DNA connection buffer solution and application thereof

Abstract

The invention relates to a DNA (deoxyribonucleic acid) ligation buffer solution and application thereof, the DNA ligation buffer solution comprises 5-15 mM of divalent metal ions, 0.5-5 mM of DTT (dithiothreitol), 10-15% of polyethylene glycol, 0.5-5 mM of ATP (adenosine triphosphate) and a Tris-HCl buffer solution, and the pH (potential of hydrogen) value of the Tris-HCl buffer solution is 7-9. The DNA ligation buffer solution can significantly improve the ligation reaction efficiency, and increase ligation products in the ligation reaction and the number of molecules capable of being sequenced during library construction.

Description

DNA ligation buffer and its application Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to a DNA ligation buffer and applications thereof. Background Art

[0002] DNA ligation reactions are widely used in the field of molecular biology, such as vector construction for gene cloning and library construction for gene sequencing. The construction of sequencing libraries usually requires high ligation efficiency in a relatively short period of time. One optimization direction of existing commercial ligation kits is to use additives that increase the crowded environment between molecules, such as the polyethylene glycol (PEG) series, which has also been used in some commercial ligation kits for TA-ligation-based library construction. The ligation efficiency determines the number of library molecules that can be sequenced, and for the TA ligation reaction during library construction, the rapid ligation kits containing PEG on the market have low reaction efficiency in a short period of time, which limits the efficiency of library construction. Therefore, optimization of ligation efficiency is an important optimization direction of library construction methods.

[0003] Therefore, there is an urgent need to develop a DNA ligation buffer that can improve the efficiency of the ligation reaction.

[0004] Summary of the Invention

[0005] The present disclosure aims to address, at least to some extent, one of the technical problems in the related art. To this end, one object of the present disclosure is to provide a DNA ligation buffer. This buffer can significantly improve ligation reaction efficiency, increasing the number of ligation products in the ligation reaction and the number of molecules that can be sequenced during library construction.

[0006] To this end, the present disclosure provides a DNA ligation buffer. According to an embodiment of the present disclosure, the buffer comprises 10-15% polyethylene glycol.

[0007] The inventors found that PEG, as an additive in the buffer of the DNA ligation reaction, can play the role of crowding the molecular environment. At the same time, by adjusting the PEG concentration, the ligation efficiency during the library construction process can be significantly improved.

[0008] According to an embodiment of the present disclosure, the polyethylene glycol includes at least one selected from PEG4000, PEG6000, and PEG8000.

[0009] According to an embodiment of the present disclosure, the DNA ligation buffer further includes divalent metal ions, DTT, and ATP.

[0010] According to an embodiment of the present disclosure, the divalent metal ions include Mg 2+ , Ca 2+ 、Mn 2+、Zn 2+ and Co 2+ At least one of .

[0011] According to an embodiment of the present disclosure, the buffer contained in the DNA ligation buffer includes at least one selected from Tris-HCl buffer and HEPES buffer.

[0012] According to an embodiment of the present disclosure, the DNA ligation buffer comprises: 5-15 mM divalent metal ions; 0.5-5 mM DTT; 10%-15% polyethylene glycol; 0.5-5 mM ATP; and Tris-HCl buffer, with a pH of 7-9. The concentrations of the components in the DNA ligation buffer are all final concentrations.

[0013] According to a preferred embodiment of the present disclosure, the DNA ligation buffer comprises: 8-12 mM divalent metal ions; 0.8-3 mM DTT; 10%-15% polyethylene glycol; 0.8-3 mM ATP; and Tris-HCL buffer with a pH value of 7-8.

[0014] According to a preferred embodiment of the present disclosure, the DNA ligation buffer comprises: 5-15 mM Mg 2+ ; 0.5-5mM DTT; 10%-15% PEG6000; 0.5-5mM ATP; Tris-HCL buffer, pH 7-9.

[0015] According to a preferred embodiment of the present disclosure, the DNA ligation buffer comprises: 8-12 mM Mg 2+ ; 0.8-3 mM DTT; 10%-15% PEG6000; 0.8-3 mM ATP; Tris-HCL buffer, pH 7-8.

[0016] According to a preferred embodiment of the present disclosure, the DNA ligation buffer comprises: 10 mM Mg 2+ ; 1 mM DTT; 10% PEG6000; 1 mM ATP; Tris-HCL buffer, pH 7.5.

