Oligonucleotide purification methods

Abstract

This application discloses a method for purifying oligonucleotides, comprising: enzymatically digesting an oligonucleotide sample after enzymatic DNA synthesis with a protease solution to obtain an enzymatic digest product; and inactivating the enzyme in the enzymatic digest product to obtain purified oligonucleotides; wherein the oligonucleotide sample contains a biological enzyme. The method of this application can effectively remove residual biological enzymes after enzymatic DNA synthesis without affecting the biological activity of the oligonucleotides, yielding high-throughput and high-purity oligonucleotide samples. Furthermore, the method is rapid, convenient, low-cost, and has a high yield, making it suitable for widespread application and providing various possibilities for the subsequent use of oligonucleotide antigens.

Description

Technical Field

[0001] This application relates to the field of biology, and more specifically, to a method for purifying oligonucleotides. Background Technology

[0002] DNA synthesis is a key common technology in life sciences, biomedicine, materials science, and information storage. Currently, DNA synthesis mainly relies on chemical synthesis methods, which have insurmountable limitations in terms of synthesis length and cost, failing to meet the growing demands of the biomanufacturing field. Enzymatic DNA synthesis, on the other hand, possesses unparalleled potential in terms of synthesis speed, length, efficiency, and cost compared to chemical synthesis, making it a cutting-edge direction in DNA synthesis technology development.

[0003] Enzymatic DNA synthesis is a method that relies on the catalytic activity of biological enzymes to assemble DNA. After enzymatic DNA synthesis, residual biological enzymes remain in the reaction system. These residual enzymes can affect the subsequent application of the synthesized DNA, so appropriate measures need to be taken to remove them. However, methods for removing residual biological enzymes after enzymatic DNA synthesis to purify nucleotides still need further research. Summary of the Invention

[0004] This application aims to at least partially address the technical problems existing in the prior art. To this end, this application proposes a method for purifying oligonucleotides. The method of this application can effectively remove residual biological enzymes after enzymatic DNA synthesis without affecting the biological activity of oligonucleotides. It can obtain oligonucleotide samples with high throughput and high purity. Furthermore, the operation is rapid, convenient, low-cost, and has a high yield, making it suitable for wide application and providing multiple possibilities for the subsequent use of oligonucleotide antigens.

[0005] It should be noted that this application is based on the following facts and findings:

[0006] To address the residual enzymes used in enzymatic DNA synthesis in crude oligonucleotide samples, the inventors of this application have explored various protein removal methods and principles, including denaturation and enzymatic digestion, both individually and in combination, in an attempt to remove these enzymes. Specifically, the inventors initially employed a molecular sieve method. Due to the significant molecular weight difference between polymerase proteins and oligonucleotides, their retention times differ after passing the sample through the sieve, thus achieving separation and collection of pure oligonucleotide samples. However, the results showed that polymerase and oligonucleotides elute simultaneously, indicating potential binding. Furthermore, the results of the His-tag purification polymerase method and buffer washing method also revealed incomplete protein removal and oligonucleotide loss, respectively. Therefore, direct removal of the polymerase is not feasible. The inventors also attempted to denature and inactivate the polymerase through high-temperature heating, followed by separation from the oligonucleotide antigen using an oligonucleotide purification kit, before removing the inactivated polymerase. However, this method is limited by the purification kit, resulting in low throughput; only 5 μg of sample can be purified at a time, making high-throughput sample purification impossible.

[0007] Therefore, in a first aspect of this application, a method for purifying oligonucleotides is proposed. According to an embodiment of this application, the method includes: enzymatically digesting an oligonucleotide sample after enzymatic DNA synthesis with a protease solution to obtain an enzymatic digestion product; and inactivating the enzyme in the enzymatic digestion product to obtain purified oligonucleotides; wherein the oligonucleotide sample contains a biological enzyme.

