Synthesis method of circLigase-based long single-stranded DNA and obtained long single-stranded DNA

Abstract

The invention provides a synthesis method of long single-stranded DNA (deoxyribonucleic acid) based on CircLigase and the obtained long single-stranded DNA. The synthesis method comprises the following steps: connecting DNA1 with the solid-phase surface of the magnetic bead; the DNA2 is connected with the solid-phase surface of the silicon ball; carrying out directional connection under the catalytic condition of CircLigase, and carrying out ultraviolet irradiation; and repeating the steps, and eluting to obtain a solution containing the long single-stranded DNA. The invention further discloses the long single-stranded DNA synthesized according to the synthesis method. The invention establishes a synthesis method of long single-stranded DNA based on cyclization ligase CircLigase, realizes synthesis of long single-stranded DNA by solid-liquid interface single-stranded template-free directional efficient connection, and has wide application prospects in the fields of gene editing, gene sequencing, gene transfection and drug delivery, DNA storage and information materials, DNA nanotechnology and the like.

Description

Technical Field

[0001] The present disclosure relates to the technical field of artificial synthesis of deoxyribonucleic acid, and in particular to a method for synthesizing long single-stranded DNA based on CircLigase and the obtained long single-stranded DNA. Background Art

[0002] DNA plays an important role as genetic material in natural organisms. Based on its physiological characteristics and various physical and chemical properties as a biomacromolecule, people synthesize and modify DNA, giving it the ability to be applied in many fields such as nanotechnology, polymer materials, health care and information storage. DNA synthesis and assembly are the underlying technologies for the application of functional nucleic acids, and the demand for them is increasing in many fields such as scientific research and medical applications. DNA synthesis usually uses solid-phase chemical synthesis, but there are still problems such as low efficiency, high error rate and high cost, making it difficult to synthesize long fragments of DNA. In order to overcome these limitations, researchers have developed alternative technologies including molecular assembly, cloning methods, template-independent enzyme synthesis, microarrays and rolling circle amplification technology. However, most technologies are limited by the efficiency of the extension cycle (the efficient synthesis of each nucleotide incorporated into the sequence), making it very difficult to synthesize DNA with more than 300 nucleotides.

[0003] The DNA fragment splicing technology based on the concept of molecular assembly can connect short-chain DNA synthesized by chemical and enzymatic methods into long-chain DNA of a specific sequence in a short period of time. It is a simple and effective way to quickly produce large fragments of DNA. The controllability of DNA enzymes makes it possible to assemble or splice DNA by enzymatic methods. In the cellular environment, DNA polymerase and DNA ligase can repair DNA with broken phosphate backbones, but they require the assistance of template chains. However, there are no special enzymes in nature to connect single-stranded DNA, and the sequence is prone to errors when connecting single-stranded DNA without a template, which brings technical challenges to the synthesis and further application of long single-stranded DNA splicing. Summary of the invention

[0004] The present disclosure provides a CircLigase-based synthesis method for long single-stranded DNA and the resulting long single-stranded DNA, so as to at least solve the above technical problems existing in the prior art.

[0005] According to a first aspect of the present disclosure, a method for synthesizing long single-stranded DNA based on CircLigase is provided, the method comprising the following steps:

[0006] S1: DNA1 is connected to the solid surface of magnetic beads to obtain magnetic beads connected with DNA1;

[0007] S2: DNA2 is connected to the solid phase surface of the silica sphere to obtain a silica sphere connected with DNA2, wherein the 5' end of the DNA2 is modified with a phosphate group, and the 3' end is modified with a protective sequence and a protective group connected with a PC bond;

[0008] S3: The magnetic beads connected with DNA1 and the silica balls connected with DNA2 are directional connected with DNA1 and DNA2 under CircLigase catalysis conditions, and the PC bond modified at the 3' end of DNA2 is broken under ultraviolet light to expose the hydroxyl group at the 3' end of DNA2, thereby obtaining magnetic beads with DNA1 and DNA2 sequentially connected on the surface;

[0009] S4: Repeat steps S2 and S3 to connect the next short single-stranded DNA to obtain magnetic beads connected with long single-stranded DNA;

[0010] S5: Eluting the magnetic beads connected with the long single-stranded DNA to obtain a solution containing the long single-stranded DNA.

[0011] In one embodiment, in the step S1, the 5' end of DNA1 is modified with biotin; and the magnetic beads are streptavidin-modified magnetic beads.

