A method for recovering biotin-modified nucleic acids using streptavidin magnetic beads

Abstract

The present application provides a method for recovering biotin-modified nucleic acids using streptavidin magnetic beads, which belongs to the field of biotechnology. The method provided by the present application can significantly improve the recovery efficiency of biotin-modified nucleic acids by reducing the salt ion concentration in the binding buffer.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for recovering biotin-modified nucleic acids using streptavidin magnetic beads. Background Art

[0002] DNA recovery is a very important step in the process of molecular biology research. Currently, common DNA recovery methods include alcohol precipitation, phenol-chloroform extraction, column extraction and magnetic bead method. The magnetic bead method uses magnetic substances (usually Fe 3 O 4 ) as the core, which has superparamagnetic properties and is able to generate magnetism in the presence of an external magnetic field. The different coatings and chemical properties on the surface of the magnetic beads give each type of beads its own binding properties, which can be used for magnetic separation of nucleic acids, proteins or other biomolecules in an easy, efficient and scalable way.

[0003] Streptavidin beads (SA beads) are SA beads covalently bonded with hydrophilic superparamagnetic polystyrene microspheres and high-purity SA. Based on the principle that the streptavidin-biotin (Biotin, Bio) system has extremely high affinity, it can quickly and efficiently bind to biotin-modified nucleic acids, antibodies and other molecules, and is suitable for nucleic acid hybridization capture and separation (Holmberg, Anders, et al. "The biotin-streptavidin interaction can bereversibly broken using water at elevated temperatures." Electrophoresis 26.3 (2010): 501-510). The basic routine process of recovering biotin-modified DNA with SA magnetic beads is as follows: (1) SA magnetic bead pretreatment: wash the SA magnetic beads twice with 1× binding buffer to activate the magnetic beads; (2) SA magnetic beads bind to biotin-modified DNA: incubate the activated magnetic beads with biotin-modified DNA at room temperature for 20 min to bind the DNA. The recovered DNA is bound to the SA magnetic beads. If DNA without magnetic beads is required, DNA release is required; (3) DNA release: use 95% formamide and heat at 65°C for 5 min to complete DNA release (attached Figure 1 ). However, the problem of DNA loss still exists when only relying on the high affinity principle of SA-Biotin to specifically recover DNA.

[0004] In order to improve the efficiency of SA-biotin specific DNA recovery, some researchers optimized and upgraded the SA protein and obtained a SA variant enzyme (traptavidin, TA) to improve its binding ability with biotin ([1] Claire E Chivers Estelle Crozat Calvin Chu Calvin Chu David J Sherratt Mark Howarth. "Astreptavidin variant with slower biotin dissociation and increased mechanostability." Nature methods 7.5 (2010)). However, there are currently no commercial TA magnetic beads, so they cannot be used in practical applications. Summary of the invention

[0005] The present application aims to provide a method for recovering biotin-modified nucleic acids using streptavidin magnetic beads, which can significantly improve the recovery efficiency of biotin-modified nucleic acids by reducing the salt ion concentration in the binding buffer.

[0006] The first aspect of the present application provides a method for recovering biotin-modified nucleic acids using streptavidin magnetic beads, the method comprising:

[0007] In the presence of a binding buffer, incubate streptavidin magnetic beads and biotin-modified nucleic acids to allow the biotin-modified nucleic acids to bind to the streptavidin magnetic beads, wherein the binding buffer comprises a buffer component and NaCl, and the working concentration of the NaCl is 0.001-1M; apply an external magnetic field, remove the supernatant; and elute the biotin-modified nucleic acids from the streptavidin magnetic beads.

[0008] In some embodiments, the buffer component is, for example, one or more of Tris, Triton, PBS and HEPES, preferably Tris, and the working concentration of Tris is 1-20 mM, including 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM and 20 mM within the range.

[0009] In some embodiments, the working concentration of NaCl is 0.001-1M, including 0.001M, 0.002M, 0.003M, 0.004M, 0.005M, 0.006M, 0.007M, 0.008M, 0.009M, 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0. ... .1M, 0.125M, 0.15M, 0.175M, 0.2M, 0.225M, 0.25M, 0.75M, 0.3M, 0.325M, 0.35M, 0.375M, 0.4M, 0.425M, 0.45M, 0.475M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M and 1M.

[0010] In some embodiments, the binding buffer further comprises a chelating agent; preferably, the chelating agent is EDTA; preferably, the working concentration of the EDTA is 0.1-5 mM, including 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM and 5 mM within the range.

