Method for obtaining pure biotin primer based on paramagnetic particle method
By combining streptavidin magnetic beads with biotin probes and subsequent processing steps, the problems of primer site uniformity and low target hit rate were solved, thus improving the accuracy and efficiency of biological experiments.
Patent Information
- Application Number
- CN202511173129.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-21
- Publication Date
- 2025-11-28
AI Technical Summary
In existing technologies, when streptavidin magnetic beads bind to probes, a small portion of the primers fail to synthesize into biotin, resulting in low site uniformity and low target hit rate.
By binding streptavidin magnetic beads to biotin probes, pure biotin primers were prepared through steps such as separation by external magnetic field, TE washing, formamide thermal elution, and vacuum concentration, ensuring primer uniformity and capture efficiency.
It improves the target hit rate and the uniformity between probe sites in hybridization capture experiments in biological experiments, reduces experimental costs, and obtains more accurate data.
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Figure CN121022983A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biomedical technology, in particular to a method for obtaining pure biotin primers based on magnetic bead method. BACKGROUND
[0002] The combination of biotin magnetic beads and biotin probes is a core technology in the field of molecular biology and biotechnology, which is derived from the unique interaction between biotin and streptavidin and its high efficiency in biomolecular labeling and separation. The specific performance is as follows:
[0003] Biotin is a small molecule vitamin, and its carboxyl group forms an irreversible complex with four binding sites of streptavidin, with a low dissociation constant, which is one of the strongest non-covalent interactions. This combination has high specificity, strong stability and high detection sensitivity.
[0004] Secondly, the surface of the magnetic beads is modified with streptavidin, which becomes a biotin-labeled molecular capture carrier. Its superparamagnetic property can be quickly separated under an external magnetic field, and the uniform particle size distribution and low non-specific adsorption ensure the repeatability of the experiment.
[0005] From the aspect of technical development, biotin probes cover nucleic acid probes and protein probes, and the existing technology has been effectively optimized for magnetic bead separation technology, including probe design and experimental process.
[0006] In addition, biotin probes are also applied in various fields such as nucleic acid research, protein analysis, diagnosis and treatment, etc.
[0007] In summary, the combination technology of biotin magnetic beads and probes relies on the advantages of the biotin-streptavidin system and has become the cornerstone of modern molecular biology and diagnostics, with applications ranging from basic research to precision medicine and industrial production. In the future, it will release greater potential in emerging fields.
[0008] Although the technology of combining streptavidin magnetic beads with probes has been widely used, there are still some problems in the use process:
[0009] The synthesized biotin probes have a small part of the primers not added with biotin during the synthesis process, resulting in low uniformity of the sites on the probes combined with streptavidin magnetic beads, and low target rate in the probe (primer) capture experiment. SUMMARY
[0010] The purpose of the present application is to solve the technical problem of low uniformity of the sites on the probes combined with streptavidin magnetic beads due to the small part of the primers not synthesized with biotin when combining streptavidin magnetic beads with probes in the prior art.
[0011] To achieve the above object, the application provides a method for obtaining pure biotin primers based on a magnetic bead method, comprising the following steps: combining streptavidin magnetic beads with a biotin probe, then separating by using an external magnetic field, then washing by using TE, then washing the biotin probe by using formamide after the washing is completed, then concentrating the probe by using a vacuum concentrator, then dissolving the probe by using TE, and finally quantifying by using Qubit.
[0012] Preferably, before the biotin probe is combined with streptavidin, TE is added to the biotin probe to dissolve the biotin probe to form a MIX solution, and the volume ratio of the MIX solution to the combination of the streptavidin magnetic beads is 1:1.
[0013] Preferably, the method for obtaining the streptavidin magnetic beads is as follows: the streptavidin magnetic beads are taken out first, then the original solution is removed, then PBS is used for washing, then a magnetic bead preservation solution with the same volume as the original solution is added, and then the magnetic bead solution is uniformly mixed without precipitation by fully shaking.
[0014] Preferably, the PBS used for washing the streptavidin magnetic beads is 1x PBS, and the washing is performed twice.
[0015] Preferably, the magnetic bead preservation solution is a mixed solution of 2x HYB and ultrapure water.
[0016] Preferably, the volume ratio of 2x HYB to ultrapure water in the mixed solution is 12:12.
[0017] Preferably, the separation by using an external magnetic field comprises the following steps: after the combination of the streptavidin magnetic beads and the biotin probe is completed, the solution is fully shaken first, then the solution is placed on a magnetic stand until the solution is clear, and then the supernatant is discarded.