[0012] In a preferred embodiment, the solid phase surface connection in step S1 is to mix the DNA1 modified with biotin at the 5' end with the magnetic beads modified with streptavidin, shake them to allow the biotin to bind to the streptavidin and adsorb the magnetic beads, and discard the supernatant to obtain the magnetic beads connected with DNA1. In a specific embodiment, in the mixed system, the final concentration of DNA1 modified with biotin at the 5' end is 0.5-1.0 μmol / L, and the magnetic beads modified with streptavidin are 0.5-1.0 mg / mL; the mixed system also includes a buffer, whose final concentration is 1×B&W buffer, wherein 1×B&W is obtained by diluting 2×B&W, and 2×B&W buffer is 2×Binding&Washing buffer, comprising the following components at each concentration: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2MNaCl, 0.01%-0.1% Tween-20; the oscillation is performed in a vertical rotating mixer at room temperature for 2-4 hours; specifically, in the mixed system, the final concentration of DNA1 modified with biotin at the 5' end is 1.0 μmol / L, and the magnetic beads modified with streptavidin are 1.0 mg / mL; the oscillation is performed in a vertical rotating mixer at room temperature for 2 hours.

[0013] In one possible embodiment, the protection sequence connected by the PC bond in step S2 is 10 cytosine deoxynucleotides and the protection group is a thiol group; and the silica sphere is an amino-modified silica sphere.

[0014] In a preferred embodiment, the solid phase surface connection in step S2 is to mix DNA2 modified with a phosphate group at the 5' end and 10 cytosine deoxynucleotides and thiol groups connected by PC bonds at the 3' end with amino-modified silica balls, shake them so that the thiol groups are combined with the amino groups, centrifuge them, and discard the supernatant to obtain silica balls connected with DNA2. In a specific embodiment, in the mixed system, the final concentration of DNA2 modified with a phosphate group at the 5' end and 10 cytosine deoxynucleotides and thiol groups connected by PC bonds at the 3' end is 0.5-1μmol / L, and the final concentration of amino-modified silica balls is 0.5-1mg / mL; the shaking is to shake in a vertical rotating mixer at room temperature for 2-4 hours; the centrifugation is 8000g centrifugation for 10 minutes. Specifically, the final concentration of DNA2 modified with a phosphate group at the 5' end and 10 cytosine deoxynucleotides and a thiol group connected by PC bonds at the 3' end was 1 μmol / L, and the final concentration of the amino-modified silica spheres was 1 mg / mL; the oscillation was performed in a vertical rotating mixer at room temperature for 2 hours.

[0015] In one possible embodiment, the directional connection in step S3 is to mix the magnetic beads connected to DNA1, the silica spheres connected to DNA2, and CircLigase, incubate and oscillate, so that the DNA1 connected to the magnetic beads and the DNA2 connected to the silica spheres are directionally connected under the catalytic conditions of CircLigase; in a preferred embodiment, the final concentration of the magnetic beads connected to DNA1 in the mixed system is 0.05-0.10 mg / mL, the final concentration of the silica spheres connected to DNA2 is 0.05-0.10 mg / mL, and the final concentration of CircLigase is 5U / μL; the incubation temperature is 60°C, and the time is 1-2h; the oscillation is to react at 10°C in a vertical rotating mixer for 10-12h to prevent precipitation of the magnetic beads. In a specific embodiment, the final concentration of the magnetic beads connected to DNA1 in the mixed system is 0.05 mg / mL, the final concentration of the silica spheres connected to DNA2 is 0.05 mg / mL, and the final concentration of CircLigase is 5 U / μL; the incubation temperature is 60°C and the time is 1 h; the oscillation is carried out in a vertical rotating mixer at 10°C for 12 h.

[0016] In one embodiment, the ultraviolet irradiation in step S3 is performed by irradiating with ultraviolet light for 10-20 minutes.

[0017] In a preferred embodiment, the ultraviolet irradiation in step S3 is performed by irradiating with ultraviolet light for 10 minutes.

[0018] In one embodiment, the lengths of DNA1, DNA2 and short single-stranded DNA are all 60 to 100 nt. In a specific embodiment, the lengths of DNA1, DNA2 and short single-stranded DNA are all 80 nt.

[0019] In one embodiment, step S4 repeats steps S2 and S3 for ≥ 3 times, so that the long single-stranded DNA is formed by connecting ≥ 5 short single-stranded DNAs, so that the length of the long single-stranded DNA is more than 400 nt. In a specific embodiment, the short single-stranded DNA is DNA3, DNA4, and DNA5.

[0020] In one embodiment, the elution in step S5 is to add a 10mM EDTA, pH 8.2, 95% formamide solution to the magnetic beads connected to the long single-stranded DNA, incubate at 95°C for 5-10 minutes, elute the biotinylated nucleic acid into the solution, adsorb the magnetic beads, collect the supernatant, and obtain a solution containing the long single-stranded DNA. In a specific embodiment, the elution in step S6 is to add a 10mM EDTA, pH 8.2, 95% formamide solution to the magnetic beads connected to the long single-stranded DNA, and incubate at 95°C for 5 minutes.

