Method for validating sequence of single-stranded nucleic acid
By preparing single-stranded nucleic acid into double-stranded nucleic acid and adding A to perform TA cloning and Sanger sequencing, the problem of high cost and complex operation of single-stranded nucleic acid sequence confirmation in the prior art is solved, and a low-cost and easy-to-operate sequence confirmation method is achieved.
Patent Information
- Application Number
- CN202311796786.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-25
- Publication Date
- 2025-06-27
AI Technical Summary
When confirming the nucleotide sequence of a single-stranded nucleic acid using HPLC in the prior art, the concentration and purity of the sample are high and the cost is high, making it difficult to meet the low-cost and easy-to-operate needs.
The single-stranded nucleic acid was prepared as a double-stranded nucleic acid, and A was added to the end of the double-stranded nucleic acid, and then Sanger sequencing was performed to confirm its sequence.
This method is cheap and simple to operate, can quickly complete the sequence confirmation of single-stranded nucleic acid, is highly practical, and is suitable for a wide range of application scenarios.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of gene sequencing, and particularly relates to a method for confirming the sequence of single-stranded nucleic acid. Background Art
[0002] Toll-like Receptor (TLR) is a type I transmembrane receptor and plays an important role in innate immunity against microbial invasion. Currently, 13 types of TLRs have been found in mammals, and among them, TLRs of types 1 to 10 exist in the human body. These TLR receptors activate the innate immune response by recognizing pathogen-associated pattern molecules (PAMPs) of different microorganisms. Among them, TLR 9 (Toll-like receptor type 9) can recognize nucleic acid microbial PAMPs and is a receptor for microbial unmethylated cytosine-guanine dinucleotide (CpG). The first DNA reported to have immunostimulatory effects is part of the DNA of Bacillus, and subsequent studies have shown that DNA containing unmethylated CpG deoxynucleotides has immunomodulatory functions.
[0003] Synthetic CpG ODN (CpG Oligonucleotide) is a single-stranded oligodeoxynucleotide with a thiol modification containing unmethylated CpG, which can mimic the immunostimulatory effects of bacterial CpG. CpG-ODN can stimulate a series of immune responses, including promoting the antigen presentation of DC cells, upregulating the expression of co-stimulatory molecules; promoting DC cells, monocytes, and macrophages to secrete inflammatory factors; activating B lymphocytes and promoting the secretion of immunoglobulin IgG; activating NK cells to secrete cytokines IFN-γ; and also activating T lymphocytes, initiating Th1-type cell-mediated immune responses and enhancing the differentiation of cytotoxic T lymphocytes (CTL).
[0004] As a TLR9 agonist, CpG regulates the immune response by activating TLR9, including activating the antigen presentation of DC cells, monocytes, macrophages, and NK cells, and promoting the secretion of cytokines. CpG-ODN can also activate and promote the proliferation of B cells through TLR9, upregulate the expression of Th1-type cytokines such as TNF-α, IL-6, IL-12, interferons and other cytokines and chemokines, thereby promoting the activation of T cells. In recent years, the immunomodulatory effects of CpG-ODN have been widely studied, and there are many clinical immunotherapy studies on CpG-ODN. At the end of 2017, the first hepatitis B vaccine with CpG-ODN as an adjuvant was approved for marketing in the United States, which means that the immunomodulatory effects of CpG-ODN can not only be used for clinical immunotherapy, but also be used as a novel adjuvant for the development of prophylactic vaccines. In China, there are also multiple vaccines developed based on CpG-ODN in the clinical research or clinical research stage.
[0005] As one of the important components of clinical immunotherapy or vaccines, the key points for the quality control of CpG-ODN include sequence accuracy, purity, endotoxin content and solubility, stability, bioactivity verification and batch consistency. The length of CpG-ODN is usually 10-30 bases, and any deviation in the nucleic acid sequence may affect its mechanism of action and efficacy. Therefore, ensuring that the synthesized CpG-ODN has an accurate nucleic acid sequence is the key to ensuring its consistency in terms of immunological activity or vaccine efficacy.
[0006] In the prior art, the confirmation of the nucleotide sequence of artificially synthesized single-stranded nucleic acids (such as CpG-ODN) is usually analyzed by high performance liquid chromatography (HPLC). The principle of this method is to determine the sequence by molecular weight, but this method has high requirements for equipment, certain requirements for the concentration and purity of samples, and relatively high costs. Therefore, it is very necessary to develop a method for determining the sequence of CpG-ODN with low cost and easy operation. Summary of the Invention
[0007] The technical problem to be solved by the present invention is to overcome the defects of high requirements for the concentration and purity of samples and high costs when using HPLC to confirm the nucleotide sequence of single-stranded nucleic acids in the prior art, and to provide a method for confirming the sequence of single-stranded nucleic acids. The method provided by the present invention has low cost, simple operation, can quickly complete the confirmation of the sequence of single-stranded nucleic acids, has strong practicability, and has a wide application prospect.
