Lysate and use method thereof
By optimizing the composition and usage of the lysis buffer, the problem of long detection time for DNA from old bones was solved, enabling rapid and simplified DNA extraction and detection.
Patent Information
- Application Number
- CN202511036251.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-26
- Publication Date
- 2025-10-28
AI Technical Summary
In existing technologies, DNA testing of old bones requires a long digestion and lysis time, which is labor-intensive and time-consuming.
A lysis buffer and its usage method are provided, including the ratio of reagent A and reagent B and the usage steps, which rapidly lyse DNA in old bone marrow through steps such as vortexing, incubation in the dark, and centrifugation.
The pre-processing steps for old skeletal specimens have been greatly simplified, enabling rapid processing and DNA testing at room temperature to identify victims.
Smart Images
Figure CN120843646A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to a lysis buffer and its method of use. Background Technology
[0002] Lysis buffers are primarily used for the lysis of cells and tissues to release internal proteins, nucleic acids, and other biomolecules, and are widely used in biomedical and molecular biology experiments.
[0003] The main function of the lysis buffer.
[0004] Cell lysis: Lysis buffers break down cell membranes, releasing intracellular components, including proteins and nucleic acids. Commonly used lysis buffers, such as RIPA lysis buffer and SDS lysis buffer, can effectively lyse cells and extract soluble proteins.
[0005] Protein extraction: Lysis buffers can dissolve intracellular proteins, facilitating subsequent analysis and experiments, such as Western blotting and immunoprecipitation (IP). Different types of lysis buffers are suitable for different experimental needs; for example, RIPA lysis buffer is suitable for extracting complex proteins, while SDS lysis buffer is suitable for strong lysis.
[0006] Inhibition of protease activity: Many lysis buffers contain protease inhibitors to prevent protein degradation during extraction and to maintain its activity and structure.
[0007] DNA testing of old bones has always been a difficult problem and challenge for forensic workers. In order to extract DNA that can be used for STR testing from old bones, the bone samples first need to be crushed, digested, lysed, and purified. In the laboratory, we usually use a stepwise lysis method to digest and lyse the bones, which is the most labor-intensive and time-consuming step, usually taking 3 days or longer. Summary of the Invention
[0008] The purpose of this invention is to provide a lysis solution and its method of use, which solves the problem that the digestion and lysis of bones requires a long time.
[0009] To achieve the above objectives, the present invention provides the following technical solution: A lysis buffer comprising reagent A and reagent B, wherein reagent A comprises the following components: 50 ml of TES buffer (10×) is required. 60ml of 0.5M EDTA is required. 25ml of EGTA 0.6M is required; The B reagent is composed of the following components: Proteinase K 20 mg / ml requires 5 ml. 10ml of DTT 1M is required. 0.5 ml of thioglycerin is needed. Sodium dodecyl sarcosinate (NLS) 1% requires 5 ml. CaCl2 110.98-10mmol (1.1098g) 2.5ml of glycerin is needed. 2.5ml is needed for 5×TES. 2.5ml is needed for 1M Tris-HCl. 40ml of ultrapure water is needed.