[0021] In a preferred embodiment, the magnetic beads are ferroferric oxide magnetic nanoparticles with a diameter of 50-100 nm; in a specific embodiment, the magnetic beads are ferroferric oxide magnetic nanoparticles with a diameter of 100 nm.

[0022] In a preferred embodiment, the silicon spheres are silicon dioxide nanoparticles with a diameter of 60-100 nm; in a specific embodiment, the silicon spheres are silicon dioxide nanoparticles with a diameter of 60 nm.

[0023] In the present disclosure, the cyclization ligase catalyzes the self-cyclization of single-stranded DNA due to its cyclization ability, and the cyclization reaction requires that the catalytic substrate single-stranded DNA has a 5'-phosphate group and a 3' hydroxyl group. The present disclosure discloses a method for synthesizing long single-stranded DNA based on cyclization ligase. After one end of the short single-stranded DNA is sealed with magnetic beads or silica balls, the cyclization ligase is used to connect, which solves the self-cyclization problem and ensures the directionality after connection. The simplicity, effectiveness and reversibility of the closure are important steps for successful connection. The present disclosure adopts two connection and closure schemes: 1) Biotin-Streptavidin: Biotin is a hapten molecule that can be combined with streptavidin by affinity. Formamide can destroy the combination of biotin and streptavidin under high temperature environment (60-95°C), and can be broken in a 95°C formamide solution; 2) Photocleavable linker (PC): PC is a non-nucleoside molecule that can connect two nucleotide chains through a short UV light-cleavable C3 spacer arm. It can be cleaved under ultraviolet light to obtain two nucleotide chains, exposing the 3' hydroxyl group. These schemes realize the template-free directional and efficient connection of single-stranded DNA at the solid-liquid interface to achieve the purpose of synthesizing long single-stranded DNA.

[0024] According to a second aspect of the present disclosure, a long single-stranded DNA synthesized according to the above synthesis method is provided.

[0025] According to an implementable embodiment of the present disclosure, at least the following beneficial effects are achieved:

[0026] The present invention discloses a method for synthesizing long single-stranded DNA based on CircLigase, which realizes the efficient and template-free directional connection of single-stranded DNA at the solid-liquid interface, improves the shortcomings of existing chemically synthesized DNA single-stranded fragments, and greatly extends the length of DNA single-stranded synthesis. In addition, the present invention discloses a single-stranded DNA template-free enzymatic connection technology, which does not require the introduction of a template chain to connect two short single-stranded DNAs end to end using an enzymatic method, has the advantages of safety and high product purity, and has broad application prospects in the fields of gene editing, gene sequencing, gene transfection and drug delivery, DNA storage and information materials, DNA nanotechnology, etc.

[0027] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present disclosure, nor is it intended to limit the scope of the present disclosure. Other features of the present disclosure will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The above and other objects, features and advantages of the exemplary embodiments of the present disclosure will become readily understood by reading the detailed description below with reference to the accompanying drawings. In the accompanying drawings, several embodiments of the present disclosure are shown in an exemplary and non-limiting manner, in which:

[0029] In the drawings, the same or corresponding reference numerals represent the same or corresponding parts.

[0030] Figure 1 A schematic diagram of the template-free enzymatic synthesis of long single-stranded DNA disclosed in the present invention is shown;

[0031] Figure 2 The electrophoresis diagram of the ligation product DNA1-2 of DNA1 and DNA2 disclosed in the present invention is shown;

[0032] Figure 3 The electrophoresis diagram of DNA1-3, a ligation product of DNA1, DNA2 and DNA3 disclosed in the present invention, is shown;

[0033] Figure 4 The electrophoresis diagram of DNA1-4, a ligation product of DNA1, DNA2, DNA3 and DNA4 of the present disclosure, is shown;

[0034] Figure 5 The electrophoresis diagram of DNA1-5, the ligation products of DNA1, DNA2, DNA3, DNA4 and DNA5 of the present disclosure, is shown;

[0035] Figure 6 Shown are electrophoretic diagrams of the PCR products of DNA1, DNA1-2, DNA1-3, DNA1-4, and DNA1-5 of the present disclosure. DETAILED DESCRIPTION

[0036] In order to make the purpose, features, and advantages of the present disclosure more obvious and easy to understand, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present disclosure.

[0037] Details of some of the materials used in the following examples of this disclosure are as follows:

[0038] 1. The magnetic beads are streptavidin-modified iron oxide magnetic nanoparticles (SA@Fe3O4) with a diameter of 100 nm;

[0039] 2. The silica spheres are amino-modified silica nanoparticles with a diameter of 60 nm;

[0040] 3. The length of DNA1 is 80 nt, and the sequence is shown in SEQ ID NO.1. Its 5' end is modified with biotin. For details, see Table 1. It was ordered from Shanghai Biotechnology Co., Ltd.