[0011] In some embodiments, the binding buffer further comprises a surfactant, such as one or more of a cationic surfactant, an anionic surfactant and a nonionic surfactant, preferably a nonionic surfactant, the nonionic surfactant being, for example, one or more of Tween, polyethylene glycol and NP40, preferably Tween 20, the working concentration (volume fraction) of the Tween 20 being 0.01%-5%, including 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% and 5% within the range.

[0012] In some embodiments, the working concentration of each component in the binding buffer is its final concentration in the reaction system of incubating streptavidin magnetic beads and biotin-modified nucleic acids.

[0013] In some embodiments, the concentration of each component in the binding buffer can be present in the form of 2-50 times the working concentration of the above-mentioned components, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 12 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times or 50 times.

[0014] For example, when the binding buffer is present in the form of 2X (2 times concentration), a volume of biotin-modified nucleic acid equal to that of the binding buffer can be added to make the binding buffer in the reaction system reach the working concentration; when the binding buffer is present in the form of 3X (3 times concentration), a volume of biotin-modified nucleic acid 2 times the volume of the diluent can be added to make the binding buffer in the reaction system reach the working concentration, and so on.

[0015] In some embodiments, the incubation is room temperature incubation, e.g., room temperature incubation for 5-30 min, e.g., room temperature incubation for 10-30 min, including 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min and 30 min within the range.

[0016] In some embodiments, the room temperature refers to 25-35°C, including 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C and 35°C within the range.

[0017] In some embodiments, the step of eluting the biotin-modified nucleic acid from the streptavidin magnetic beads comprises adding an elution solution, incubating, and collecting the elution solution.

[0018] In some embodiments, the eluent comprises formamide, preferably 80%-98% formamide, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% and 98% within the range.

[0019] In some embodiments, the step of eluting the biotin-modified nucleic acid from the streptavidin magnetic beads further comprises thermal elution, such as heating at 50-95°C for 1-20 min, such as heating at 60-95°C for 1-20 min, such as heating at 60-95°C for 1-10 min, such as heating at 65-90°C for 1-10 min, such as heating at 65-80°C for 1-10 min, such as heating at 65-70°C for 1-10 min, such as heating at 65-70°C for 5-10 min, such as heating at 65°C for 5 min.

[0020] In some embodiments, the method further comprises pre-treating the streptavidin magnetic beads before incubating, and the step of pre-treating the streptavidin magnetic beads comprises resuspending the streptavidin magnetic beads using the binding buffer to obtain resuspended streptavidin magnetic beads.

[0021] In some embodiments, the method further comprises adding biotin-modified nucleic acid to the resuspended streptavidin magnetic beads.

[0022] In some embodiments, the biotin-modified nucleic acid refers to a nucleic acid containing biotin modification on one or more nucleotides, wherein the biotin is covalently linked to the base.

[0023] In some embodiments, the position of biotin modification is at the nucleotide at the 3' terminus and / or the 5' terminus of the nucleic acid strand.

[0024] In some embodiments, the number of biotin modifications is at least 1 nucleotide, for example, 1 biotin molecule is modified at the 3' end or 5' end of a single-stranded DNA; for example, 2 biotin molecules are modified at the 3' end and 5' end of a single-stranded DNA; for example, 2 biotin molecules are modified at the 3' end of both strands of a double-stranded DNA; for example, 2 biotin molecules are modified at the 5' end of both strands of a double-stranded DNA; for example, 2 biotin molecules are modified at the 3' end and 5' end of the sense strand of a double-stranded DNA; for example, 2 biotin molecules are modified at the 5' end of the sense strand of a double-stranded DNA and the 3' end of the antisense strand; for example, 3 biotin molecules are modified at the two ends of the sense strand of a double-stranded DNA and the 5' end of the antisense strand; for example, 4 biotin molecules are modified at the 3' end and 5' end of both strands of a double-stranded DNA. When the biotin-modified nucleic acid is RNA or a DNA-RNA hybrid, the same can be applied.

[0025] In some embodiments, the biotin-modified nucleic acid is a biotin-modified single-stranded DNA, a biotin-modified double-stranded DNA, a biotin-modified RNA, or a biotin-modified DNA-RNA hybrid.

[0026] In some embodiments, the biotin-modified nucleic acid is, for example, a biotin-modified probe, a biotin-modified transposon, a biotin-modified primer, a biotin-modified amplicon, and a biotin-modified library.

