-OH end, allowing the DNA polymerase to start synthesizing a new DNA chain from there. A nucleic acid containing a sequence selected to be substantially complementary to each specific sequence to be amplified. The starting primer chain is (T)n, (A)n, (C)n, (G)n, where T represents deoxythymidine nucleotide (abbreviated as deoxythymidine), A represents deoxyadenine nucleotide (abbreviated as deoxyadenosine), C represents deoxycytosine nucleotide (abbreviated as deoxycytidine), and G represents deoxyguanine nucleotide (abbreviated as deoxyguanosine).

[0029] The method for extending one or more natural polynucleotides with an initial primer chain comprises providing one or more natural polynucleotides in a reaction mixture under TdT reaction conditions, wherein the natural polynucleotides have a 3'-terminal nucleotide whose 5' end is connected to a solid support and has a free 3'-hydroxyl group; and extending the one or more natural polynucleotides with nucleotides of a predetermined sequence by repeated cycles of the following steps: (i) contacting the natural polynucleotide with a free 3'-O-hydroxyl group or an extended natural polynucleotide with a 3'-O-blocked nucleoside triphosphate and a TdT variant, so that the natural polynucleotide or the extended natural polynucleotide is extended by incorporating 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 with a free 3'-hydroxyl group, thereby synthesizing an oligonucleotide of a predetermined sequence on the natural polynucleotide.

[0030] The above-mentioned polynucleotides include modified nucleotides, such as 3'-O modified nucleotides, including nucleoside triphosphates carrying 3'-O-methyl, 3'-azido, 3'-O-azidomethyl, 3'-O-amino, 3'-aminooxy or 3'-O-allyl groups at the 3' end. For example, 3'-O blocked nucleoside triphosphates include being blocked by 3'-O-azidomethyl, 3'-aminooxy or 3'-O-allyl groups, and the 3'-O-blocking group has a molecular weight of 100 or less. For example, 3'-O modified nucleotides include nucleoside triphosphates carrying esters, ethers, nitrites, phosphates, carbonates, carbamates, hydroxylamines, borates, nitrates, sugars, phosphoramides, phosphoramidates, benzenesulfonates, sulfates, sulfones or amino acids at the 3' end.

[0031] The terms "modified" and "blocked" are used interchangeably and are intended to refer to a moiety covalently linked to a specific group that prevents chemical changes to that group during a chemical or enzymatic process. As long as the designated group is the 3'-hydroxyl group of a nucleoside triphosphate, or an extended segment (or "extension intermediate") into which a 3'-modified / protected / or blocked-nucleoside triphosphate is incorporated, the chemical change that is prevented is further or subsequent extension of the extended segment (or "extension intermediate") by an enzymatic ligation reaction.

[0032] The term "terminal deoxynucleotidyl transferase (TdT)" as used herein includes references to purified and recombinant forms of the enzyme. TdT enzyme synthesis does not rely on a template, and DNA synthesis can be performed using only single-stranded DNA, so it has the function of creating genomic materials from scratch, making it a key biological enzyme for the synthesis of third-generation DNA. In terms of synthesis strategy, the reaction is terminated mainly by modifying nucleotides, adding reagents with specific chemical groups, or coupling TdT enzymes with nucleotides, etc. The TdT enzyme in the present invention includes but is not limited to an enzymatic DNA synthesis kit purchased from Tianjin Zhonghe Gene Technology Co., Ltd., or an expression cassette comprising all elements for expressing TdT enzyme disclosed in patents CN112746063B and CN110331136B. The bases may be conventional bases (A, G, C, T), non-natural nucleotides such as isocytosine and isoguanine and their analogs (e.g., inosine, derivatives of purine and pyrimidine bases, such as N4-methyldeoxyguanosine, aza or azapurine, aza or azapyrimidine, purines or pyrimidines having substituents altered or substituted at any of a range of chemical positions, such as 2-amino-6-methylaminopurine, O6-methylguanine, 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.