[0041] 4. The chain lengths of DNA2, DNA3, DNA4 and DNA5 are all 80 nt, and the sequences are represented by SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively. The 5' end is modified with a phosphate group, and the 3' end is modified with 10 cytosine deoxynucleotides and a thiol group connected by PC bonds at the C3 position of deoxyribose. The length of the protection sequence is 10 nt. For detailed information, see Table 1. The order was placed at Shanghai Bioengineering.

[0042] Table 1 Sequences and uses of short single-stranded DNA

[0043]

[0044]

[0045] The preparation methods of some materials in the following examples of this disclosure are as follows:

[0046] 1. Preparation of streptavidin-modified Fe3O4 magnetic nanoparticles linked to DNA1 (DNA1-SA@Fe3O4)

[0047] Establish a solid surface connection between magnetic beads and DNA1 to obtain SA@Fe3O4 connected with DNA1 (DNA1-SA@Fe3O4). The specific steps are as follows:

[0048] The components were mixed according to Table 2 and shaken on a vertical rotating mixer for 2 h (to prevent the magnetic beads from settling). After the reaction, the magnetic beads were adsorbed using a magnet, the supernatant was discarded, and the beads were resuspended with 50 μL of ultrapure water to obtain SA@Fe3O4 connected with DNA1 (DNA1-SA@Fe3O4).

[0049] Table 2 Solid-phase surface connection system of magnetic beads and DNA1

[0050]

[0051] The 2×B&W buffer (Binding&Washing buffer (2×)) is made of the following components at different concentrations: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl, and 0.01%-0.1% Tween-20.

[0052] 2. Preparation of silica spheres connected with DNA2 / DNA3 / DNA4 / DNA5 (DNA2 / DNA3 / DNA4 / DNA5-SiO2)

[0053] Establishing the connection between the silicon ball and the solid surface of DNA2, DNA3, DNA4 and DNA5 to obtain the silicon dioxide ball connected with DNA2 / DNA3 / DNA4 / DNA5 (DNA2 / DNA3 / DNA4 / DNA5-SiO2), the specific steps are as follows:

[0054] The components were mixed according to Table 3, shaken on a vertical rotating mixer for 2 h (to prevent the silica spheres from settling), so that the thiol group was combined with the amino group, centrifuged at 8000 g for 10 min, the supernatant was discarded, and the precipitate was resuspended with 50 μL of ultrapure water to obtain silica spheres connected with DNA2 / DNA3 / DNA4 / DNA5 (DNA2 / DNA3 / DNA4 / DNA5-SiO2), wherein the 5' end of DNA2 / DNA3 / DNA4 / DNA5 was modified with a phosphate group, and the 3' end was modified with 10 cytosine deoxynucleotides connected by PC bonds and a thiol group.

[0055] Table 3 Solid-phase surface connection system of silica spheres and DNA2, DNA3, DNA4 and DNA5

[0056]

[0057] The present invention discloses a schematic diagram of a method for synthesizing a long single-stranded DNA based on a circular ligase CircLigase, such as Figure 1As shown, the main steps are as follows:

[0058] The magnetic beads connected with DNA1 and the silica balls connected with DNA2 were directionally connected under CircLigase catalysis conditions, and UV light was irradiated to break the PC bond modified at the 3' end of DNA2, exposing the hydroxyl group at the 3' end, and obtaining magnetic beads with DNA1 and DNA2 sequentially connected on the surface;

[0059] Repeat the above steps three times, connect DNA3, DNA4, and DNA5 in sequence, and obtain magnetic beads connected with DNA1-5;

[0060] After elution, long single-stranded DNA 1-5 was obtained.

[0061] The following is a detailed description through the following specific embodiments.

[0062] Example 1

[0063] This example explores the effects of three DNA ligases (T4 DNA ligase, T4 RNA ligase, and CircLigase) on the template-free enzymatic ligation of DNA1 and DNA2. The specific steps are as follows:

[0064] 1. Prepare the following three systems: a1. CircLigase reaction system as shown in Table 4-1, the final concentration of each component is: DNA1-SA@Fe3O4 with a final concentration of 0.05 mg / mL, DNA2-SiO2 with a final concentration of 0.05 mg / mL, 1×CircLigase buffer with a final concentration of 1.5 mM MnCl2, and CircLigase with a final concentration of 5 U / μL; a2. T4 DNA ligase reaction system as shown in Table 4-2, the final concentration of each component is: DNA1-SA@Fe3O4 with a final concentration of 0.05 mg / mL, DNA2-SiO2 with a final concentration of 0.05 mg / mL, 1×T4 DNA buffer with a final concentration of 15% (v / v) PEG8000, and T4 DNA ligase with a final concentration of 5 U / μL; a3. T4 The RNA ligase reaction system is shown in Table 4-3. The final concentrations of each component are: DNA1-SA@Fe3O4 with a final concentration of 0.05 mg / mL, DNA2-SiO2 with a final concentration of 0.05 mg / mL, 1×T4 RNA buffer with a final concentration of 15% (v / v) PEG8000, ATP with a final concentration of 0.5 mM and T4 RNA ligase with a final concentration of 5 U / μL.