[0018] Preferably, the TE washing step is as follows: after TE solution (a commonly used nucleic acid dissolving solution) is added and fully shaken, the solution is placed on a magnetic stand until the solution is clear, and then the supernatant is discarded.
[0019] The above washing step is repeated at least twice.
[0020] Preferably, 95% formamide is used in the step of washing the biotin probe by using formamide, the formamide is fully shaken and mixed for 2 min after being added, and the solution is kept at 75℃ for 10 min in a metal bath.
[0021] Preferably, the probe is concentrated by using a vacuum concentrator until the probe is in the form of dry powder or is purified by using a column.
[0022] The one or more technical solutions in the embodiments of the application have at least one or more of the following technical effects:
[0023] 1、The principle of streptavidin binding to biotin is used in the application to bind the synthesized biotin probe (primer) with streptavidin magnetic beads to obtain pure biotin probes;
[0024] 2、The amount of streptavidin magnetic beads combined with biotin probes (primers) can be obtained by controlling the amount of streptavidin magnetic beads added to obtain the desired total amount of probes (primers), which facilitates the adjustment of the ratio between the sites of the probes (primers);
[0025] 3、The application can effectively improve the target rate in hybrid capture experiments in biological experiment operations, thereby obtaining more accurate and effective data and reducing experimental costs;
[0026] 4、The application can effectively improve the uniformity of the sites of the probes (primers) in hybrid capture experiments in biological experiment operations. BRIEF DESCRIPTION OF DRAWINGS
[0027] Figure 1 is a flowchart of a method for obtaining pure biotin primers based on a magnetic bead method in an embodiment of the application;
[0028] Figure 2 is a flowchart of the preparation of pure biotin probes in a method for obtaining pure biotin primers based on a magnetic bead method in an embodiment of the application. DETAILED DESCRIPTION
[0029] Some English abbreviations in the application and their interpretations are as follows:
[0030]
[0031] The application provides a method for obtaining pure biotin primers based on a magnetic bead method, please refer to Figure 1 , which comprises the following steps:
[0032] S1: Preparation of streptavidin magnetic beads:
[0033] First, remove the original solution after taking out the streptavidin magnetic beads, wash twice with 1x PBS, add a volume of magnetic bead preservation solution as the original solution, shake well to mix, and make the magnetic bead solution uniform without precipitation.
[0034] In an embodiment, the 1x PBS is prepared using 20x PBS and ultrapure water in a ratio of 1:19 by volume (if not specified, the ratio of the prepared solution in the application is by volume).
[0035] The magnetic bead preservation solution is prepared using 2x HYB and ultrapure water in a ratio of 12:12 by volume.
[0036]
[0037] S2: Preparation of pure biotin probe, please refer to Figure 2 , the specific steps are as follows:
[0038] a. Combine the streptavidin magnetic beads obtained in S1 with the biotin probe,
[0039] b. Separate using an external magnetic field,
[0040] c. Wash three times using TE,
[0041] d. Heat elute the biotin probe using 75°C, 95% formamide,
[0042] e. Concentrate the probe into a dry powder using a vacuum concentrator / purify through a column
[0043] f. Dissolve the probe using TE
[0044] g. Qubit quantification
[0045] S3: Hybrid capture experiment using the probe:
[0046] In an embodiment, the probe that has not been subjected to magnetic bead binding and purified through streptavidin magnetic beads, and each hybrid takes 0.2 pmol, and two hybrids are performed for each probe. The hybridization experiment is performed according to the Hejian Bio "Hybrid Capture Instruction Manual - 10X Wash Solution Standard Edition".
[0047] S4: Next-generation sequencing
[0048] S5: Data analysis
[0049] Based on the above preparation method, the application also provides a biotin probe, which is prepared using the above preparation method.
[0050] To make the purpose, technical solutions and advantages of the present disclosure clearer, the embodiments of the present disclosure will be described in further detail below with reference to the drawings.
[0051] Example 1:
[0052] 1. Preparation of streptavidin magnetic beads:
[0053] 1.1 Take 100 μL of streptavidin magnetic beads and place them on a magnetic stand to adsorb to the solution. Discard the supernatant.
[0054] 1.2 Add 100 μL of 1x PBS and mix well. Shake for 2 min, then place on a magnetic stand and magnetically adsorb to the clear solution. Discard the supernatant.
[0055] 1.3 Repeat step 1.2 once.