[0033] Cleavage is deprotection. When the solid phase phosphoramidite triester method synthesizes DNA, each nucleotide monomer needs to remove the protecting group (usually dimethoxytrityl, DMT) on the 5'-hydroxyl before being connected to the growing oligonucleotide chain. This protecting group protects the 5'-hydroxyl in each step of the synthesis to prevent it from reacting with the next monomer added. It will be appreciated by those skilled in the art that the selection of cleavage agent depends on the type of 3'-nucleotide blocking group used. For example, tris (2-carboxyethyl) phosphine (TCEP) can be used for cracking 3'O-azidomethyl groups, palladium complexes can be used for cracking 3'-O-allyl groups, or sodium nitrite can be used for cracking 3'-O-amino groups. In a specific embodiment, the cleavage reaction includes: TCEP, palladium complexes or sodium nitrite.

[0034] The term "solid phase carrier" is defined as a fixed support for the synthesis reaction in DNA synthesis, which enables the enzymatic DNA synthesis process to be carried out on a fixed surface. The starting primer and the solid phase carrier are combined by amide bond formation, ester bond formation, carbon-carbon bond coupling, Schiff base reaction, biotin-streptavidin coupling, click chemistry reaction, thiol-gold interaction, and any one of the linker molecules. The nucleotide monomer forms a 3'→5' phosphodiester bond through a coupling reaction and is connected to the solid phase support, thereby achieving the step-by-step extension of the DNA chain. The embodiment of the present invention uses streptavidin magnetic beads (SAbead, beaver, particle size of 300 nm) as a solid phase carrier, which is only for the purpose of example and not intended to be limiting. Those skilled in the art can make a reasonable choice of the solid phase carrier according to actual needs and based on experience. Still other solid phase carriers are suitable for the operation of the present invention, including product carriers synthesized in situ on silicon-based materials, glass, resins, and metal carriers; preferably, the product synthesized in situ on silicon-based materials includes silicon wafers or nanowire carriers; preferably, the product synthesized in situ on glass includes microporous plate carriers; preferably, the product synthesized in situ on resins includes insoluble particles of polystyrene or polyacrylate; preferably, the product synthesized in situ on metal carriers includes precious metals of gold or silver. The product includes microspheres, the microspheres include polymer microspheres, preferably, polyvinyl alcohol PVA or polylactic acid-glycolic acid copolymer; also includes magnetic microspheres, preferably, streptavidin magnetic beads; also includes inorganic microspheres, preferably, silica microspheres or Fe3O4 microspheres or TiO2 microspheres or ZnO microspheres; also includes natural polymer microspheres, preferably, gelatin microspheres or starch microspheres or chitosan microspheres. The present invention is further illustrated in the following examples, but the scope of the present invention is not limited thereto. Some details of molecular cloning methods vary depending on the reagents, enzymes or kits provided by different vendors and should be performed according to the product instructions. They will not be described in detail in the examples.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] Example 1 (16 nt, 4-point method) 1. Fixation of the probe The present invention uses streptavidin magnetic beads (SA beads, beaver, particle size of 300 nm) as solid phase carriers to synthesize 16 N, and then performs enzymatic DNA synthesis through the combination of SA magnetic beads and initiator, namely primer chain (5'-biotin).

[0038] 1. Take 800 µL SA magnetic beads into a low-adsorption EP tube, put the low-adsorption EP tube into the magnetic rack, and aspirate the supernatant when the magnetic beads are completely adsorbed to the tube wall; 2. The concentration of SA magnetic beads is 10 mg / ml, and the final binding concentration is 2 mg / ml. Add 2000 µL 2* binding buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 M NaCl, 0.01%~0.1% Tween-20), 80 µL 20 µM primer chain, and 1920 µL ddH2O; 3. Mix the added liquid and magnetic beads by blowing, and place them on an oscillating metal bath at 900 rpm and 30°C for 30 min; 4. After binding, place the low-adsorption EP tube into the magnetic rack, wait for the magnetic beads to be completely adsorbed to the tube wall, aspirate the liquid, add PBS buffer (50 mM KH2PO4, 100 mM NaCl, 1% Triton, pH 6.8), and wash twice by blowing.