[0065] Table 4-1 Components of the CircLigase reaction system for template-free enzymatic ligation of DNA1 and DNA2

[0066]

[0067] Table 4-2 Components of T4 DNA ligase reaction system for template-free enzymatic ligation of DNA1 and DNA2

[0068]

[0069] Table 4-3 Components of T4 RNA ligase reaction system for template-free enzymatic ligation of DNA1 and DNA2

[0070]

[0071] 2. The three reaction systems were incubated at 60°C for 60 min, and incubated in a vertical rotating mixer at 10°C overnight to allow DNA1 and DNA2 to be directionally connected under the catalytic action of the three DNA ligases. The magnetic beads were adsorbed using a magnet, the supernatant was discarded, and the beads were resuspended in 100 μL of deionized water and irradiated with ultraviolet light for 10 min to break the PC bond modified at the 3' end of DNA2 and expose the hydroxyl group at the 3' end. The supernatant was discarded to obtain magnetic beads (DNA1-2-SA@Fe3O4) with DNA1 and DNA2 sequentially connected on the surface.

[0072] 3. Resuspend with 20 μL 10 mM EDTA, pH 8.2, 95% formamide solution and incubate at 95°C for 5 min to elute the biotinylated nucleic acid into the solution. Use a magnet to adsorb the magnetic beads and collect the supernatant to obtain a solution containing DNA 1-2.

[0073] 4. The synthesis of DNA 1-2 was verified by 12% polyacrylamide gel electrophoresis. Electrophoresis conditions: voltage, 120 V; time, 120 min.

[0074] When DNA1 and DNA2 are connected under the action of three DNA ligases (T4 DNA ligase, T4 RNA ligase and CircLigase), the gel electrophoresis diagram of DNA1-2 is as follows: Figure 2 As shown, the results showed that under the action of three DNA ligases, some fragments of DNA1 and DNA2 could be connected.

[0075] Example 2

[0076] This example explores the effects of three DNA ligases (T4 DNA ligase, T4 RNA ligase, and CircLigase) on the template-free enzymatic ligation of DNA1-2 and DNA3. The specific steps are as follows:

[0077] 1. Prepare the following three systems: a1. CircLigase reaction system as shown in Table 5-1, the final concentration of each component is: DNA1-2-SA@Fe3O4 with a final concentration of 0.05 mg / mL (obtained in step 3 of Example 1), DNA3-SiO2 with a final concentration of 0.05 mg / mL, 1×CircLigase buffer with a final concentration, MnCl2 with a final concentration of 2.5 mM, and CircLigase with a final concentration of 5 U / μL; a2. T4 DNA ligase reaction system as shown in Table 5-2, the final concentration of each component is: DNA1-2-SA@Fe3O4 with a final concentration of 0.05 mg / mL, DNA3-SiO2 with a final concentration of 0.05 mg / mL, 1×T4 DNA buffer with a final concentration, PEG8000 with a final concentration of 15% (v / v), and T4 DNA ligase with a final concentration of 5 U / μL; a3. T4 The RNA ligase reaction system is shown in Table 5-3. The final concentrations of each component are: DNA1-2-SA@Fe3O4 with a final concentration of 0.05 mg / mL, DNA3-SiO2 with a final concentration of 0.05 mg / mL, 1×T4 RNA buffer with a final concentration of 15% (v / v) PEG8000, ATP with a final concentration of 0.5 mM and T4 RNA ligase with a final concentration of 5 U / μL.

[0078] Table 5-1 Components of the CircLigase reaction system for template-free enzymatic ligation of DNA1-2 and DNA3

[0079]

[0080] Table 5-2 Components of T4 DNA ligase reaction system for template-free enzymatic ligation of DNA1-2 and DNA3

[0081]

[0082] Table 5-3 Components of T4 RNA ligase reaction system for template-free enzymatic ligation of DNA1-2 and DNA3

[0083]

[0084] 2. The three reaction systems were incubated at 60°C for 60 min, and then incubated in a vertical rotating mixer at 10°C overnight to allow DNA1-2 and DNA3 to be directionally connected under the catalytic action of the three DNA ligases. The magnetic beads were adsorbed using a magnet, the supernatant was discarded, and the beads were resuspended in 100 μL of deionized water and irradiated with ultraviolet light for 10 min to break the PC bond modified at the 3' end of DNA3 and expose the hydroxyl group at the 3' end. The supernatant was discarded to obtain magnetic beads (DNA1-3-SA@Fe3O4) with DNA1-2 and DNA3 sequentially connected on the surface.