[0008] The present invention solves the above technical problems through the following technical solutions.
[0009] The first aspect of the present invention provides a method for confirming the sequence of single-stranded nucleic acids, the method comprising the following steps:
[0010] (1) Preparing double-stranded nucleic acids from the single-stranded nucleic acids;
[0011] (2) Adding A to the ends of the double-stranded nucleic acids and performing TA cloning;
[0012] (3) Sequencing, such as Sanger sequencing.
[0013] In some embodiments of the present invention, the single-stranded nucleic acids have 10-30 nucleotide residues.
[0014] In some preferred embodiments of the present invention, the single-stranded nucleic acids are CpG oligodeoxynucleotides.
[0015] In some specific embodiments of the present invention, the CpG oligodeoxynucleotides have the nucleotide sequences shown in SEQ ID NO:1 or SEQ ID NO:3.
[0016] In some embodiments of the present invention, step (1) includes obtaining a complementary sequence of the single-stranded oligonucleic acid and synthesizing a double-stranded nucleic acid.
[0017] In some preferred embodiments of the present invention, the starting temperature of the synthesis is 90 - 98 °C, the temperature decreases as the synthesis progresses, and the ending temperature is 35 - 40 °C; and / or, the reaction time of the synthesis is 120 - 160 min.
[0018] In some specific embodiments of the present invention, the conditions for the synthesis are to carry out the reaction according to the following conditions and sequence: at 95 °C for 5 min; 90 °C for 10 min; 85 °C for 10 min; 80 °C for 10 min; 75 °C for 10 min; 70 °C for 10 min; 65 °C for 10 min; 60 °C for 10 min; 55 °C for 10 min; 50 °C for 10 min; 45 °C for 10 min; 37 °C for 30 min.
[0019] In some embodiments of the present invention, step (2) includes the following steps:
[0020] (a) Adding a single A tail to the 3' end of the double-stranded nucleic acid obtained in step (1);
[0021] (b) Ligating the product obtained in step (a) to a T-vector;
[0022] (c) Transforming the ligation product obtained in step (b) into Escherichia coli competent cells;
[0023] (d) Plating and performing blue-white screening.
[0024] In some embodiments of the present invention, step (a) satisfies one or more of the following conditions:
[0025] In the reaction system, double-stranded nucleic acid:DNA polymerase = (10 - 20):1;
[0026] The reaction temperature is 60 - 68 °C;
[0027] The reaction time is 8 - 15 min.
[0028] In some embodiments of the present invention, the T-vector in step (b) is pMD18-T, pMD19-T, pMD20-T or pGEM-T; and / or,
[0029] The Escherichia coli competent cells in step (c) are mach-T1, DH5α, Stbl3, TOP10, JM109, DB3.1 or DH10B.
[0030] In some embodiments of the present invention, the transformation in step (c) includes the steps of mixing the ligation product with competent Escherichia coli cells, heat shock, and shaking culture.
[0031] In some specific embodiments of the present invention, the temperature of the heat shock is 40 - 42 °C; and / or, the time of the heat shock is 80 - 120 s.
[0032] In some specific embodiments of the present invention, the temperature of the shaking culture is 32 - 38 °C, such as 32, 33, 34, 35, 36, 37, and 38 °C; and / or, the time is 0.5 - 3 h, such as 0.5, 1, 1.5, 2, 2.5, and 3 h; and / or, the plate obtained by plating is cultured at 32 - 38 °C for 8 - 16 h.
[0033] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.
[0034] The reagents and raw materials used in the present invention are all commercially available.
[0035] The positive and progressive effects of the present invention are as follows:
[0036] The method provided by the present invention for confirming the sequence of single-stranded nucleic acid is low in cost, simple in operation, can quickly complete the confirmation of the sequence of single-stranded nucleic acid, has strong practicability, and has a wide application prospect. BRIEF DESCRIPTION OF THE DRAWINGS
[0037] Figure 1 It is a detection diagram of double-stranded ZJ001 by agarose gel electrophoresis.
[0038] M: DL2000 DNA Marker;
[0039] 1: Double-stranded ZJ001.
[0040] Figure 2 It is a forward sequencing signal peak diagram of ZJ001.
[0041] Figure 3 It is a reverse sequencing signal peak diagram of ZJ001.
[0042] Figure 4 It is a sequencing result comparison diagram of ZJ001.
[0043] Figure 5 It is a forward sequencing signal peak diagram and sequencing result comparison diagram of CpG1826.