[0008] This application purifies the sample by using a protease to enzymatically digest oligonucleotide samples after enzymatic DNA synthesis to remove residual enzymes, followed by enzyme inactivation treatment of the digestion products to remove the protease itself. This method not only effectively removes enzyme residues but also supports high-throughput sample purification processes.

[0009] According to embodiments of this application, the bioenzyme includes at least one of DNA polymerase, terminal transferase, reverse transcriptase, and DNA ligase.

[0010] According to embodiments of this application, the protease includes at least one of proteinase K, trypsin, pepsin, and glycosaminoglycanase.

[0011] According to embodiments of this application, the molar ratio of the protease in the protease solution to the oligonucleotides in the oligonucleotide sample is 10% to 60%.

[0012] According to the embodiments of this application, the enzymatic hydrolysis treatment is carried out at a temperature of 55°C to 65°C for a time of 30 min to 120 min.

[0013] According to an embodiment of this application, the enzyme inactivation treatment includes heating the enzymatic hydrolysis product;

[0014] The heat treatment is performed at a temperature of 85℃ to 95℃ for a time of 20 min to 40 min.

[0015] According to an embodiment of this application, the enzymatic hydrolysis product is cooled to room temperature before the enzyme inactivation treatment.

[0016] According to an embodiment of this application, the oligonucleotide has a partially complementary first strand and a second strand, and the non-complementary region includes the 5' end of the first strand; the length of the first strand is greater than the length of the second strand, and the 3' end of the second strand is a reversible termination nucleotide with a blocking group.

[0017] According to an embodiment of this application, the first chain is 1 to 5 nucleotides longer than the second chain.

[0018] According to an embodiment of this application, the enzymatic DNA synthesis method includes: extending double-stranded DNA to be extended, single-stranded nucleic acid, DNA polymerase, and a reversible termination nucleotide with a blocking group to generate oligonucleotides.

[0019] According to embodiments of this application, the blocking group includes at least one selected from amino, allyl, thiol, phosphate ester, azide, azidomethyl, and methyl ester.

[0020] According to embodiments of this application, the reversible terminating nucleotide with a blocking group is selected from at least one of reversible terminating adenine nucleotide, reversible terminating thymine nucleotide, reversible terminating cytosine nucleotide, and reversible terminating guanine nucleotide.

[0021] According to an embodiment of this application, the nucleotides in the second strand, except for the first nucleotide at the 3' end, are the same as those in the single-stranded nucleic acid.

[0022] According to an embodiment of this application, the 5' end of the double-stranded DNA to be extended is modified with biotin.

[0023] According to an embodiment of this application, the double-stranded DNA to be extended includes at least partially complementary template strands and amplification strands; the length of the template strand is greater than the length of the amplification strand, preferably, the template strand is 1 to 5 bases longer than the amplification strand.

[0024] According to an embodiment of this application, the 3' end of the template strand and the 5' end of the amplification strand are biotin-modified.

[0025] According to an embodiment of this application, the double-stranded DNA to be extended is obtained by annealing the template strand and the amplification strand.

[0026] According to an embodiment of this application, the annealing treatment temperature is 80℃~95℃ and the time is 20min~40min.

[0027] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0028] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0029] Figure 1 The image shows an electrophoretic diagram of oligonucleotide antigen synthesis according to an embodiment of this application;

[0030] Figure 2 This diagram illustrates an oligonucleotide antigen purification-molecular sieve method according to an embodiment of this application.

[0031] Figure 3 Electrophoresis images of oligonucleotide antigen purification using the His tagging method, buffer washing method, and purification kit method according to one embodiment of this application are shown.

[0032] Figure 4 The image shows an oligonucleotide antigen purification-proteinase K electrophoresis diagram according to an embodiment of this application. Detailed Implementation

[0033] The embodiments of the technical solution of this application are described in detail below. The following embodiments are only used to illustrate the technical solution of this application more clearly, and are therefore only examples, and should not be used to limit the scope of protection of this application.