[0039] 2. Biosynthetic oligo 1. Divide the mixed 800 µL magnetic beads into 4 tubes, 200 µL in each tube, and place them in the magnetic rack. After the magnetic beads are completely adsorbed to the tube wall, aspirate the liquid; 2. Enzymatic DNA synthesis. The enzymatic DNA synthesis system is shown in Table 1; Table 1: Enzymatic DNA synthesis system

[0040] (1) Add the corresponding reaction solution according to the reaction system, and add four different modified nucleotides to the four tubes respectively; (2) Oscillate the reaction at 40°C for 1 min, place in a magnetic rack, and aspirate the liquid after the magnetic beads are completely adsorbed to the tube wall; (3) Add 200 µL PBS buffer to resuspend and mix the four tubes of magnetic beads in the same tube. (4) Add deprotection solution (sodium nitrite), shake and deprotect at 40°C for 30 seconds, place in a magnetic rack, and aspirate the liquid after the magnetic beads are completely adsorbed to the tube wall to remove the blocking group at the nucleotide terminal and restore the 3'-end modification group to -OH; (5) After mixing and washing twice with PBS buffer, divide the tubes equally into four tubes; (6) Repeat the above steps for a total of 16 times. When synthesizing 4, 8, 12, and 16 nt, the deprotected product obtained in each step (5) is resuspended in a buffer with the same volume as the magnetic beads.

[0041] 3. Sample Testing 1. Urea-PAGE verification When synthesizing the 4th, 8th, 12th, and 16th nt, resuspend the beads in the same volume of buffer, take 10 µL of the sample to prepare the sample and run the gel, remove the buffer that preserves the beads, add 10 µL of 0.1% SDS, heat at 98℃ for 5 min, take the liquid and add 10 µL of 2* loading buffer to mix, and run 20% urea-PAGE. The PAGE gel electrophoresis results of the SA magnetic bead synthesis sequence are shown in Figure 1. Figure 2 shown.

[0042] 2. Next-generation sequencing verification (1) Add poly A tail to the sample, referring to the instruction manual of terminal transferase (M0315S, NEB).

[0043] (2) Index + product enrichment: PCR amplification Table 2: PCR amplification system

[0044] 1) After the magnetic beads are added with polyA tails, add PCR amplification liquid; 2) Perform PCR according to the PCR program: 95°C, 3 min; 95°C, 15 s, 50°C, 15 s, 72°C, 15 s, 26X; 72°C, 5 min; 12°C, ∞ 3) The amplified liquid is sent out for library construction and sequencing 4) Analysis of sequencing results: Sequence the synthesis results, filter the sequencing results by the front target and the back target (polyA), and then count the number of bases at each position and calculate the ratio, such as Figure 3 As shown, it can be seen that the random distribution of bases is relatively uniform, so our synthesis mode can ensure the diversity and uniformity of the sequence.

[0045] Example 2 (20 nt, last round of dichotomy, only C / G) 1. Fixation of the probe The present invention uses streptavidin magnetic beads (SA beads, beaver, particle size of 500 nm) as solid phase carriers to synthesize 20 N, and then performs enzymatic DNA synthesis through the combination of SA magnetic beads and initiator, namely primer chain (5'-biotin).

[0046] 1. Take 800 µL SA magnetic beads into a low-adsorption EP tube, put the low-adsorption EP tube into the magnetic rack, and aspirate the supernatant when the magnetic beads are completely adsorbed to the tube wall; 2. The concentration of SA magnetic beads is 10 mg / ml, and the final binding concentration is 2 mg / ml. Add 2000 µL 2* binding buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 M NaCl, 0.01%~0.1% Tween-20), 80 µL 20 µM primer chain, and 1920 µL ddH2O; 3. Mix the added liquid and magnetic beads by blowing, and place them on an oscillating metal bath at 900 rpm and 30°C for 30 min; 4. After the binding is completed, place the low-adsorption EP tube into the magnetic rack, wait for the magnetic beads to be completely adsorbed to the tube wall, aspirate the liquid, add PBS buffer (50 mM KH2PO4, 100 mM NaCl, 1% Triton, pH6.8) and wash twice.