[0017] Another aspect of the present disclosure provides use of the aforementioned DNA ligation buffer in preparing a DNA ligation kit.

[0018] According to an embodiment of the present disclosure, the DNA ligation kit further includes a ligase.

[0019] According to a preferred embodiment of the present disclosure, the ligase comprises at least one selected from Quick T4 DNA Ligase, T7 DNA ligase and Taq DNA ligase.

[0020] Another aspect of the present disclosure provides a kit comprising the aforementioned DNA ligation buffer.

[0021] According to an embodiment of the present disclosure, the kit further comprises a ligase.

[0022] According to a preferred embodiment of the present disclosure, the ligase comprises at least one selected from Quick T4 DNA Ligase, T7 DNA ligase and Taq DNA ligase.

[0023] Another aspect of the present disclosure provides use of the aforementioned buffer and / or the aforementioned kit in constructing a nucleic acid library.

[0024] Another aspect of the present disclosure provides use of the aforementioned buffer and / or the aforementioned kit in constructing a sequencing library.

[0025] Another aspect of the present disclosure provides a method for constructing a sequencing library, comprising connecting adapters to sequencing reads using the aforementioned buffer and / or the aforementioned kit to construct a sequencing library.

[0026] Additional aspects and advantages of the present disclosure will be given in part in the description that follows and, in part, will be obvious from the description that follows, or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0028] FIG1 shows the ligation reaction results in Example 4 of the present disclosure;

[0029] FIG2 shows a sequencing current diagram in Example 7 of the present disclosure;

[0030] FIG3 shows the protein control results after purification in Example 5 of the present disclosure;

[0031] FIG4 shows a comparison of the effective sequencing times of reaction 1 and reaction 2 in Example 7 of the present disclosure.

[0032] Detailed Description of the Invention

[0033] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure.

[0034] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. Throughout the present disclosure, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0035] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0036] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present disclosure belongs.

[0037] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present disclosure, but not excluding other contents.

[0038] According to a specific embodiment of the present disclosure, a DNA ligation buffer is provided, comprising 10-15% polyethylene glycol. Adding a specific concentration of polyethylene glycol to the DNA ligation buffer acts as a molecular crowding environment, thereby improving ligation reaction efficiency, increasing the number of ligation products, and increasing the number of sequenced molecules during subsequent library construction.

[0039] The type of polyethylene glycol is not particularly limited. Any polyethylene glycol known in the art that can function as a crowded molecular environment is within the scope of this disclosure. The concentration of polyethylene glycol herein should be understood as mass-volume concentration, i.e., 10-15% polyethylene glycol should be understood as 10-15% (m / V) polyethylene glycol. The units of mass-volume concentration can be g / mL or kg / L.

[0040] The above “10-15% polyethylene glycol” should be understood as the polyethylene glycol concentration may be 10%, 11%, 12%, 13%, 14% and 15% as well as any other concentration within the concentration range of 10-15%.

[0041] According to a preferred embodiment of the present disclosure, the polyethylene glycol includes at least one selected from PEG4000, PEG6000, and PEG8000.

[0042] According to a specific embodiment of the present disclosure, the DNA ligation buffer further includes ATP, DTT, and divalent metal ions. ATP provides energy for the reaction, and its hydrolysis products participate in the formation of reaction intermediates; DTT is used to stabilize the ligase; and the divalent metal ions form coordination bonds between the reactants.

[0043] According to a specific embodiment of the present disclosure, the divalent metal ions include Mg 2+ , Ca 2+ 、Mn 2+ 、Zn 2+ and Co 2+ At least one of.

[0044] According to a specific embodiment of the present disclosure, the buffer contained in the DNA ligation buffer includes at least one selected from Tris-HCl buffer and HEPES buffer.

[0045] According to a specific embodiment of the present disclosure, the DNA ligation buffer comprises: 5-15 mM divalent metal ions; 0.5-5 mM DTT; 10%-15% polyethylene glycol; 0.5-5 mM ATP; and Tris-HCL buffer with a pH value of 7-9.