[0034] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0035] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is also expected that ranges of 60-110 and 80-120 are also included. Furthermore, if minimum range values ​​1 and 2 are listed, and if maximum range values ​​3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers, and ranges defined in this way can include endpoints a and b. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0036] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0037] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0038] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0039] In a first aspect, this application provides a method for purifying oligonucleotides. According to an embodiment of this application, the method includes: enzymatically digesting an oligonucleotide sample after enzymatic DNA synthesis with a protease solution to obtain an enzymatic digestion product; and inactivating the enzyme in the enzymatic digestion product to obtain purified oligonucleotides; wherein the oligonucleotide sample contains a biological enzyme.

[0040] This application purifies the sample by using a protease to enzymatically digest oligonucleotide samples after enzymatic DNA synthesis to remove residual enzymes, followed by enzyme inactivation treatment of the digestion products to remove the protease itself. This method not only effectively removes enzyme residues but also supports high-throughput sample purification processes.

[0041] According to embodiments of this application, the bioenzyme includes at least one of DNA polymerase, terminal transferase, reverse transcriptase, and DNA ligase.

[0042] According to embodiments of this application, the protease includes at least one of proteinase K, trypsin, pepsin, and glycosaminoglycanase.

[0043] According to embodiments of this application, the molar ratio of protease in the protease solution to oligonucleotides in the oligonucleotide sample is 10%-60%, for example, it can be 10%, 20%, 30%, 40%, 50%, or 60%. Therefore, the protease can efficiently remove residual enzymes from the sample after enzymatic DNA synthesis, ensuring sample purity and the accuracy of subsequent experiments. This avoids unnecessary cost increases and protease residues in the sample due to excessive dosage, which could affect experimental results. It also avoids insufficient dosage, which could lead to incomplete removal of residual enzymes, causing enzyme activity interference in subsequent applications and affecting the reliability and repeatability of the experiment.

[0044] According to embodiments of this application, the enzymatic hydrolysis temperature is 55℃~65℃, for example, 55℃, 56℃, 58℃, 60℃, 62℃, 64℃, or 65℃, and the time is 30min~120min, for example, 30min, 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min, or 120min. Therefore, residual enzymes in the sample after enzymatic DNA synthesis can be effectively removed, improving reaction efficiency, reducing non-specific degradation, and maintaining the integrity of the DNA sample. This avoids excessive degradation of the DNA chain or inactivation of the enzyme due to excessively high hydrolysis temperature or time, which would affect sample quality and the accuracy of subsequent experiments; it also avoids incomplete hydrolysis due to excessively low temperature or time, where residual enzyme activity is not completely removed, thus affecting sample purity and the reliability of subsequent experimental results.

[0045] According to embodiments of this application, the enzyme inactivation treatment includes heating the enzymatic hydrolysis product; the heating temperature is 85℃~95℃, for example, 85℃, 86℃, 88℃, 90℃, 92℃, 94℃, or 95℃, and the time is 20min~40min, for example, 20min, 25min, 30min, 35min, or 40min. This effectively removes proteases, reduces non-specific degradation, and maintains the integrity of the DNA sample, avoiding excessive degradation of the DNA chain due to excessively high temperatures or prolonged times, which would affect sample quality and the accuracy of subsequent experiments; it also avoids incomplete enzyme inactivation due to excessively low temperatures or short times, where residual protease activity is not completely removed, thus affecting sample purity and the reliability of subsequent experimental results.

[0046] For example, enzyme inactivation treatment can be carried out in a water bath or metal bath to provide a heat source.

[0047] According to an embodiment of this application, the enzymatic hydrolysis product is cooled to room temperature before the enzyme inactivation treatment. This terminates the enzymatic hydrolysis reaction.