[0085] 3. Resuspend DNA1-3-SA@Fe3O4 with 20 μL 10 mM EDTA, pH 8.2, 95% formamide solution and incubate at 95°C for 5 min to elute the biotinylated nucleic acid into the solution. Use a magnetic stand to adsorb the magnetic beads and collect the supernatant to obtain a solution containing DNA1-3.

[0086] 4. The synthesis of DNA 1-3 was verified by 12% polyacrylamide gel electrophoresis. Electrophoresis conditions: voltage, 120 V; time, 120 min.

[0087] When DNA1-2 and DNA3 were connected under the action of three DNA ligases (T4 DNA ligase, T4 RNA ligase and CircLigase), the gel electrophoresis diagram of DNA1-3 was as follows: Figure 3 As shown, the results showed that under the action of CircLigase, some fragments of DNA1-2 and DNA3 could be connected, under the action of T4 DNA ligase, DNA1-2 and DNA3 were not obviously connected, and under the action of T4RNA ligase, DNA1-2 and DNA3 were not connected.

[0088] Example 3

[0089] In this example, T4 RNA ligase was abandoned, and the effects of two DNA ligases (T4 DNA ligase and CircLigase) on the template-free enzymatic ligation of DNA1-3 and DNA4 were explored. The specific steps are as follows:

[0090] 1. Prepare the following two systems: a1. CircLigase reaction system as shown in Table 6, the final concentration of each component is: DNA1-3-SA@Fe3O4 with a final concentration of 0.05 mg / mL (obtained in step 3 of Example 2), DNA4-SiO2 with a final concentration of 0.05 mg / mL, 1×CircLigase buffer with a final concentration, MnCl2 with a final concentration of 2.5 mM and CircLigase with a final concentration of 5 U / μL; a2. T4 DNA ligase reaction system as shown in Table 5-2, the final concentration of each component is: DNA1-2-SA@Fe3O4 with a final concentration of 0.05 mg / mL, DNA3-SiO2 with a final concentration of 0.05 mg / mL, 1×T4 DNA buffer with a final concentration, 15% (v / v) PEG8000 and 5 U / μL T4 DNA ligase.

[0091] Table 6-1 Components of the CircLigase reaction system for template-free enzymatic ligation of DNA1-3 and DNA4

[0092]

[0093] Table 6-2 Components of T4 DNA ligase reaction system for template-free enzymatic ligation of DNA1-3 and DNA4

[0094]

[0095] 2. The two reaction systems were incubated at 60°C for 60 min respectively, and then incubated in a vertical rotating mixer at 10°C overnight to allow DNA1-3 and DNA4 to be directionally connected under the catalytic action of the two DNA ligases. The magnetic beads were adsorbed using a magnet, the supernatant was discarded, and the beads were resuspended in 100 μL of deionized water and irradiated with ultraviolet light for 10 min to break the PC bond modified at the 3' end of DNA4 and expose the hydroxyl group at the 3' end. The supernatant was discarded to obtain magnetic beads (DNA1-4-SA@Fe3O4) with DNA1-3 and DNA4 sequentially connected on the surface.

[0096] 3. Resuspend DNA1-4-SA@Fe3O4 with 100 μL deionized water and irradiate with ultraviolet light for 10 min to break the PC bond modified at the 3' end of DNA4 and expose the hydroxyl group at the 3' end. Discard the supernatant to obtain DNA1-4-SA@Fe3O4.

[0097] 4. Resuspend DNA1-4-SA@Fe3O4 with 20 μL 10 mM EDTA, pH 8.2, 95% formamide solution and incubate at 95°C for 5 min to elute the biotinylated nucleic acid into the solution. Use a magnetic stand to adsorb the magnetic beads and collect the supernatant to obtain a solution containing DNA1-4.

[0098] 5. The synthesis of DNA 1-4 was verified by 12% polyacrylamide gel electrophoresis. Electrophoresis conditions: voltage, 120 V; time, 120 min.

[0099] When DNA1-3 and DNA4 were connected under the action of two DNA ligases (T4 DNA ligase and CircLigase), the gel electrophoresis of DNA1-4 was as follows: Figure 4 As shown, the results showed that only under the action of CircLigase, some fragments of DNA1-3 and DNA4 could be connected.

[0100] Example 4

[0101] In this example, T4 DNA ligase was abandoned and the effect of CircLigase on the template-free enzymatic ligation of DNA1-4 and DNA5 was explored. The specific steps are as follows:

[0102] 1. Prepare the following CircLigase reaction system as shown in Table 7: DNA1-4-SA@Fe3O4 with a final concentration of 0.05 mg / mL (obtained in step 3 of Example 3), DNA5-SiO2 with a final concentration of 0.05 mg / mL, 1×CircLigase buffer with a final concentration, MnCl2 with a final concentration of 2.5 mM, and CircLigase with a final concentration of 5 U / μL.