[0027] In some embodiments, the length of the biotin-modified nucleic acid does not affect the non-covalent binding interaction between biotin and streptavidin, or the length of the biotin-modified nucleic acid can be any length conventional in the art that can be recovered by streptavidin magnetic beads.

[0028] In some embodiments, the length of the biotin modified nucleic acid is, for example, 10-2000bp, including 10bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1100bp, 1200bp, 1300bp, 1400bp, 1500bp, 1600bp, 1700bp, 1800bp, 1900bp and 2000bp within the range.

[0029] In some embodiments, the method further comprises washing the streptavidin magnetic beads bound to the biotin-modified nucleic acid before elution, wherein the washing comprises incubating the streptavidin magnetic beads bound to the biotin-modified nucleic acid with the binding buffer in an external magnetic field and removing the supernatant.

[0030] In some embodiments, the method further comprises repeating the washing step, for example 1, 2 or 3 times.

[0031] In some embodiments, the method further comprises purifying the eluted biotin-modified nucleic acid, preferably by magnetic bead purification, wherein the magnetic bead purification comprises magnetic bead adsorption, washing and elution steps.

[0032] In some embodiments, the methods provided herein can be used to recover biotin-modified libraries, such as CUT&Tag libraries, methylation sequencing libraries, and amplicon libraries.

[0033] The second aspect of the present application provides the application of the method described in the first aspect in improving the recovery efficiency of biotin-modified nucleic acids.

[0034] The third aspect of the present application provides a kit, which is used to implement the method described in the first aspect of the present application.

[0035] In some embodiments, the kit comprises a binding buffer comprising a buffer component and NaCl, wherein the working concentration of NaCl is 0.001-1M. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 : The conventional process of recovering biotin-modified DNA using SA magnetic beads in the prior art;

[0037] Figure 2 :DNA yields under different pH conditions when the working concentrations of NaCl in the binding buffer were 0.125M, 0.25M, 0.5M, 0.75M and 1M respectively;

[0038] Figure 3 : DNA yields under different NaCl working concentration conditions when the pH of the binding buffer is 6, 6.5, 7, 7.4 and 8 respectively;

[0039] Figure 4 : DNA yields under different NaCl working concentration conditions when the pH of the binding buffer is 7, 7.2, 7.4, 7.6, 7.8 and 8 respectively;

[0040] Figure 5 : DNA yield under different Tris concentrations in binding buffer;

[0041] Figure 6 : DNA yield under different EDTA concentrations in binding buffer;

[0042] Figure 7 : Effect of binding buffer containing different concentrations of Tween20 on DNA yield.

[0043] Specific implementation (embodiment)

[0044] The technical solution of the present application is further explained below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are merely simple examples of the present application and do not represent or limit the scope of protection of the present application. The scope of protection of the present application shall be subject to the claims.

[0045] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0046] Example 1

[0047] 1. Preparation of SA magnetic bead binding buffer with different pH and NaCl concentration

[0048] Prepare different 2×SA magnetic bead binding buffers according to Table 1. When using in the DNA recovery process, you need to add an equal volume of ddH 2 O, dilute to 1× SA magnetic bead binding buffer:

[0049] Table 1

[0050] Components Final concentration Tris 10mM EDTA 1mM NaCl 0.25M / 0.5M / 1M / 1.5M / 2M

[0051] Adjust the pH to 6.0, 6.5, 7.0, 7.4 and 8.0 respectively (Note: dilution has almost no effect on the pH of the buffer, the same below).

[0052] 2. Preparation of Biotin-modified DNA Template

[0053] The biotin-modified double-stranded DNAs of 300 bp with different synthetic sequences are mixed in proportion and set aside. The double-stranded DNA contains two biotin molecules, which are respectively located at the 5' end of each DNA single strand.

[0054] 3. SA magnetic bead recovery efficiency detection

[0055] (1) SA magnetic bead pretreatment

[0056] A. Use 5μl SA magnetic beads to recover DNA from each sample. Use a pipette to fully resuspend SA magnetic beads (Vazyme#N512), take 5μl SA magnetic beads to a 1.5ml centrifuge tube, add 200μl 1× SA magnetic bead binding buffer with different pH and NaCl concentrations, mix gently with a pipette, place on a magnetic stand, and discard the supernatant after the solution is clarified (about 2 minutes).

[0057] B. Remove the 1.5 ml centrifuge tube from the magnetic stand, add 200 μl of 1× SA magnetic bead binding buffer with different pH and NaCl concentrations, and mix gently with a pipette.