[0048] In this application, the term "oligonucleotide" refers to a polymer formed by two or more nucleotides through covalent bonds. The nucleotides may be ribonucleotides (RNA) or deoxyribonucleotides (DNA), preferably deoxyribonucleotides (DNA). In some embodiments, the oligonucleotide has, for example, 6-200 nucleotides, 6-150 nucleotides, 6-100 nucleotides, 6-50 nucleotides, 10-30 nucleotides, 18-24 nucleotides, or 20-24 nucleotides.

[0049] According to embodiments of this application, the oligonucleotide has a partially complementary first and second strand, with the non-complementary region including the 5' end of the first strand; the length of the first strand is greater than the length of the second strand, and the 3' end of the second strand is a reversibly terminated nucleotide with a blocking group. In some embodiments, the first strand is 1 to 5 nucleotides longer than the second strand. Therefore, the oligonucleotide of this application can be used in sequencing cycles, and base recognition can be performed using fluorescently labeled antibodies with base specificity that can recognize 3' blocking dNTPs. After collecting the fluorescence signal, the bound antibody and the 3' blocking group on the dNTP can be removed to regenerate the natural nucleotide for the next round of sequencing. Therefore, the "oligonucleotide" of this application can also be called an "oligonucleotide antigen" or an "antigen".

[0050] According to embodiments of this application, the enzymatic DNA synthesis method includes: extending double-stranded DNA to be extended, single-stranded nucleic acid, DNA polymerase, and a reversible terminating nucleotide with a blocking group (hereinafter referred to as "blocking dNTP") to generate oligonucleotides. The single-stranded nucleic acid can be extended using one strand of the double-stranded DNA to be extended (also called the "template strand") as a template. The reaction is terminated when a reversible terminating nucleotide with a blocking group is incorporated, so that the 3' end of the generated amplified strand is a reversible terminating nucleotide with a blocking group.

[0051] For example, using a dual-color fluorescent labeling method, the specific sequencing process is described as follows: Antibodies A (an antibody or its antigen-binding fragment targeting reversibly terminating adenine nucleotides) and G (an antibody or its antigen-binding fragment targeting reversibly terminating guanine nucleotides) are labeled with a fluorescent dye excited by green light, while antibodies C (an antibody or its antigen-binding fragment targeting reversibly terminating cytosine nucleotides) and T (an antibody or its antigen-binding fragment targeting reversibly terminating thymine nucleotides) are labeled with a fluorescent dye excited by red light. During sequencing, a first hybridization is performed, and antibodies A and C obtain two signals. After washing, a second hybridization is performed, and antibodies G and T obtain two signals. Based on the basecall processing and analysis of the sequencer's built-in software, the four bases are identified.

[0052] According to embodiments of this application, the blocking group includes at least one selected from amino, allyl, thiol, phosphate ester, azide, azidomethyl, and methyl ester.

[0053] According to embodiments of this application, the reversible terminating nucleotide with a blocking group is selected from at least one of reversible terminating adenine nucleotide, reversible terminating thymine nucleotide, reversible terminating cytosine nucleotide, and reversible terminating guanine nucleotide.

[0054] According to embodiments of this application, the DNA polymerase has the activity of extending bases with blocking groups.

[0055] According to an embodiment of this application, the nucleotides in the second strand, except for the first nucleotide at the 3' end, are the same as those in the single-stranded nucleic acid.

[0056] According to embodiments of this application, the 5' end of the double-stranded DNA to be extended is modified with biotin. Thus, the strong affinity between biotin and streptavidin or avidin allows for the specific capture of DNA molecules.

[0057] According to embodiments of this application, the double-stranded DNA to be extended includes at least partially complementary template strands and amplification strands.

[0058] According to an embodiment of this application, the length of the template chain is greater than the length of the amplification chain. This facilitates amplification.