[0103] Table 7 Components of the CircLigase reaction system for template-free enzymatic ligation of DNA1-4 and DNA5

[0104]

[0105] 2. Incubate the CircLigase reaction system at 60°C for 60 min and incubate in a vertical rotating mixer at 10°C overnight to allow DNA1-4 and DNA5 to be directionally connected under the catalytic action of CircLigase. Use a magnet to adsorb the magnetic beads, discard the supernatant, resuspend with 100 μL of deionized water, and irradiate with ultraviolet light for 10 min to break the PC bond modified at the 3' end of DNA5 and expose the hydroxyl group at the 3' end. Discard the supernatant to obtain magnetic beads (DNA1-5-SA@Fe3O4) with DNA1-3 and DNA5 sequentially connected on the surface.

[0106] 3. Resuspend DNA1-5-SA@Fe3O4 with 20 μL 10 mM EDTA, pH 8.2, 95% formamide solution and incubate at 95°C for 5 min to elute the biotinylated nucleic acid into the solution. Use a magnetic stand to adsorb the magnetic beads and collect the supernatant to obtain a solution containing DNA1-5.

[0107] 4. The synthesis of DNA 1-5 was verified by 12% polyacrylamide gel electrophoresis. Electrophoresis conditions: voltage, 120 V; time, 120 min.

[0108] When DNA1-4 and DNA5 were connected under the action of CircLigase, the gel electrophoresis of DNA1-5 was as follows: Figure 5 As shown, the results showed that under the action of CircLigase, some fragments of DNA1-4 and DNA5 could be connected.

[0109] Test example

[0110] This test example verifies the DNA ligation product obtained under the action of CircLigase in Examples 1-4, and the specific steps are as follows:

[0111] 1. Primers capable of amplifying DNA1, DNA1-2, DNA1-3, DNA1-4 and DNA1-5 obtained under the action of CircLigase in Example 1-4 were designed based on DNA1, DNA2, DNA3, DNA4 and DNA5, respectively, as shown in Table 8.

[0112] Table 8 Primer sequences and uses

[0113]

[0114] 2. Mix the components according to Tables 9-13 to prepare PCR systems of DNA1, DNA1-2, DNA1-3, DNA1-4 and DNA1-5 respectively.

[0115] Table 9 PCR system of DNA1

[0116]

[0117] Table 10 PCR system of DNA1-2

[0118]

[0119] Table 11 PCR system of DNA1-3

[0120]

[0121] Table 12 PCR system of DNA1-4

[0122]

[0123] Table 13 PCR system of DNA1-5

[0124]

[0125] 3. Place the prepared PCR systems of DNA1, DNA1-2, DNA1-3, DNA1-4 and DNA1-5 in a PCR instrument for PCR amplification to obtain PCR products. The PCR amplification procedures of each DNA are as follows:

[0126] PCR amplification procedure for DNA1

[0127] ①94℃ 3min

[0128] ②94℃ 30s

[0129] ③55℃ 30s

[0130] ④72℃ 10s

[0131] ⑤72℃ 5min

[0132] Step 4 returns to step 2 and repeats for 35 times.

[0133] PCR amplification procedure for DNA1-2

[0134] ①94℃ 3min

[0135] ②94℃ 30s

[0136] ③55℃ 30s

[0137] ④72℃ 20s

[0138] ⑤72℃ 5min

[0139] Step 4 returns to step 2 and repeats for 35 times.

[0140] PCR amplification procedure for DNA1-3

[0141] ①94℃ 3min

[0142] ②94℃ 30s

[0143] ③55℃ 30s

[0144] ④72℃ 30s

[0145] ⑤72℃ 5min

[0146] Step 4 returns to step 2 and repeats for 35 times.

[0147] PCR amplification procedure for DNA1-4

[0148] ①94℃ 3min

[0149] ②94℃ 30s

[0150] ③55℃ 30s

[0151] ④72℃ 40s

[0152] ⑤72℃ 5min

[0153] Step 4 returns to step 2 and repeats for 35 times.

[0154] PCR amplification procedure for DNA 1-5

[0155] ①94℃ 3min

[0156] ②94℃ 30s

[0157] ③55℃ 30s

[0158] ④72℃ 50s

[0159] ⑤72℃ 5min

[0160] Step 4 returns to step 2 and repeats for 35 times.

[0161] 4. The PCR product was verified by 2% agarose gel electrophoresis, and the target band was excised and purified. Then the purified target product was verified again by 2% agarose gel electrophoresis. The results are as follows: Figure 6 As shown, the results showed that the positions of the corresponding target bands of DNA1-2, DNA1-3, DNA1-4 and DNA1-5 were all displayed, proving that DNA1-2, DNA1-3, DNA1-4 and DNA1-5 were successfully connected under the action of CircLigase.