[0058] C. Place a 1.5 ml centrifuge tube on a magnetic rack. After the liquid becomes clear (about 2 minutes), discard the supernatant and add 50 μl of 2×SA magnetic bead binding buffer with different pH and NaCl concentrations to resuspend the SA magnetic beads for later use.

[0059] (2) SA magnetic beads bind to biotin-modified DNA

[0060] A. Take 150ng of prepared biotin-modified DNA template and put it into eight tubes. 2 Make up to 50 μl with 5% HO.

[0061] B. Add the SA magnetic beads treated in step (1)-C to the template in step (2)-A and mix thoroughly. At this time, the template and magnetic beads are mixed in equal volumes, and the working concentration of the magnetic beads is 1×. Incubate at room temperature for 20 minutes, inverting and mixing 2-3 times.

[0062] C. Centrifuge briefly, place the 8-tube strip on a magnetic rack, let stand for about 2-3 minutes, and carefully remove the supernatant.

[0063] D. Keep the PCR tube on the magnetic rack, add 200 μl of 1×SA magnetic bead binding buffer with different pH and NaCl concentrations, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0064] E. Repeat washing step D once.

[0065] F. Open the lid and let it air in the room temperature for 2-5 minutes until there is no liquid left in the tube and the surface of the magnetic beads is not reflective.

[0066] (3) DNA release

[0067] A. Remove the eight-tube strip from step (2)-F from the magnetic rack, add 50 μl of 95% (volume fraction) formamide, and heat at 65°C for 5 min to release DNA.

[0068] B. Vortex and mix VAHTS DNA Clean Beads (Vazyme#N411) and pipette 100 μl into the eight-tube strip (3)-A after heating. Vortex or pipette 10 times to mix thoroughly and incubate at room temperature for 5 min.

[0069] C. Briefly centrifuge the reaction tube and place it on a magnetic rack to separate the magnetic beads from the liquid. After the solution becomes clear (about 5 minutes), carefully remove the supernatant.

[0070] D. Keep the reaction tube on the magnetic rack, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0071] E. Repeat step D for a total of two rinses.

[0072] F. Keep the reaction tube on the magnetic rack and air dry it for about 5 minutes with the lid open.

[0073] G. Remove the reaction tube from the magnetic rack and add 22 μl of sterile ultrapure water for elution. Vortex or pipette 10 times to mix thoroughly and incubate at room temperature for 5 minutes.

[0074] H. Briefly centrifuge the reaction tube and place it on a magnetic rack to separate the magnetic beads from the liquid. After the solution becomes clear (about 5 minutes), carefully pipette 20 μl of the supernatant into a new sterilized PCR tube.

[0075] (4) DNA concentration detection

[0076] The DNA concentration was determined using a double-stranded DNA (dsDNA) fluorescent quantitative detection kit (Vazyme #EQ121). The specific operation was performed according to the kit instructions.

[0077] (5) Calculation of DNA recovery efficiency

[0078] The DNA yield recovered in each group was counted, and the data were analyzed by T-Test in GraphPadPrism8 to calculate the p-value between the groups to determine whether there was a significant difference between the groups. At the same time, the average value between the groups was used to calculate the recovery efficiency between different groups.

[0079] Result analysis:

[0080] like Figure 2As shown in the figure, the recovery efficiency of SA magnetic beads when the working concentration of NaCl is 1M and the pH is 8.0 is significantly higher than that of pH 6.0, pH 6.5, pH 7.0, and pH 7.4, which are 1.3 times, 1.2 times, 1.2 times, and 1.2 times, respectively; while the recovery efficiency of SA magnetic beads when the working concentration of NaCl is 0.125M and the pH is 8.0 is significantly lower than that of pH 6.0, pH 6.5, pH 7.0, and pH 7.4. That is, in a low-salt environment, the different pH of the binding buffer has no significant effect on the recovery efficiency of SA magnetic beads, while in a high-salt environment, the recovery efficiency of SA magnetic beads for biotin-modified DNA is greatly affected by the pH of the binding buffer. At the same time, an alkaline environment is more conducive to the recovery efficiency of SA magnetic beads, and the recovery efficiency of SA magnetic beads tends to be stable when the pH is between 7.0 and 8.0.