[0046] According to a specific embodiment of the present disclosure, the DNA ligation buffer comprises: 8-12 mM divalent metal ions; 0.8-3 mM DTT; 10%-15% polyethylene glycol; 0.8-3 mM ATP; and Tris-HCL buffer with a pH value of 7-8.

[0047] The above-mentioned "divalent metal ions" may include Mg 2+ , Ca 2+ 、Mn 2+ 、Zn 2+ and Co 2+At least one of the following: The phrase "8-12 mM divalent metal ion" should be understood to mean that the divalent metal ion concentration may be 8 mM, 9 mM, 10 mM, 11 mM, and 12 mM, as well as any other concentration within the 8-12 mM concentration range. The phrase "0.8-3 mM DTT" should be understood to mean that the DTT concentration may be 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, and 3 mM, as well as any other concentration within the 0.8-3 mM concentration range. The above “0.8-3 mM ATP” should be understood as ATP concentrations of 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, and 3 mM, as well as any other concentration within the range of 0.8-3 mM. The above “pH value of 7-8” should be understood as pH value of 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8, as well as any other pH value within the range of 7-8.

[0048] According to a specific embodiment of the present disclosure, the DNA ligation buffer comprises: 5-15 mM Mg 2+ ; 0.5-5mM DTT; 10%-15% PEG6000; 0.5-5mM ATP; Tris-HCL buffer, pH 7-9.

[0049] According to a specific embodiment of the present disclosure, the DNA ligation buffer comprises: 8-12 mM Mg 2+ ; 0.8-3mM DTT; 10%-15% PEG6000; 0.8-3mM ATP; Tris-HCL buffer, pH 7-8.

[0050] According to a preferred embodiment of the present disclosure, the DNA ligation buffer comprises: 10 mM Mg 2+ ; 1 mM DTT; 10% PEG6000; 1 mM ATP; Tris-HCL buffer, pH 7.5.

[0051] According to a specific embodiment of the present disclosure, another aspect of the present disclosure provides the use of the aforementioned DNA ligation buffer in preparing a DNA ligation kit. The DNA ligation buffer disclosed herein is used in preparing a DNA ligation kit to improve the ligation reaction efficiency and substrate conversion rate of the DNA ligation kit.

[0052] According to a specific embodiment of the present disclosure, the DNA ligation kit further comprises a ligase.

[0053] According to a specific embodiment of the present disclosure, the ligase comprises at least one selected from Quick T4 DNA Ligase, T7 DNA ligase and Taq DNA ligase.

[0054] According to a specific embodiment of the present disclosure, another aspect of the present disclosure provides a kit comprising the aforementioned DNA ligation buffer. The kit disclosed herein can improve the ligation reaction efficiency and substrate conversion rate of the DNA ligation kit.

[0055] According to a specific embodiment of the present disclosure, the kit further comprises a ligase.

[0056] According to a specific embodiment of the present disclosure, the ligase comprises at least one selected from Quick T4 DNA Ligase, T7 DNA ligase and Taq DNA ligase.

[0057] According to a specific embodiment of the present disclosure, another aspect of the present disclosure provides use of the aforementioned buffer and / or the aforementioned kit in constructing a nucleic acid library.

[0058] According to a specific embodiment of the present disclosure, another aspect of the present disclosure provides use of the aforementioned buffer and / or the aforementioned kit in constructing a sequencing library.

[0059] According to a specific embodiment of the present disclosure, another aspect of the present disclosure provides a method for constructing a sequencing library, comprising connecting adapters to sequencing reads using the aforementioned buffer and / or the aforementioned kit to construct a sequencing library.

[0060] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially. Example

[0061] Example 1. Preparation of linker sequence Ad3

[0062] (1) Order SEQ ID NO. 1 and SEQ ID NO. 2 from Sangon Biotechnology. Prepare a 100 μM stock solution of each using TE buffer (pH = 8). Then, take 10 μL of each stock solution and add 40 μL of TE buffer (pH = 8) to dilute it to a 20 μM working solution.

[0063] Among them, SEQ ID NO.1:

[0064] 5'-XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTTTTTTTTTTYYYYGGTTGTTTCTGTTGGTGCTGATATTGCT 3' (X=iSpC3, specifically a carbon chain containing three hydroxyl groups; Y=iSp18, specifically an 18-atom hexaethylene glycol chain. Both iSpC3 and iSp18 are commonly used as spacers in oligonucleotide chains)

[0065] SEQ ID NO.2:

[0066] 5'pho-GCAATATCAGCACCAACAGAAACAACCTTTGAGGCGAGCGGTCAA-3'

[0067] pho refers to the 5' end of the primer having phosphorylation modification, and the same applies below.