[0162] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps recorded in this disclosure can be executed in parallel, sequentially or in different orders, as long as the desired results of the technical solution of this disclosure can be achieved, and this document is not limited here.

[0163] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present disclosure, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0164] The above is only a specific embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present disclosure, which should be included in the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be based on the protection scope of the claims.

Claims

1. A method for synthesizing long single-stranded DNA based on CircLigase, characterized in that: The synthesis method comprises the following steps: S1: DNA1 is connected to the solid surface of magnetic beads to obtain magnetic beads connected with DNA1; S2: DNA2 is connected to the solid phase surface of the silica sphere to obtain a silica sphere connected with DNA2, wherein the 5' end of the DNA2 is modified with a phosphate group, and the 3' end is modified with a protective sequence and a protective group connected with a PC bond; S3: The magnetic beads connected with DNA1 and the silica balls connected with DNA2 are directional connected with DNA1 and DNA2 under CircLigase catalysis conditions, and the PC bond modified at the 3' end of DNA2 is broken under ultraviolet light to expose the hydroxyl group at the 3' end of DNA2, thereby obtaining magnetic beads with DNA1 and DNA2 sequentially connected on the surface; S4: Repeat steps S2 and S3 to connect the next short single-stranded DNA to obtain magnetic beads connected with long single-stranded DNA; S5: Eluting the magnetic beads connected with the long single-stranded DNA to obtain a solution containing the long single-stranded DNA.

2. The synthesis method according to claim 1, characterized in that In step S1, the 5' end of DNA1 is modified with biotin; the magnetic beads are streptavidin-modified magnetic beads; Preferably, in step S1, solid phase surface connection is to mix the DNA1 modified with biotin at the 5' end with the magnetic beads modified with streptavidin, shake them to allow the biotin to bind to the streptavidin and adsorb the magnetic beads, and discard the supernatant to obtain the magnetic beads connected with the DNA1.

3. The synthesis method according to claim 2, characterized in that In the mixed system, the final concentration of DNA1 modified with biotin at the 5' end is 0.5-1.0 μmol / L, and the final concentration of magnetic beads modified with streptavidin is 0.5-1.0 mg / mL; Preferably, the shaking is performed in a vertical rotary mixer for 2-4 hours.

4. The synthesis method according to claim 1, characterized in that The protection sequence connected by the PC bond in step S2 is 10 cytosine deoxynucleotides and the protection group is a thiol group; the silica ball is an amino-modified silica ball; Preferably, in step S2, solid-phase surface connection is to mix DNA2 whose 5' end is modified with a phosphate group and whose 3' end is modified with 10 cytosine deoxynucleotides and a thiol group connected by PC bonds with amino-modified silica balls, shake them so that the thiol groups are combined with the amino groups, centrifuge them, discard the supernatant, and obtain silica balls connected with DNA2.

5. The synthesis method according to claim 4, characterized in that In the mixed system, the final concentration of DNA2 modified with a phosphate group at the 5' end and a protection sequence and a thiol group connected by a PC bond at the 3' end is 0.5-1 μmol / L, and the final concentration of the amino-modified silica sphere is 0.5-1.0 mg / mL; Preferably, the shaking is performed in a vertical rotary mixer at room temperature for 2-4 hours; Preferably, the centrifugation is performed at 8000 g for 10 min.

6. The synthesis method according to claim 1, characterized in that Step S3 directional connection is to mix the magnetic beads connected with DNA1, the silica balls connected with DNA2, and CircLigase, incubate, and shake, so that the DNA1 connected with the magnetic beads and the DNA2 connected with the silica balls are directionally connected under the catalytic conditions of CircLigase; Preferably, in the mixed system, the final concentration of the magnetic beads connected to DNA1 is 0.05-0.1 mg / mL, the final concentration of the silica spheres connected to DNA2 is 0.05-0.1 mg / mL, and the final concentration of CircLigase is 5 U / μL; Preferably, the incubation temperature is 60°C and the time is 1-2h; Preferably, the shaking is carried out in a vertical rotary mixer at 10°C for 10-12 hours.

7. The synthesis method according to claim 1, characterized in that In step S3, ultraviolet light irradiation is performed for 10-20 minutes.

8. The synthesis method according to claim 1, characterized in that The lengths of the DNA1, DNA2 and short single-stranded DNA are all 60 to 100 nt; Preferably, step S4 repeats steps S2 and S3 for a number of times ≥ 3.

9. The synthesis method according to claim 1, characterized in that In step S5, elution is performed by adding 10 mM EDTA, pH 8.2, 95% formamide solution to the magnetic beads connected with the long single-stranded DNA, incubating at 95° C. for 5-10 min, adsorbing the magnetic beads, collecting the supernatant, and obtaining a solution containing the long single-stranded DNA.

10. The long single-stranded DNA synthesized according to the synthesis method according to any one of claims 1 to 9.

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