[0081] like Figure 3 As shown, under different pH environments, when the NaCl working concentration in the binding buffer was 0.125M, the recovery efficiency of SA magnetic beads was the highest, significantly higher than other test groups. When the pH was 6.0, the library yield of the NaCl working concentration of 0.125M was 1.2 times, 1.3 times, and 1.9 times that of the three groups with NaCl working concentrations of 0.5M, 0.75M, and 1M, respectively. When the pH was 6.5, 7.0, and 7.4, the library yield of the NaCl working concentration of 0.125M was 1.2 times, 1.3 times, and 1.7 times that of the three groups with NaCl working concentrations of 0.5M, 0.75M, and 1M, respectively. When the pH is 8.0, the library yield of the NaCl working concentration of 0.125 M is 1.2 times, 1.3 times, and 1.5 times that of the three groups of NaCl working concentrations of 0.5 M, 0.75 M, and 1 M. That is, under different pH environments, as the salt ion concentration increases, the recovery efficiency of SA magnetic beads gradually decreases, and the binding buffer with low NaCl concentration significantly improves the recovery efficiency of SA magnetic beads for biotin-modified DNA.

[0082] Example 2

[0083] 1. Preparation of SA magnetic bead binding buffer with different pH and NaCl concentration

[0084] Prepare different 2×SA magnetic bead binding buffers according to Table 2 below. When using in the DNA recovery process, you need to add an equal volume of ddH 2 O, dilute to 1× SA magnetic bead binding buffer:

[0085] Table 2

[0086]

[0087] The pH was adjusted to 7.0, 7.2, 7.4, 7.6, 7.8 and 8.0 respectively.

[0088] The other steps were the same as in Example 1, and the recovery rate of biotin-modified DNA was determined. The results were as follows: Figure 4 shown.

[0089] Result analysis:

[0090] like Figure 4 , further adjust the NaCl concentration and pH range in the binding buffer. As the salt ion concentration decreases, the recovery efficiency further increases. At the same time, the recovery efficiency of SA magnetic beads in an alkaline environment is more stable. When the pH is 7.0-7.6 and the NaCl working concentration is between 0.005mM and 0.025mM, the recovery efficiency of SA magnetic beads tends to be stable and the efficiency is the highest. Among them, when the pH is 7.0, 7.2 and 7.4, the DNA yield when the NaCl working concentration is 0.005mM is 1.5 times that of the NaCl working concentration of 0.25mM. When the pH is 7.6, the DNA yield when the NaCl working concentration is 0.005mM is 1.4 times that of the NaCl working concentration of 0.25mM. When the pH is 7.8 and the NaCl working concentration is between 0.005mM and 0.05mM, the recovery efficiency of SA magnetic beads tends to be stable, and the DNA yield is 1.4 times that of the NaCl working concentration of 0.25mM. When the pH is 8.0 and the NaCl working concentration is between 0.005mM and 0.125mM, the recovery efficiency of the SA magnetic beads tends to be stable, and the DNA yield is 1.3 times that of the NaCl working concentration of 0.25mM. Based on the results of Example 1, it can be seen that when the NaCl working concentration is in the range of 0.005M to 0.125M, compared with the binding buffer containing 2MNaCl commonly used in the prior art, the magnetic bead recovery buffer used in this application can significantly improve the recovery efficiency of the SA magnetic beads for biotin-modified DNA, and can reach up to 2-3 times.

[0091] Example 3

[0092] 1. Preparation of SA magnetic bead binding buffer with different Tris concentrations

[0093] Prepare different 2×SA magnetic bead binding buffers according to Table 3 below. When using in the DNA recovery process, you need to add an equal volume of ddH 2 O, dilute to 1× SA magnetic bead binding buffer:

[0094] Components Final concentration Tris 5mM / 10mM / 15mM / 20mM EDTA 1mM NaCl 0.05mM

[0095] Adjust pH to 7.4.

[0096] The other steps were the same as in Example 1, and the recovery rate of biotin-modified DNA was determined. The results were as follows: Figure 5 shown.

[0097] Result analysis:

[0098] like Figure 5 Adjusting the concentration of Tris in the binding buffer has no significant effect on the recovery efficiency of SA magnetic beads for biotin-modified DNA. The role of Tris in the binding buffer is to provide buffer for SA protein and biotin-modified DNA. When the pH value does not exceed the buffer range of Tris, it will not affect the recovery efficiency of SA magnetic beads.

[0099] Example 4

[0100] 1. Preparation of SA magnetic bead binding buffer with different EDTA concentrations

[0101] Prepare different 2×SA magnetic bead binding buffers according to Table 1.