[0047] 2. Biosynthetic oligo 1. Divide the mixed 800 µL magnetic beads into 4 tubes, 200 µL in each tube, and place them in the magnetic rack. After the magnetic beads are completely adsorbed to the tube wall, aspirate the liquid; 2. Enzymatic DNA synthesis: the enzymatic DNA synthesis system is the same as that in Table 1; (1) Add the corresponding reaction solution according to the reaction system, and add four different modified nucleotides to the four tubes respectively; (2) Oscillate the reaction at 40°C for 1 min, place in a magnetic rack, and aspirate the liquid after the magnetic beads are completely adsorbed to the tube wall; (3) Add 200 µL PBS buffer to resuspend and mix the four tubes of magnetic beads in the same tube. (4) Add deprotection solution (sodium nitrite), shake and deprotect at 40°C for 30 seconds, place in a magnetic rack, and aspirate the liquid after the magnetic beads are completely adsorbed to the tube wall to remove the blocking group at the nucleotide terminal and restore the 3'-end modification group to -OH; (5) After mixing and washing twice with PBS buffer, divide the tubes equally into four tubes; (6) Repeat the above steps 19 times. When synthesizing the 20th nt, only the base C / G is present. Divide the beads into two tubes and resuspend them in the same volume of buffer when synthesizing the 5th, 10th, 15th, and 20th nt.

[0048] 3. Sample Testing Urea-PAGE verification When synthesizing the 4th, 8th, 12th, and 16th nt, resuspend the beads in the same volume of buffer, take 10 µL of the sample to prepare the sample and run the gel, remove the buffer that preserves the beads, add 10 µL of 0.1% SDS, heat at 98℃ for 5 min, take the liquid and add 10 µL of 2* loading buffer to mix, and run 20% urea-PAGE. The PAGE gel electrophoresis results of the SA magnetic bead synthesis sequence are shown in Figure 1. Figure 2 As shown, the gel image shows that the reaction efficiency is very high.

[0049] Example 3 (15 nt, last round of one-step method) 1. Fixation of the probe The present invention uses streptavidin magnetic beads (SA beads, beaver, particle size of 5 μm) as a solid phase carrier to synthesize 15N, and then performs enzymatic DNA synthesis through the combination of SA magnetic beads and initiator, namely primer chain (5'-biotin).

[0050] 1. Take 800 µL SA magnetic beads into a low-adsorption EP tube, put the low-adsorption EP tube into the magnetic rack, and aspirate the supernatant when the magnetic beads are completely adsorbed to the tube wall; 2. The concentration of SA magnetic beads is 10 mg / ml, and the final binding concentration is 2 mg / ml. Add 2000 µL 2* binding buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 M NaCl, 0.01%~0.1% Tween-20), 80 µL 20 µM primer chain, and 1920 µL ddH2O; 3. Mix the added liquid and magnetic beads by blowing, and place them on an oscillating metal bath at 900 rpm and 30°C for 30 min; 4. After the binding is completed, place the low-adsorption EP tube into the magnetic rack, wait for the magnetic beads to be completely adsorbed to the tube wall, aspirate the liquid, add PBS buffer (50 mM KH2PO4, 100 mM NaCl, 1% Triton, pH6.8) and wash twice.