[0010] Furthermore, the normal usage ratio of reagent A to reagent B is 2:1; The storage methods for reagent A and reagent B are as follows: for short-term storage, the storage temperature is 4°C, and the external conditions are sealed and protected from light for one week; for long-term storage, the storage temperature is -20°C and the container is sealed. The method for using the lysis buffer includes the following steps: S1 Preparation of lysis buffer, S2 Cleaning and grinding, S3 Weighing and placing, S4 Adding reagent A, S5 Vortexing, S6 Incubation in the dark, S7 Second vortexing, S8 Centrifugation, S9 Adding reagent B, S10 Third vortexing, S11 Second incubation in the dark, S12 Fourth vortexing, S13 Second centrifugation, S14 Transfer, S15 Detection, purification, and storage; S1 lysis buffer preparation: Pretreatment solution A reagent: 5.1 ml Pretreatment solution B reagent: 2.4 ml; S2 Cleaning and Grinding: Select bone or teeth of suitable size, clean them, crush them by hand or grind them with a freeze grinder, and the standard size of the fragments should be 1-3 mm. S3 Weighing and Placement: Weigh 400-500 mg of the ground bone fragments or bone powder and place them in a 2 ml microsample tube; Add reagent A to S4: After shaking reagent A well, add it to the sample tube containing bone fragments / bone powder, the amount added is 1250 μl; S5 Vortex Oscillation: Vortex the sample tube for 15 seconds to ensure the mixture is thoroughly mixed. S6 incubation in the dark: Place the sample tube on a shaker, set the temperature to 37℃, incubate in the dark for 3.0 hours, and rotate at 1000 rpm. S7 Secondary Vortex Oscillation: Remove the sample tube and vortex for about 15 seconds to mix thoroughly. S8 centrifugation treatment: After cooling to room temperature, place it in a centrifuge, set the centrifugal force to 13000×g, centrifuge for 2 minutes, and discard the supernatant. Add reagent B to S9: Add 600 μl of shaken reagent B to the precipitate in the sample tube; S10 three-stage vortex oscillation: Vortex the sample tube containing reagent B for 10 seconds to mix thoroughly. S11 secondary light-protected incubation: Place the sample tube on a shaker, set the temperature to 56℃, and incubate overnight (4.0-12 hours) in the dark at a speed of 500 rpm. S12 Four-fold Vortex Oscillation: Remove the sample tube and vortex to mix for 10 seconds; S13 secondary centrifugation treatment: After cooling to room temperature, place it in a centrifuge, set the centrifugal force to 13000×g, and centrifuge for 4 minutes; S14 Transfer: Without disturbing the lower layer of precipitate, transfer the supernatant to a new 2ml sample tube and discard the sample tube containing the precipitate. S15 detection, purification, and storage: The obtained supernatant can be directly used for loading onto a bone DNA rapid detection instrument; it can also be further purified using DNA purification methods, such as magnetic bead method, silica bead method, Chelex method, organic method, DNA purification kit, etc.; the supernatant can also be stored at -20℃ for later use.
[0011] Further, the supernatant was transferred to a 2 ml spiral sample tube, and an equal volume of binding buffer (5.0 M guanidine isothiocyanate (GuSCN), 60% isopropanol) was added and mixed well. 8 µl of magnetic beads were added to bind DNA at room temperature in the dark for 90 min. After DNA binding, the sample was washed once each with 1.0 ml of 75%, 75%, and 100% ethanol. Finally, the sample DNA bound to the magnetic beads was eluted with 30 µl of 0.5×TE. The sample was stored at -20°C in a sealed container in the dark.
[0012] The beneficial effects of this invention are as follows: This application, through its specially optimized design for DNA samples such as old bones and teeth, can greatly simplify the pre-processing steps of old bone samples. Through practical testing in domestic cases, it can quickly process bone samples involved in cases, and, in conjunction with the Andi rapid DNA testing instrument, determine the identity of the victims. The samples can be stored and used at room temperature.
[0013] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description
[0014] Figure 1 This is a detection DNA map of a practical example shown in an embodiment of the present invention. Detailed Implementation
[0015] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0016] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.
[0017] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.
[0018] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.
[0019] Please see Figure 1 A preferred embodiment of this application shows a lysis buffer comprising reagent A and reagent B, wherein reagent A is composed of the following components: 50 ml of TES buffer (10×) is required. 60ml of 0.5M EDTA is required. 25ml of EGTA 0.6M is required; The B reagent is composed of the following components: Proteinase K 20 mg / ml requires 5 ml. 10ml of DTT 1M is required. 0.5 ml of thioglycerin is needed. Sodium dodecyl sarcosinate (NLS) 1% requires 5 ml. CaCl2 110.98-10mmol (1.1098g) 2.5ml of glycerin is needed. 2.5ml is needed for 5×TES. 2.5ml is needed for 1M Tris-HCl. 40ml of ultrapure water is needed.