[0102] Table 1

[0103] Components Final concentration Tris 10mM EDTA 0mM / 0.5mM / 1mM / 1.5mM / 2mM NaCl 0.05mM

[0104] Adjust pH to 7.4.

[0105] The other steps were the same as in Example 1, and the recovery rate of biotin-modified DNA was determined. The results were as follows: Figure 6 shown.

[0106] Result analysis:

[0107] like Figure 6 Adjusting the concentration of EDTA in the binding buffer, or even excluding EDTA in the binding buffer, will not affect the recovery efficiency of SA magnetic beads for biotin-modified DNA. The role of EDTA in the binding buffer is to chelate the nuclease introduced into the environment to prevent it from degrading DNA. Therefore, EDTA itself does not affect the recovery efficiency of SA magnetic beads.

[0108] Example 5

[0109] 1. Preparation of SA magnetic bead binding buffer with different Tween20 concentrations

[0110] Prepare different 2×SA magnetic bead binding buffers according to the table below. When using in the DNA recovery process, you need to add an equal volume of ddH 2 O, dilute to 1× SA magnetic bead binding buffer:

[0111] Components Final concentration Tris 10mM EDTA 1mM NaCl 0.05mM Tween20 0% / 0.05% / 0.1% / 0.2% / 0.3% / 0.5% (volume fraction)

[0112] Adjust pH to 7.4.

[0113] The other steps were the same as in Example 1, and the recovery rate of biotin-modified DNA was determined. The results were as follows: Figure 7shown.

[0114] Result analysis:

[0115] like Figure 7 Adding different concentrations of Tween20 to the binding buffer has little effect on the results of SA magnetic beads recovering biotin-modified DNA. The role of Tween20 in the binding buffer is to protect the SA protein and prevent the front-end biotin-modified DNA from introducing substances that destroy the protein structure, such as SDS, etc. Therefore, Tween20 can also be included in the binding buffer in this application.

Claims

1. A method for recovering biotin-modified nucleic acids using streptavidin magnetic beads, the method comprising: The streptavidin magnetic beads are resuspended using a binding buffer, and the streptavidin magnetic beads and the biotin-modified nucleic acid are incubated in the presence of the binding buffer so that the biotin-modified nucleic acid is bound to the streptavidin magnetic beads, wherein the binding buffer comprises Tris, EDTA and NaCl, the working concentration of the NaCl is 0.005-0.125 M, the working concentration of the Tris is 2.5-10 mM, the working concentration of the EDTA is 0-1 mM, and the pH of the binding solution is 7-8; an external magnetic field is applied, and the supernatant is removed; the streptavidin magnetic beads are washed, and the washing includes incubating the streptavidin magnetic beads with the binding buffer in an external magnetic field environment, and removing the supernatant; and the biotin-modified nucleic acid is eluted from the streptavidin magnetic beads.

2. The method of claim 1, wherein the working concentration of NaCl is 0.005-0.1M.

3. The method of claim 1, wherein the binding buffer further comprises a surfactant.

4. The method of claim 3, wherein the surfactant is a nonionic surfactant.

5. The method of claim 4, wherein the nonionic surfactant is Tween 20.

6. The method of claim 5, wherein the working concentration of Tween 20 is 0.01%-5%. The method of claim 1 , wherein the incubation is room temperature incubation.

8. The method according to claim 1, wherein the incubation is performed at room temperature (25-35°C) for 5-30 min.

9. The method of claim 1, wherein the step of eluting the biotin-modified nucleic acid from the streptavidin magnetic beads comprises adding an eluent, incubating, and collecting the eluent.

10. The method of claim 9, wherein the eluent comprises formamide.

11. The method of claim 10, wherein the formamide is 80%-98% formamide.

12. The method of claim 9, wherein the step of eluting the biotin-modified nucleic acid from the streptavidin magnetic beads further comprises heat elution, wherein the heat elution comprises heating at 50-95°C for 1-20 min.

13. The method of claim 1, further comprising repeating the washing step.

14. The method of claim 1, further comprising repeating the washing step once, twice or three times.

15. The method of claim 1, wherein the biotin-modified nucleic acid is a biotin-modified single-stranded DNA, a biotin-modified double-stranded DNA, a biotin-modified RNA, or a biotin-modified DNA-RNA hybrid.

16. Use of the method according to any one of claims 1 to 15 in improving the recovery efficiency of biotin-modified nucleic acids.

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