[0059] For example, the template strand is complementary to the amplification strand starting from the first to third bases at the 3' end and starting from the first to third bases at the 5' end. The 5' end of the template strand has a non-complementary region, the length of which is the number of bases by which the template strand is longer than the amplification strand, specifically 1 to 5 bases, for example, 1, 2, 3, 4, or 5 bases. The 3' end of the amplification strand is a reversible termination nucleotide with a blocking group. If this nucleotide is an A base, the oligonucleotide is called antigen A; if it is a T base, it is called antigen T; if it is a C base, it is called antigen C; and if it is a G base, it is called antigen G.

[0060] According to embodiments of this application, the 3' end of the template strand and the 5' end of the amplification strand are modified with biotin. Thus, the strong affinity between biotin and streptavidin or avidin allows for the specific capture of DNA molecules.

[0061] According to an embodiment of this application, the double-stranded DNA to be extended is obtained by annealing the template strand and the amplification strand.

[0062] According to embodiments of this application, the annealing temperature is 80℃~95℃, for example, 80℃, 82℃, 85℃, 88℃, 90℃, 92℃, or 95℃, and the time is 20min~40min, for example, 20min, 22min, 25min, 28min, 30min, 32min, 35min, 38min, or 40min. This facilitates the effective binding of the template strand and the amplification strand to form a stable double-stranded structure, while avoiding non-specific binding.

[0063] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0064] Example 1

[0065] In this embodiment, crude oligonucleotide samples were prepared according to the following method:

[0066] 1. Mix equal amounts of strand 1 (template strand) and strand 2 (amplification strand) as shown in Table 1, and anneal at 85℃ for 10 min to obtain double-stranded DNA.

[0067] Table 1. Sequence information for chain 1 and chain 2

[0068] Oligonucleotide antigen chain Sequence 5'-3' 1-strand-template-antigen A CGGATGTAGCCATGT (SEQ ID NO: 1) 1-strand-template-antigen T TCGGAGTAGCCATGT (SEQ ID NO: 2) 1-strand-template-antigen C TCGGGTAGCCATGT(SEQ ID NO: 3) 1-strand-template-antigen G GATCGTAGCCATGT(SEQ ID NO: 4) 2-strand amplification strand ACATGGCTAC (SEQ ID NO: 5)

[0069] 2. Using the CoolMPS high-throughput sequencing reagent kit (MGISEQ-2000RS FCL PE100), double-stranded DNA was prepared according to the reaction system in Table 2 and incubated at 40℃ for 4 hours to obtain oligonucleotide samples.

[0070] Table 2 Reaction System

[0071] Components Volume (μL) 1mM dNTP-3'Azido 2.5 500μM DNA double strand 2 2×PBS buffer 25 5 mg / ml DNA polymerase dominant mutant 5 <![CDATA[ddH2O]]> 15.5 Total volume 50

[0072] Note: dNTP-3'Azido is a reversible termination nucleotide with a blocking group at the 3' end.

[0073] 3. Oligonucleotide samples were analyzed by 20% μREA-PAGE gel electrophoresis. The results are as follows: Figure 1 As shown in the diagram. Taking T as an example, lane 1 is the control group, without the addition of the DNA polymerase dominant mutant; lane 2 is the experimental group, with the addition of the DNA polymerase dominant mutant for polymerase extension. The upper band in each lane represents the template 2 strand, 14-15 nt in length; the lower band represents the extension 1 strand primer, 10 nt in length. After polymerase extension, the experimental group's extension 1 strand primer length is 11 nt (modified with an Azido blocking group at the 3' end). C, G, and A are the same as T, as shown in the diagram. Figure 1 As shown, all amplified chains underwent a single-step extension reaction, and the length of almost all amplified chains changed from 10nt to 11nt, indicating that the enzymatic oligonucleotide synthesis was completed.

[0074] Furthermore, the concentration of the obtained oligonucleotide sample was measured to be 82.2 ng / μL, and the volume was 1300 μL, which translates to an oligonucleotide quantity of 82.2 × 1300 / 1000 = 107 μg. Therefore, the enzymatic DNA synthesis method of this application can be used to obtain high-throughput oligonucleotides.