[0056] 1.4 Add 100 μL magnetic bead storage solution, mix well for 2 min until the solution is uniform and there are no precipitates or large particles.
[0057] 2. Streptavidin magnetic bead and biotin probe binding
[0058] 2.1 Add 1 tube of synthesized biotin probe M0027 to 500 μL TE to 2 pmol / μL. Mix well to dissolve.
[0059] 2.2 Take 100 μL (200 pmol) from the dissolved MIX and combine with the prepared 100 μL streptavidin magnetic beads (SA).
[0060] 2.3 Mix well for 30 min, place on a magnetic stand until the solution is clear, and discard the supernatant.
[0061] 2.4 Add 100 μL TE solution, mix well for 2 min, place on a magnetic stand, and discard the supernatant.
[0062] 2.5 Repeat step 2.4 twice.
[0063] 2.6 Add 100 μL of 95% formamide, mix well for 2 min, and incubate in a 75°C metal bath for 10 min.
[0064] 2.7 After mixing well, place on a magnetic stand. Transfer the solution probe solution to a new 1.5 mL tube or PCR tube.
[0065] 2.8 Vacuum concentrate to dry powder, add 50 μL TE Buffer and mix well to dissolve.
[0066] 2.9 Qubit (quantitative instrument name) quantification: 22.8 ng / μL.
[0067] 3. Probe hybridization
[0068] Take 0.2 pmol of each hybridization for probes that have not been subjected to magnetic bead binding and probes that have been purified by streptavidin magnetic bead binding, respectively, and perform two hybridizations for each probe. Perform the hybridization experiment according to the He Fain Biological "Hybridization Capture Instruction Manual - 10X Wash Solution Standard Edition". After mixing the library, add blocking reagent and concentrate, add hybridization solution and probes, denature at 95°C, hybridize at 65°C for 2 h, combine with 50 μL streptavidin magnetic beads at 65°C for 35 min, perform 65°C heat washing, room temperature washing, POST-PCR amplification for 17 cycles, and after library purification and recovery, elute with 30 μL TE solution to obtain the final library, and quantify the final library.
[0069] 4. Next-generation sequencing and data analysis
[0070] Results Display:
[0071]
[0072] Based on the table above, the results are analyzed as follows:
[0073] 1. The probe purified by magnetic bead binding has a 50% or higher homogeneity after hybridization that is 3-5 points higher than that synthesized without magnetic bead binding purification.
[0074] 2. In terms of target capture rate, the purified product is 6-10 points higher than the untreated product.
[0075] Implementation Case 2:
[0076] 1. Preparation of streptavidin magnetic beads:
[0077] 1.1 Take 1.5 mL of streptavidin magnetic beads, place them on a magnetic rack to adsorb until the solution becomes clear, and discard the supernatant.
[0078] 1.2 Add 500 μL of 1x PBS for washing, mix and vortex for 2 min, place on a magnetic rack, and magnetically adsorb until clear. Discard the supernatant.
[0079] 1.3 Repeat step 1.2 once.
[0080] 1.4 Add 1.5 mL of magnetic bead preservation solution and shake thoroughly for 2 min until the solution is homogeneous and free of precipitate and large particles.
[0081] 2. Binding of streptavidin magnetic beads to biotin probes
[0082] 2.1 Add the synthesized biotin probe M0024 from one tube to 500 μL TE and dissolve to 2 pmol / μL. Mix thoroughly to dissolve.
[0083] 2.2 M0024 has 34 probes. 40 μL of streptavidin magnetic beads were added to each probe that was synthesized separately.
[0084] 2.3 After shaking thoroughly for 30 min, mix the magnetic beads from the 34 probe tubes together, place them on a magnetic rack until the solution becomes clear, and discard the supernatant.
[0085] 2.4 Add 500 μL of TE solution, shake well for 2 min, place on a magnetic rack, and discard the supernatant.
[0086] 2.5 Repeat step 2.4 twice.
[0087] 2.6 Add 100 μL of 95% formamide, shake thoroughly for 2 min, and incubate in a 75℃ metal bath for 10 min.
[0088] 2.7 After oscillating and mixing evenly, place it on a magnetic stand. Transfer the solution probe solution to a new 1.5 mL tube or PCR tube.
[0089] 2.8 Concentrate to dry powder under vacuum, add 50 μL of TE Buffer, and dissolve and mix evenly.
[0090] 2.9 Qubit quantification: 27.2 ng / μL.