[0068] (2) 30 μL of the SEQ ID NO. 1 working solution and 30 μL of the SEQ ID NO. 2 working solution obtained by dilution in the previous step were mixed and thoroughly vortexed. The mixture was heated to 70°C and incubated for 10 minutes using a thermal cycler. The temperature was then lowered to 25°C at a rate of 0.1°C / s and incubated for another half an hour. This resulted in a 10 μM annealed linker solution. The linker product was named Ad3.

[0069] Example 2. Preparation of 500 bp insert

[0070] (1) Order SEQ ID NO. 3 and SEQ ID NO. 4 from Sangon Biotechnology. Prepare a 100 μM stock solution of each using TE buffer (pH = 8). Then, take 10 μL of each stock solution and add 90 μL of TE buffer (pH = 8) to dilute it to a 10 μM working solution.

[0071] Among them, SEQ ID NO.3:

[0072] 5'pho-AGCCACATCGCTCAGACAC-3'

[0073] SEQ ID NO.4:

[0074] 5'pho-GAGGCATTGCTGATGATCTTG-3'

[0075] (2) Perform a PCR reaction using the pcDNA3.1-C-(k)DYK plasmid (Genescript, OHu20465) containing the GADPH sequence as a template. Prepare the PCR mixture on ice according to the recipe in Table 1. After thorough vortexing, place the mixture in a PCR instrument and run the program in Table 2.

[0076] Table 1 PCR mixture formula

[0077] Reagent volume DNA template (100ng / μL) 1μL 10X PCR Buffer (Mg 2+ plus) 5 μL

[0078] dNTPs (25mM each dNTP) 0.4μSEQ ID NO.3 (10μM) 1μSEQ ID NO.4 (10μM) 1μLTaq DNA polymerase (TaKaRa) 1μL H2O Make up to 50μL

[0079] Table 2 PCR program

[0080]

[0081] (3) Remove Ampure XP magnetic beads (Beckman Coulter, A63882) from the refrigerator in advance, vortex to mix, and then equilibrate at room temperature for half an hour. Add 50 μL of the equilibrated magnetic beads to the double enzyme digestion system, vortex to mix, centrifuge briefly, and let stand at room temperature for 10 minutes.

[0082] (4) Place the centrifuge tube on the magnetic stand for 10 minutes. After the magnetic beads are completely adsorbed to the side of the magnetic stand and the solution becomes completely clear, carefully remove the supernatant.

[0083] (5) Resuspend the magnetic beads in 200 μL of 75% ethanol solution and wash them with a pipette. Place the centrifuge tube on a magnetic stand for 10 minutes. After the magnetic beads are completely adsorbed to the side of the magnetic stand and the solution becomes completely clear, carefully remove the supernatant.

[0084] (6) Repeat the ethanol solution washing step once. After removing the supernatant, place the centrifuge tube on a magnetic stand and let it stand. After the surface of the magnetic beads becomes dry, add 22 μL TE buffer (pH = 8) to resuspend the magnetic beads and let it stand at room temperature for 10 minutes.

[0085] (7) Place the centrifuge tube on a magnetic stand. When all the magnetic beads are adsorbed to the side of the magnetic stand, transfer the supernatant to a new centrifuge tube. The amplified insert sequence SEQ ID NO. 5 with 5' phosphorylated end and 3' A added is obtained and diluted to 0.2 μM with TE buffer.

[0086] Among them, SEQ ID NO.5:

[0087]

[0088] In this example, a 500 bp sequence of the GAPDH gene was amplified by polymerase chain reaction (PCR) to serve as an insert substrate for the ligation reaction.

[0089] Example 3. Preparation of ligation buffer

[0090] Prepare ligation buffers 1, 2, 3, and 4 according to the recipes in Tables 3, 4, 5, and 6, respectively.