[0075] Example 2

[0076] 2 μL of proteinase K (20 mg / mL) was added to 100 μL of the oligonucleotide sample (10 μM) prepared in Example 1, with a molar ratio of proteinase to oligonucleotides in the proteinase solution of 27%. The sample was heated at 58 °C for 2 h, then allowed to cool naturally to room temperature before being heated at 85 °C for 30 min in a metal bath to obtain a purified oligonucleotide sample with a purity ≥95% and a yield ≥90%.

[0077] Comparative Example 1

[0078] The purification method was adopted using molecular sieves. The AKAT pμre T25 (cytiva) purification instrument was used with 24 mL Superdex 200 increase 10 / 300GL as the molecular sieve column (cytiva) and PBS as the buffer. When the oligonucleotide sample prepared in Example 1 flowed through the column, the samples with absorption peaks of 280 nm and 254 nm were collected respectively.

[0079] Samples with absorption peaks of 280 nm and 254 nm were subjected to electrophoretic analysis, and the results are as follows: Figure 2 As shown, the top left image is the molecular sieve peak diagram, the bottom left image is the sample (protein) with an absorption peak at 254 nm, and the top right image is the sample (nucleic acid) with an absorption peak at 280 nm. The results show that nucleic acid and protein elute simultaneously in the molecular sieve diagram, and the nucleic acid gel and protein gel detection results are consistent with those of the molecular sieve method. This indicates that proteases and oligonucleotides may be bound, preventing complete removal of the protease.

[0080] Comparative Example 2

[0081] Mix 100 μg of the oligonucleotide sample prepared in Example 1 with 200 μl of His column material and incubate at room temperature for 15 minutes. Then centrifuge at 12,000 rpm for 10 minutes and collect the supernatant as the purified sample.

[0082] Comparative Example 3

[0083] The purification method using buffer washing involves directly adding 50 μl of crude oligonucleotide sample to the ZYMO purification kit (catalog number: D4060). The washing buffer is then replaced with self-prepared buffer WB1 (sequencing reagent tank, CPAS elution reagent 1, CoolMPS high-throughput sequencing reagent kit (MGISEQ-2000RS FCL PE100), catalog number: 1000018238) or SPR regeneration buffer (50 mM NaOH, 10 mM NaCl). The purified sample is obtained by following the instructions.

[0084] Comparative Example 4

[0085] The proposed method involves first denaturing and inactivating the polymerase to separate it from the oligonucleotide antigen, followed by removal of the inactivated polymerase. Specifically, the crude oligonucleotide sample is first heated at 90°C for 30 minutes to denature the polymerase. Then, the sample is purified using an oligonucleotide antigen purification kit (Zymo, catalog number D4060) according to the manufacturer's instructions to obtain the purified oligonucleotide sample.

[0086] Comparative Example 5

[0087] The difference from Example 2 is that proteinase K treatment was not performed. Instead, the crude oligonucleotide sample prepared in Example 1 was directly heated in a metal bath at 95°C for 30 minutes to obtain the purified oligonucleotide sample.

[0088] Comparative Example 6

[0089] 2 μL of proteinase K (20 mg / mL) was added to 100 μL of the oligonucleotide sample (10 μM) prepared in Example 1, with a molar ratio of proteinase to oligonucleotide in the proteinase solution of 27%. The sample was heated at 58 °C for 2 h, and then the treated sample was removed and allowed to cool naturally to room temperature to obtain the purified oligonucleotide sample.