[0020] The normal mixing ratio of reagent A to reagent B is 2:1; The storage methods for reagent A and reagent B are as follows: for short-term storage, the storage temperature is 4°C, and the external conditions are sealed and protected from light for one week; for long-term storage, the storage temperature is -20°C and the container is sealed. The method for using the lysis buffer includes the following steps: S1 Preparation of lysis buffer, S2 Cleaning and grinding, S3 Weighing and placing, S4 Adding reagent A, S5 Vortexing, S6 Incubation in the dark, S7 Second vortexing, S8 Centrifugation, S9 Adding reagent B, S10 Third vortexing, S11 Second incubation in the dark, S12 Fourth vortexing, S13 Second centrifugation, S14 Transfer, S15 Detection, purification, and storage; S1 lysis buffer preparation: Pretreatment solution A reagent: 5.1 ml Pretreatment solution B reagent: 2.4 ml; S2 Cleaning and Grinding: Select bone or teeth of suitable size, clean them, crush them by hand or grind them with a freeze grinder, and the standard size of the fragments should be 1-3 mm. S3 Weighing and Placement: Weigh 400-500 mg of the ground bone fragments or bone powder and place them in a 2 ml microsample tube; Add reagent A to S4: After shaking reagent A well, add it to the sample tube containing bone fragments / bone powder, the amount added is 1250 μl; S5 Vortex Oscillation: Vortex the sample tube for 15 seconds to ensure the mixture is thoroughly mixed. S6 incubation in the dark: Place the sample tube on a shaker, set the temperature to 37℃, incubate in the dark for 3.0 hours, and rotate at 1000 rpm. S7 Secondary Vortex Oscillation: Remove the sample tube and vortex for about 15 seconds to mix thoroughly. S8 centrifugation treatment: After cooling to room temperature, place it in a centrifuge, set the centrifugal force to 13000×g, centrifuge for 2 minutes, and discard the supernatant. Add reagent B to S9: Add 600 μl of shaken reagent B to the precipitate in the sample tube; S10 three-stage vortex oscillation: Vortex the sample tube containing reagent B for 10 seconds to mix thoroughly. S11 secondary light-protected incubation: Place the sample tube on a shaker, set the temperature to 56℃, and incubate overnight (4.0-12 hours) in the dark at a speed of 500 rpm. S12 Four-fold Vortex Oscillation: Remove the sample tube and vortex to mix for 10 seconds; S13 secondary centrifugation treatment: After cooling to room temperature, place it in a centrifuge, set the centrifugal force to 13000×g, and centrifuge for 4 minutes; S14 Transfer: Without disturbing the lower layer of precipitate, transfer the supernatant to a new 2ml sample tube and discard the sample tube containing the precipitate. S15 detection, purification, and storage: The obtained supernatant can be directly used for loading onto a bone DNA rapid detection instrument; it can also be further purified using DNA purification methods, such as magnetic bead method, silica bead method, Chelex method, organic method, DNA purification kit, etc.; the supernatant can also be stored at -20℃ for later use.
[0021] DNA purification method: Transfer the supernatant to a 2ml spiral sample tube, add an equal volume of binding buffer (5.0M guanidine isothiocyanate (GuSCN), 60% isopropanol) and mix well. Add 8µl of magnetic beads and bind DNA at room temperature in the dark for 90min. After DNA binding, wash once each with 1.0ml of 75%, 75%, and 100% ethanol. Finally, elute the sample DNA bound to the magnetic beads with 30µl of 0.5×TE. Store at -20℃ in a sealed container in the dark.
[0022] Real-world case study: Source of the old bone sample: The sample was submitted for testing earlier this month by a subordinate unit of a municipal public security bureau. The sample was the ulna of a corpse found on August 16, 2018, in a bungalow. Analysis indicated the deceased had been dead for two years at the time of discovery. Conventional laboratory methods failed to produce STR typing. A rapid bone chromatogram was used to identify some loci. The instrument used was the Andi DNA rapid chromatogram, and the reagent used was the lysis buffer of this invention. The test results (number of STR detection sites) were superior to those obtained in the laboratory.
[0023] Testing time: 9:30 AM, June 13, 2019 Detection method: 1. Grinding the sample: Crush the sample and grind some of the fragments to a size of 1-3 mm; 2. Preparation of supernatant: Place an appropriate amount of fragments into a 2ml centrifuge tube and add approximately 200µl of [unspecified liquid]. Bone lysis fluid was incubated overnight at 56°C with low-speed shaking in a constant-temperature mixer. 3. Prepare the swab: Add 100-150 μL of supernatant to the swab; 4. Sample loading and testing: Place the swab scan into the I-CHIP kit and run the test; 5. After the operation is complete, export the test data and view the DNA profile.