[0044] Figure 6 It is a reverse sequencing signal peak diagram and sequencing result comparison diagram of CpG1826. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0045] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.
[0046] The present invention is used for sequence confirmation of CpG-ODN (taking ZJ001 and CpG1826 as examples).
[0047] Example 1 Synthesis of Complementary Sequence of ZJ001 and Preparation of Double-stranded ZJ001
[0048] According to the theoretical sequence of ZJ001 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO:1), a complementary sequence 202A-S (5'-AACGACAAAACGACAAAACGACGAA-3', SEQ ID NO:2) with an A added at the 3' end was designed.
[0049] Take ZJ001 powder and dissolve it in sterile deionized water with a final concentration of 10 mg / mL. Take 202A-S powder and dissolve it in sterile deionized water with a final concentration of 1 mg / mL. Take 10 μg each of ZJ001 and 202A-S, mix well and add 2×Phanta Max Buffer, and complete the preparation of double-stranded ZJ001 in a PCR instrument. The reaction system is shown in Table 1, and the reaction proceeds in the order shown in Table 2.
[0050] Table 1 Double-stranded DNA Reaction System
[0051] Component Volume ZJ001 1 μL 202A-S 10 μL 2×Phanta Max Buffer 15 μL <![CDATA[ddH2O]]> 4 μL
[0052] Table 2 Double-stranded DNA Preparation Conditions
[0053] Temperature Time 95℃ 5 min 90℃ 10 min 85℃ 10 min 80℃ 10 min 75℃ 10 min 70℃ 10 min 65℃ 10 min 60℃ 10 min 55℃ 10 min 50℃ 10 min 45℃ 10 min 37℃ 30 min
[0054] After incubating the ZJ001 / 202A-S mixture at 95°C for 5 min, the temperature was gradually decreased at a gradient of 10°C to 37°C and incubated for 30 min. Then, 1 μL of the sample was used for double-stranded DNA detection with 2% agarose gel. The electrophoresis results are as Figure 1 shown. Under this condition, ZJ001 and the complementary sequence 202A-S formed double-stranded DNA.
[0055] After identifying the double-stranded ZJ001 by 2% agarose gel electrophoresis, an A-overhang enzyme of Mighty TA-Cloning kit (Takara Biotechnology Co., Ltd.) was used for the A-addition reaction. The reaction system is shown in Table 3, and it was incubated at 65°C for 10 min.
[0056] Table 3 Double-stranded ZJ001 A-addition Reaction
[0057] Component Volume Double-stranded ZJ001 8 μL 10×Buffer 1 μL dATP 0.5 μL A-overhang enzyme 0.5 μL
[0058] Example 2 Construction of pMD20-ZJ001 Vector
[0059] The double-stranded ZJ001 with A added and the control provided by the Mighty TA-Cloning kit were respectively ligated to the pMD20-T vector, where the control was a positive control. The reaction system is shown in Table 4, and the ligation reaction was carried out at room temperature for 1 h.
[0060] Table 4 Ligation Reaction System of pMD20-ZJ001
[0061] Component Volume Double-stranded ZJ001 / control 4 μL / 1 μL pMD20-T vector 1 μL Ligation Mighty Mix 5 μL <![CDATA[ddH2O]]> 0 / 3 μL
[0062] After the ligation reaction, the ligation product was transformed into Trans1-T1 competent cells (Beijing TransGen Biotech Co., Ltd.; namely mach-T1 competent). The specific steps are as follows:
[0063] (1) The ligation product and Trans1-T1 competent cells were ice-bathed for 30 min;
[0064] (2) The ligation product / Trans1-T1 mixture was water-bathed at 42 °C for 90 s;
[0065] (3) 500 μL of antibiotic-free LB liquid medium was added, and the cells were cultured with shaking at 37 °C for 1 h at 150 rpm;
[0066] (4) Centrifuge at 8000 rpm for 1 min, discard the supernatant, resuspend the cells with 200 μL of LB, and spread them on an LB plate containing 24 μg / mL IPTG, 40 μg / mL X-Gal, and 100 μg / mL sodium ampicillin, and then invert and culture overnight in a 37 °C incubator for blue-white screening.
[0067] The results of blue-white screening on the LB plate containing IPTG / X-Gal showed that all the colonies on the positive control pMD20-control plate were white colonies, indicating that the enzyme ligation and transformation in this experiment were successful. There were obvious blue and white colonies on the pMD20-ZJ001 plate, and the white colonies were clones containing the target gene ZJ001.