[0091] Table 3 Ligation Buffer 1 Recipe

[0092] Reagents 3X1XMgCl230mM10mMTris-HCl pH 7.5198Mm66mMDTT3mM1mMATP3mM1mMPEG600018%6%

[0093] Table 4 Ligation Buffer 2 Recipe

[0094] Reagents 3X1XMgCl230mM10mMTris-HCl pH 7.5198Mm66mMDTT3mM1mMATP3mM1mMPEG600030%10%

[0095] Table 5 Ligation Buffer 3 Recipe

[0096] Reagents 3X1XMgCl230mM10mMTris-HCl pH 7.5198Mm66mMDTT3mM1mMATP3mM1mMPEG600045%15%

[0097] Table 6 Ligation Buffer 4 Recipe

[0098] Reagent 3X1X

[0099] MgCl230mM10mMTris-HCL pH 7.5198Mm66mMDTT3mM1mMATP3mM1mMPEG600060%20%

[0100] Example 4. Ligation reaction of 500 bp insert fragment and Ad3 linker

[0101] (1) The 500 bp fragment prepared in Example 2 and the Ad3 linker prepared in Example 1 were mixed according to the reaction system in Tables 7-10 and placed on ice;

[0102] Table 7 Reaction system 1

[0103] Reagent volume 0.2μM 500bp 20μL 10μM Ad3 4μL 3x ligation buffer 120μL Quick T4 DNA Ligase (NEB, E6057AVIAL) 6μL H2O 10μL

[0104] Table 8 Reaction system 2

[0105] Reagent volume 0.2μM 500bp 20μL 10μM Ad3 4μL 3x ligation buffer 220μL Quick T4 DNA Ligase (NEB, E6057AVIAL) 6μL H2O 10μL

[0106] Table 9 Reaction system 3

[0107] Reagent volume 0.2μM 500bp 20μL 10μM Ad3 4μL 3x ligation buffer 320μL Quick T4 DNA Ligase (NEB, E6057AVIAL) 6μL H2O 10μL

[0108] Table 10 Reaction system 4

[0109] Reagent volume 0.2μM 500bp 20μL 10μM Ad3 4μL 3x ligation buffer 420μL Quick T4 DNA Ligase (NEB, E6057AVIAL) 6μL H2O 10μL

[0110] (2) The reactants were thoroughly mixed and then reacted at 25°C for 10 minutes;

[0111] (3) Remove the Ampure XP magnetic beads from the refrigerator in advance, vortex to mix, and then equilibrate at room temperature for half an hour. Add 60 μL of the equilibrated magnetic beads to the connection system in step (2), vortex to mix, centrifuge briefly, and let it stand at room temperature for 10 minutes.

[0112] (4) Place the centrifuge tube on the magnetic stand for 10 minutes. After the magnetic beads are completely adsorbed to the side of the magnetic stand and the solution becomes completely clear, carefully remove the supernatant.

[0113] (5) Resuspend the magnetic beads in 200 μL of 75% ethanol solution and wash them by pipetting. Place the centrifuge tube on a magnetic stand for 10 minutes. After the magnetic beads are completely adsorbed to the side of the magnetic stand and the solution becomes completely clear, carefully remove the supernatant.

[0114] (6) Repeat the ethanol solution washing step once. After removing the supernatant, place the centrifuge tube on a magnetic stand and let it stand. After the surface of the magnetic beads becomes dry, add 22 μL TE buffer (pH = 8) to resuspend the magnetic beads and let it stand at room temperature for 10 minutes.

[0115] (7) Place the centrifuge tube on a magnetic rack. When all the magnetic beads are adsorbed to the side of the magnetic rack, transfer the supernatant to a new centrifuge tube to obtain the purified ligation product.

[0116] (8) The ligation products were detected by non-denaturing polyacrylamide gel electrophoresis, and the detection results are shown in Figure 1. The results showed that the ligation products of reaction 2 and reaction 3 were significantly increased compared with the other reactions, and the reaction substrate was also significantly reduced compared with the other reactions. In particular, the ligation product of reaction 2 was more than that of reactions 1 and 4.

[0117] Example 5. Cloning, expression and purification of helicase Dda

[0118] In this example, helicase Dda (SEQ ID NO. 6) was prepared by recombinant expression in Escherichia coli, and the helicase was used as a motor protein.