[0051] 2. Biosynthetic oligo 1. Divide the mixed 800 µL magnetic beads into 4 tubes, 200 µL in each tube, and place them in the magnetic rack. After the magnetic beads are completely adsorbed to the tube wall, aspirate the liquid; 2. Enzymatic DNA synthesis: the enzymatic DNA synthesis system is the same as that in Table 1; (1) Add the corresponding reaction solution according to the reaction system, and add four different modified nucleotides to the four tubes respectively; (2) Oscillate the reaction at 40°C for 1 min, place in a magnetic rack, and aspirate the liquid after the magnetic beads are completely adsorbed to the tube wall; (3) Add 200 µL PBS buffer to resuspend and mix the four tubes of magnetic beads in the same tube. (4) Add deprotection solution (sodium nitrite), shake and deprotect at 40°C for 30 seconds, place in a magnetic rack, and aspirate the liquid after the magnetic beads are completely adsorbed to the tube wall to remove the blocking group at the nucleotide terminal and restore the 3'-end modification group to -OH; (5) After mixing and washing twice with PBS buffer, divide the tubes equally into four tubes; (6) Repeat the above steps 14 times. When synthesizing the 15th nt, only one tube of base C is used. When synthesizing the 5th, 10th, and 15th nt, resuspend the beads in the same volume of buffer.

[0052] 3. Sample Testing The steps are the same as in Example 1.

[0053] Example 4 (15 nt, penultimate three-step method) The steps are the same as those in Example 3, except that in the step of biosynthesizing oligos, (6) the above steps are repeated 13 times, and when synthesizing the 14th nt, there are three tubes of bases A / T / C. When synthesizing the 15th nt, the tubes are combined, and the 5th, 10th, and 15th nt are resuspended in the same volume of buffer as the magnetic beads.

[0054] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which 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 attached claims.

Claims

1. A method for biosynthesis of an oligo pool, characterized in that: The method comprises the following steps: fixation of the probe, cyclic synthesis to regulate the nucleotide sequence; distribution of the solid phase carrier as required, chain extension, cloning and deprotection.

2. The method for biosynthesis of oligo pool according to claim 1, characterized in that: 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 chain is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and the primer chain is single-stranded or double-stranded; 3) Solid phase carriers are distributed as needed: solid phase carriers or deprotected products are distributed as needed into M tubes according to volume, where M is selected from any natural number of 1, 2, 3, and 4; 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, 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.

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

4. The method for biosynthesis of oligo pool according to claim 2, 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.

5. The method for biosynthesis of oligo pool according to claim 2, characterized in that: The removal of the solution in step 5) includes at least one of centrifugation, filtration or adsorption.

6. The method for biosynthesis of oligo pool according to claim 2, characterized in that: The solid phase carrier includes a product carrier synthesized in situ on a silicon-based material, glass, resin or metal carrier.

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

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

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

10. The method for biosynthesis of oligo pool according to claim 2, 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.

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

12. The method for biosynthesis of oligo pool according to claim 2, 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.

13. The method for biosynthesis of oligo pool according to claim 2, 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.

14. A molecular diagnostic system, characterized in that The system includes: a. designing and synthesizing molecular markers for diagnosis using the biosynthesis method of the oligo pool as described in any one of claims 1 to 13; b. Use the molecular markers selected in a for molecular diagnosis of diseases.

15. A high throughput screening system, characterized in that: The system includes: a. using the biosynthesis method of the oligo pool as described in any one of claims 1 to 13 to design a library required for high-throughput screening; b. Use the library in a to perform high-throughput screening of target molecules or compounds.

16. A protein engineering construction process, characterized in that: The process includes: a. Designing and constructing protein variants using the biosynthetic method of the oligo pool as described in any one of claims 1 to 13; b. Use the protein variants in a for functional analysis or optimization.

17. A mutant library construction process, characterized in that: The process includes: a. using the oligo pool biosynthesis method as described in any one of claims 1-13 to design mutations in the mutant library; b. Use the mutant library in a for screening and analysis.

18. A single-cell or spatial omics sequencing system, characterized in that: Use the biosynthesis method of the oligo pool as described in any one of claims 1 to 13 to synthesize oligonucleotides for single-cell multi-omics sequencing, targeted sequencing or spatial-omics sequencing.

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