[0090] Example 3

[0091] The oligonucleotide samples (original DNA) from Example 1, and the purified oligonucleotide samples prepared in Examples 2 and Comparative Examples 1-6 were subjected to gel electrophoresis for detection. The results are as follows: Figure 3 and Figure 4 As shown. Among them, Figure 3 The left and right images are DNA electrophoresis and protein electrophoresis images, respectively. In each image, G-DNA original sample and C-DNA original sample correspond to Example 1, G-DNA-his purification and G-DNA-his purification-1 comparative example 2 (2 parallel samples), G-DNA-WB1 washing comparative example 3 (WB1 washing), G-DNA-SPR regeneration solution washing comparative example 3 (SPR regeneration solution washing), and C-DNA-high temperature heating corresponds to Example 4. Figure 4 The left and right images are DNA electrophoresis and protein electrophoresis images, respectively. In each image, "A-DNA-original sample" corresponds to Example 1, "A-DNA-high temperature denaturation" corresponds to Example 4, "A-DNA-proteinase K" corresponds to Example 6, and "A-DNA-proteinase K + high temperature treatment" corresponds to Example 2. It can be seen that the purified oligonucleotide sample obtained by the method of this application has the least residual protein and the best purification effect.

[0092] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A method for purifying oligonucleotides, characterized in that, include: The oligonucleotide sample after enzymatic DNA synthesis was subjected to enzymatic hydrolysis with protease solution to obtain the enzymatic hydrolysis product. The enzymatic hydrolysis product was subjected to enzyme inactivation treatment to obtain purified oligonucleotides. The oligonucleotide sample contains biological enzymes.

2. The method according to claim 1, characterized in that, The biological enzyme includes at least one of DNA polymerase, terminal transferase, reverse transcriptase, and DNA ligase. Optionally, the protease includes at least one of proteinase K, trypsin, pepsin, and glycosaminoglycanase.

3. The method according to claim 1, characterized in that, The molar ratio of protease in the protease solution to oligonucleotides in the oligonucleotide sample is 10%-60%. Optionally, the enzymatic hydrolysis treatment is carried out at a temperature of 55℃ to 65℃ for a time of 30 min to 120 min.

4. The method according to claim 1, characterized in that, The enzyme inactivation treatment includes heating the enzymatic hydrolysis product. The heat treatment is performed at a temperature of 85℃ to 95℃ for a time of 20 min to 40 min. Optionally, the enzymatic hydrolysis product is cooled to room temperature before the enzyme inactivation treatment.

5. The method according to claim 1, characterized in that, The oligonucleotide has a partially complementary first strand and a second strand, and the non-complementary region includes the 5' end of the first strand; The length of the first chain is greater than the length of the second chain, and the 3' end of the second chain is a reversible termination nucleotide with a blocking group; Preferably, the first chain is 1 to 5 nucleotides longer than the second chain.

6. The method according to claim 5, characterized in that, The method for enzymatic DNA synthesis includes: The double-stranded DNA to be extended, single-stranded nucleic acid, DNA polymerase, and reversible termination nucleotides with blocking groups are subjected to extension treatment to generate oligonucleotides.

7. The method according to claim 5 or 6, characterized in that, The blocking group includes at least one of amino, allyl, thiol, phosphate ester, azide, azide methyl, and methyl ester; Optionally, the reversible terminating nucleotide with a blocking group is selected from at least one of reversible terminating adenine nucleotide, reversible terminating thymine nucleotide, reversible terminating cytosine nucleotide, and reversible terminating guanine nucleotide. Optionally, the nucleotides in the second strand, except for the first nucleotide at the 3' end, are identical to those in the single-stranded nucleic acid.

8. The method according to claim 6, characterized in that, The 5' end of the double-stranded DNA to be extended is modified with biotin.

9. The method according to claim 6, characterized in that, The double-stranded DNA to be extended includes at least partially complementary template strands and amplification strands; The template strand is longer than the amplification strand; preferably, the template strand is 1 to 5 bases longer than the amplification strand. Optionally, the 3' end of the template strand and the 5' end of the amplification strand are biotin-modified.

10. The method according to claim 6 or 9, characterized in that, The double-stranded DNA to be extended is obtained by annealing the template strand and the amplification strand. Optionally, the annealing treatment is performed at a temperature of 80°C to 95°C for a time of 20 min to 40 min.

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