[0119] (1) Order the full-length cDNA sequence of Dda (SEQ ID NO.7), ligate it into the PET.28a(+) plasmid, and use the double restriction sites Nde1 and Xho1. Thus, the expressed Dda protein has a 6*His tag and a thrombin cleavage site at the N-terminus;

[0120] (2) The cloned PET.28a(+)-Dda plasmid was transformed into ArcticExpress (DE3) competent bacteria (Tolo Biotech., 96183-02) or its derivatives. A single colony was picked and inoculated into 5 mL of LB medium containing kanamycin, and cultured at 37°C with shaking overnight. Then, the colony was transferred into 1 L of LB medium containing kanamycin, cultured at 37°C with shaking until OD600 = 0.6-0.8, cooled to 16°C, and IPTG was added at a final concentration of 500 μM to induce Dda expression overnight;

[0121] (3) Prepare five buffer solutions according to the following formula:

[0122] Buffer A: 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 20 mM imidazole;

[0123] Buffer B: 20 ​​mM Tris-HCl pH 7.5, 250 mM NaCl, 300 mM imidazole;

[0124] Buffer C: 20 mM Tris-HCl pH 7.5, 50 mM NaCl;

[0125] Buffer D: 20 mM Tris-HCl pH 7.5, 1000 mM NaCl;

[0126] Buffer E: 20 mM Tris-HCl pH 7.5, 100 mM NaCl;

[0127] (4) Collect the bacteria expressing Dda, resuspend the bacteria with buffer A, disrupt the bacteria with a cell disruptor, and then centrifuge to obtain the supernatant. Mix the supernatant with Ni-NTA filler that has been equilibrated with buffer A in advance and bind for 1 hour. Collect the filler and wash the filler with buffer A until no impurities are washed out. Then add buffer B to the filler to elute Dda. The eluted Dda is passed through a desalting column equilibrated with buffer C for buffer exchange. Then add an appropriate amount of thrombin (Yishen Bio) and then add it to the ssDNA cellulose filler (Sigma, D8273-10G) equilibrated with buffer C, and digest and bind overnight at 4°C. Collect the ssDNA cellulose filler, wash it with buffer C 3-4 times, and then elute it with buffer D. The protein purified from the ssDNA cellulose is concentrated and applied to a molecular sieve Superdex 200 (Sigma, GE28-9909-44). The molecular sieve buffer used is buffer E. Collect the target protein peak, concentrate it, and freeze it. The purified protein was quantified using Nanodrop. The purity of the protein was also tested using HPLC and SDS-PAGE electrophoresis. The results are shown in Figure 3.

[0128] Nucleotide sequence of helicase Dda (SEQ ID NO.6):

[0129]

[0130] Amino acid sequence of helicase Dda protein (SEQ ID NO.7):

[0131] (* indicates termination).

[0132] Example 6. Preparation of sequencing library

[0133] In this example, the ligation products of reaction 1 and reaction 2 in Example 4 were incubated with the prepared helicase Dda to prepare a library for sequencing.

[0134] (1) Add 100 mL of 1 M Tris-HCl pH 7.5 buffer and 100 mL of 1 M KCl solution to a volumetric flask and dilute to 1 L with ultrapure water to prepare 2X binding buffer.

[0135] (2) Prepare a mixed solution of helicase Dda (prepared in Example 5) and the library on ice according to Table 11, and then incubate at 30°C for one hour;

[0136] Table 11 Motor protein and library binding system

[0137] Reagent volume 2X binding buffer 50μL helicase Dda 20μL purified ligation product 20μL H2O 10μL

[0138] (3) Use the Qubit DNA HS kit to quantify the library, clearly mark the concentration, and store the product in a 4°C refrigerator for later use.

[0139] Example 7. Nanopore sequencing

[0140] In this example, a nanopore detection platform based on a patch clamp platform was constructed to perform nanopore sequencing on the target sequencing library prepared in Example 6.