[0068] Example 3 Confirmation of ZJ001 Sequence
[0069] Observe the colonies on the pMD20-ZJ001 or pMD20-control plates cultured overnight. The control plate as the positive control mainly shows white colonies, which is the main parameter for evaluating the enzyme ligation efficiency and an important reference in the development of this method. Randomly pick white colonies from the pMD20-ZJ001 plate into LB liquid medium (containing 100 μg / mL sodium ampicillin) and shake culture for 8 h, and perform forward and reverse sequencing with M13 forward and reverse primers (provided by Genewiz or Sangon Biotech).
[0070] Twelve white colonies were picked from the pMD20-ZJ001 plate and cultured overnight by shaking in LB liquid medium containing 100 μg / mL sodium ampicillin. The overnight cultured bacterial solutions were sent to Genewiz and Sangon Biotech for forward (M13 forward primer 5’-CAGGAAACAGCTATGAC-3, SEQ ID NO:3; M13 reverse primer sequence is 5’-CGCCAGGGTTTTCCCAGTCACGAC-3’, SEQ ID NO:4) direction sequencing respectively. Among them, 6 clones failed to sequence due to non-clonal colonies. The forward and reverse sequencing peak maps of the successfully sequenced clone ZJ001 are as shown in Figure 2 and Figure 3 shown. The signal peaks at each site are single, and the signal peaks of each clone are consistent. The comparison results of the sequences obtained by sequencing each clone with the ZJ001 theoretical sequence are as shown in Figure 4 shown, and the sequences of each clone are consistent with the theoretical sequence.
[0071] Example 4 Sequence confirmation of CpG1826
[0072] According to the theoretical sequence of CpG1826 5'-TCCATGACGTTCCTGACGTT-3’ (SEQ ID NO:5), a complementary sequence 1826-A with an A added at the 3’ end was designed (1826-A: 5’-AACGTCAGGAACGTCATGGAA-3’, SEQ ID NO:6).
[0073] The sequence confirmation of CpG1826 was carried out according to the methods described in Examples 1-3. Five clones were successfully sequenced. The forward and reverse sequencing peak maps and the comparison results of the sequences obtained by sequencing each clone with the CpG1826 theoretical sequence are as shown in Figure 5 and Figure 6 shown. The signal peaks at each site are single, the signal peaks of each clone are consistent, and the sequences of each clone are consistent with the theoretical sequence.
Claims
1. A method for verifying the sequence of a single-stranded nucleic acid, characterized in that, The method includes the following steps: (1) Prepare double-stranded nucleic acid from the single-stranded nucleic acid; (2) Add A to the ends of the double-stranded nucleic acid and perform TA cloning; (3) Sequence, such as by Sanger sequencing.
2. The method according to claim 1, characterized in that, The single-stranded nucleic acid has 10 - 30 nucleotide residues; Preferably, the single-stranded nucleic acid is a CpG oligodeoxynucleotide.
3. The method according to claim 1 or 2, characterized in that Step (1) includes the steps of obtaining the complementary sequence of the single-stranded nucleic acid and synthesizing the double-stranded nucleic acid.
4. The method according to claim 3, characterized in that, The starting temperature of the synthesis is 90 - 98 °C, the temperature decreases as the synthesis proceeds, and the ending temperature is 35 - 40 °C; and / or, the reaction time of the synthesis is 120 - 160 min.
5. The method according to any one of claims 1-4, characterized in that, Step (2) includes the following steps: (a) Add a single A end to the 3' end of the double-stranded nucleic acid obtained in step (1); (b) Ligate the product obtained in step (a) with a T-vector; (c) Transform the ligation product obtained in step (b) into competent Escherichia coli cells; (d) Plate and perform blue-white screening.
6. The method according to claim 5, wherein Step (a) satisfies one or more of the following conditions: In the reaction system, double-stranded nucleic acid:DNA polymerase = (10 - 20):1; The reaction temperature is 60 - 68 °C; The reaction time is 8 - 15 min.
7. The method according to claim 5 or 6, characterized in that, In step (b), the T-vector is pMD18-T, pMD19-T, pMD20-T or pGEM-T; and / or, In step (c), the competent Escherichia coli cells are mach-T1, DH5α, Stbl3, TOP10, JM109, DB3.1 or DH10B.
8. The method according to any one of claims 5 to 7, characterized in that The transformation in step (c) includes the steps of mixing the ligation product with competent Escherichia coli cells, heat shock, and shaking culture.
9. The method according to claim 8, wherein The temperature of the heat shock is 40 - 42 °C; and / or, the time of the heat shock is 80 - 120 s.
10. The method according to claim 8 or 9, characterized in that, The temperature of the shaking culture is 32 - 38 °C; and / or, the time is 0.5 - 3 h; and / or, the plate obtained by plating is cultured at 32 - 38 °C for 8 - 16 h.