[0091] 3. Probe hybridization
[0092] Take 1.36 pmol of each probe that has not been bead-bound and each probe that has been purified by binding to an equal amount of streptavidin magnetic beads, and perform hybridization experiment operations according to Heyin Biology's "Hybridization Capture Operation Manual - 10X Wash Solution Standard Edition". After library mixing, add blocking reagent for concentration, add hybridization solution and add probes, perform denaturation at 95 °C, hybridize at 65 °C for 2H, bind to 50 μL of streptavidin magnetic beads at 65 °C for 35 min, perform hot washing at 65 °C, room temperature washing, and POST-PCR amplification for 17 cycles. After library purification and recovery, elute with 30 μL of TE solution to obtain the final library, and quantify the final library.
[0093] 4. Second-generation sequencing and data analysis
[0094] Result display:
[0095]
[0096] For result analysis, please refer to the above table. Based on the preparation method of this application, the biotin probes obtained have the following characteristics
[0097] 1. Homogeneity: For each probe with < unf50, the probe purified by bead-binding is about 3 points higher than the untreated probe. For each probe with ≥ unf50, the probe purified by bead-binding is about 9 - 10 points higher than the untreated probe.
[0098] 2. Capture on-target rate: For each probe purified by bead-binding, it is about 5 points higher than the untreated probe on average.
[0099] In summary, this application utilizes the principle of streptavidin's efficient, specific, and stable binding to biotin. Streptavidin magnetic beads are used to bind and purify the synthesized biotin probes (primers), ensuring that the final purified probes all contain biotin, thus effectively reducing the off-target rate in subsequent capture experiments. By utilizing the principle that a fixed concentration and amount of streptavidin magnetic beads can bind a fixed amount of biotin probe, the amount of biotin probe (primer) can be obtained by adjusting the amount of streptavidin magnetic beads added. This facilitates adjustment of the ratio between sites within the probe and also helps improve the uniformity between sites in the probe capture experiment.
Claims
1. A method for obtaining pure biotin primers based on magnetic beads, characterized in that: The procedure includes the following steps: binding streptavidin magnetic beads to a biotin probe, then separating them using an external magnetic field, followed by TE washing, followed by hot elution of the biotin probe with formamide, followed by concentration of the probe using a vacuum concentrator, followed by dissolution of the probe using TE, and finally quantification using Qubit.
2. The method for obtaining pure biotin primers based on magnetic beads according to claim 1, characterized in that: Before the biotin probe binds to streptavidin, TE is added to the biotin probe to dissolve it and form a MIX solution. The volume ratio of the MIX solution to the streptavidin magnetic beads is 1:
1.
3. The method for obtaining pure biotin primers based on magnetic beads according to claim 2, characterized in that: The method for obtaining the streptavidin magnetic beads is as follows: First, remove the streptavidin magnetic beads and remove the original solution. Wash with PBS, add an equal volume of magnetic bead preservation solution, and shake thoroughly to mix until the magnetic bead solution is uniform and free of precipitate.
4. The method for obtaining pure biotin primers based on magnetic beads according to claim 3, characterized in that: The streptavidin beads were washed with 1x PBS twice.
5. The method for obtaining pure biotin primers based on magnetic beads according to claim 4, characterized in that: The magnetic bead preservation solution is a mixed solution of 2x HYB and ultrapure water.
6. The method for obtaining pure biotin primers based on magnetic beads according to claim 5, characterized in that: The volume ratio of 2x HYB to ultrapure water in the mixed solution is 12:
12.
7. The method for obtaining pure biotin primers based on magnetic beads according to claim 1, characterized in that: Separation using an external magnetic field includes the following steps: after the streptavidin magnetic beads have bound to the biotin probe, they are first shaken thoroughly, then placed on a magnetic rack until the solution becomes clear, and the supernatant is discarded.
8. The method for obtaining pure biotin primers based on magnetic beads according to claim 1, characterized in that: The TE washing procedure is as follows: Add TE solution, shake thoroughly to mix, then place on a magnetic rack until the solution becomes clear. Discard the supernatant. Repeat the washing steps above at least twice.
9. The method for obtaining pure biotin primers based on magnetic beads according to claim 1, characterized in that: The formamide thermal elution biotin probe step uses 95% formamide. After adding formamide, the mixture is thoroughly shaken and mixed for 2 minutes, and then kept in a 75°C metal bath for 10 minutes.
10. The method for obtaining pure biotin primers based on magnetic beads according to claim 1, characterized in that: The probe is concentrated using a vacuum concentrator until it becomes a dry powder or purified by column chromatography.