[0141] (1) With reference to the single-channel electrophysiological detection system in Geng Jia and Guo Peixuan (“Application of phage phi29 DNA packaging motor phospholipid membrane chimera in single molecule detection and nanomedicine”, Life Science, 2011, 23(11): 1114-1129), a nanopore detection platform based on the patch clamp platform was constructed, and porin (Sigma-Aldrich, H9395-5mg) was inserted into the phospholipid bilayer membrane to form a single-channel nanopore;

[0142] (2) adding the sequencing library obtained in Example 6 to the single-channel system, and detecting and recording the current amplitude changes using a patch clamp system;

[0143] (3) The sequencing current graph of SEQ ID NO. 5 is shown in FIG2 . The effective sequencing times of reaction 1 and reaction 2 were also compared, and the comparison results are shown in FIG4 . This indicates that the effective sequencing time of the sequencing library constructed by nanopore sequencing using a ligation buffer containing 10% PEG6000 for fragment ligation is significantly better than that of the sequencing library constructed using a ligation buffer containing 6% PEG6000. The ligation buffer provided by the present disclosure is applied to library construction in the sequencing field, can improve the ligation efficiency during library construction, and increase the number of molecules that can be sequenced.

[0144] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and features of different embodiments or examples, unless they are mutually inconsistent.

[0145] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A DNA ligation buffer, wherein: Includes 10%-15% polyethylene glycol.

2. The DNA ligation buffer according to claim 1, wherein The polyethylene glycol includes at least one selected from PEG4000, PEG6000, and PEG8000.

3. The DNA ligation buffer according to claim 1 or 2, wherein The DNA ligation buffer further comprises divalent metal ions, DTT, and ATP.

4. The DNA ligation buffer according to claim 3, wherein The divalent metal ions include Mg 2+ , Ca 2+ , Mn 2+ 、Zn 2+ and Co 2+ At least one of .

5. The DNA ligation buffer according to any one of claims 1 to 4, wherein The buffer contained in the DNA ligation buffer includes at least one selected from Tris-HCL buffer and HEPES buffer.

6. The DNA ligation buffer according to any one of claims 1 to 5, wherein The DNA ligation buffer comprises: 5-15mM divalent metal ions; 0.5-5mM DTT; 10%-15% polyethylene glycol; 0.5-5mM ATP; Tris-HCL buffer, pH 7-9.

7. The DNA ligation buffer according to any one of claims 1 to 6, wherein The DNA ligation buffer comprises: 8-12mM divalent metal ions; 0.8-3mM DTT; 10%-15% polyethylene glycol; 0.8-3mM ATP; Tris-HCL buffer, pH 7-8.

8. The DNA ligation buffer according to any one of claims 1 to 6, wherein The DNA ligation buffer comprises: 5-15mM Mg 2+ ; 0.5-5mM DTT; 10%-15% PEG6000; 0.5-5mM ATP; Tris-HCL buffer, pH 7-9.

9. The DNA ligation buffer according to any one of claims 1 to 8, wherein The DNA ligation buffer comprises: 8-12mM Mg 2+ ; 0.8-3mM DTT; 10%-15% PEG6000; 0.8-3mM ATP; Tris-HCL buffer, pH 7-8.

10. The DNA ligation buffer according to any one of claims 1 to 9, wherein The DNA ligation buffer comprises: 10mM Mg 2+ ; 1mM DTT; 10% PEG6000; 1mM ATP; Tris-HCl buffer, pH 7.

5.

11. Use of the DNA ligation buffer according to any one of claims 1 to 10 in preparing a DNA ligation kit.

12. The use according to claim 11, wherein The DNA ligation kit further comprises a ligase.

13. The use according to claim 12, wherein The ligase comprises at least one selected from Quick T4 DNA Ligase, T7 DNA ligase and Taq DNA ligase.

14. A kit, wherein: The kit comprises the DNA ligation buffer according to any one of claims 1 to 10.

15. The kit according to claim 14, wherein The kit further comprises a ligase.

16. The kit according to claim 15, wherein The ligase includes at least one selected from Quick T4 DNA Ligase, T7 DNA ligase and Taq DNA ligase.

17. Use of the buffer according to any one of claims 1 to 10 and / or the kit according to any one of claims 14 to 16 in constructing a nucleic acid library.

18. Use of the buffer according to any one of claims 1 to 10 and / or the kit according to any one of claims 14 to 16 in constructing a sequencing library.

19. A method for constructing a sequencing library, wherein: The method comprises connecting the adapter to the sequencing read segment using the buffer described in any one of claims 1 to 10 and / or the kit described in any one of claims 14 to 16 to construct a sequencing library.

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