[0024] Some precipitation may occur between reagents A and B, which is normal. Shake well before use. The optimal operating conditions for these reagents are room temperature; avoid light exposure. When pipetting, it is recommended to remove a small section from the tip of the pipette to increase the orifice diameter and prevent clogging.
[0025] The detector used in this embodiment is the Andi GAC-eba Bone DNA Rapid Detection Instrument. DNA purification methods include magnetic bead method, silica bead method, Chelex method, organic method, DNA purification kit, etc.
[0026] In summary, this invention provides a lysis buffer and its usage method. By being specially optimized for DNA samples such as old bones and teeth, this invention can significantly simplify the pretreatment steps for old bone samples. Through practical testing in domestic cases, it can quickly process bone samples involved in cases, and, in conjunction with the Andi rapid DNA testing instrument, determine the identity of the victims. It can be stored and used at room temperature.
[0027] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
Claims
1. A lysis buffer, characterized in that, The lysis buffer includes reagent A and reagent B, wherein reagent A is composed of the following components: 50 ml of TES buffer (10×) is required. 60ml of 0.5M EDTA is required. 25ml of EGTA 0.6M is required; The B reagent is composed of the following components: Proteinase K 20 mg / ml requires 5 ml. 10ml of DTT 1M is required. 0.5 ml of thioglycerin is needed. Sodium dodecyl sarcosinate (NLS) 1% requires 5 ml. CaCl2 110.98-10mmol (1.1098g) 2.5ml of glycerin is needed. 2.5ml is needed for 5×TES. 2.5ml is needed for 1M Tris-HCl. 40ml of ultrapure water is needed.
2. The lysis buffer as described in claim 1, characterized in that, The normal mixing ratio of reagent A to reagent B is 2:
1. The storage methods for reagent A and reagent B are as follows: for short-term storage, the storage temperature is 4°C, and the external conditions are sealed and protected from light for one week; for long-term storage, the storage temperature is -20°C and the container is sealed.
3. The method of using the lysis buffer as described in claim 1, characterized in that, The process includes the following steps: S1 Preparation of lysis buffer, S2 Cleaning and grinding, S3 Weighing and placing, S4 Adding reagent A, S5 Vortexing, S6 Incubation in the dark, S7 Second vortexing, S8 Centrifugation, S9 Adding reagent B, S10 Third vortexing, S11 Second incubation in the dark, S12 Fourth vortexing, S13 Second centrifugation, S14 Transfer, S15 Detection, purification, and storage. S1 lysis buffer preparation: Pretreatment solution A reagent: 5.1 ml Pretreatment solution B reagent: 2.4 ml; S2 Cleaning and Grinding: Select bone or teeth of suitable size, clean them, crush them by hand or grind them with a freeze grinder, and the standard size of the fragments should be 1-3 mm. S3 Weighing and Placement: Weigh 400-500 mg of the ground bone fragments or bone powder and place them in a 2 ml microsample tube; Add reagent A to S4: After shaking reagent A well, add it to the sample tube containing bone fragments / bone powder, the amount added is 1250 μl; S5 Vortex Oscillation: Vortex the sample tube for 15 seconds to ensure the mixture is thoroughly mixed. S6 incubation in the dark: Place the sample tube on a shaker, set the temperature to 37℃, incubate in the dark for 3.0 hours, and rotate at 1000 rpm. S7 Secondary Vortex Oscillation: Remove the sample tube and vortex for about 15 seconds to mix thoroughly. S8 centrifugation treatment: After cooling to room temperature, place it in a centrifuge, set the centrifugal force to 13000×g, centrifuge for 2 minutes, and discard the supernatant. Add reagent B to S9: Add 600 μl of shaken reagent B to the precipitate in the sample tube; S10 three-stage vortex oscillation: Vortex the sample tube containing reagent B for 10 seconds to mix thoroughly. S11 secondary light-protected incubation: Place the sample tube on a shaker, set the temperature to 56℃, and incubate overnight (4.0-12 hours) in the dark at a speed of 500 rpm. S12 Four-fold Vortex Oscillation: Remove the sample tube and vortex to mix for 10 seconds; S13 secondary centrifugation treatment: After cooling to room temperature, place it in a centrifuge, set the centrifugal force to 13000×g, and centrifuge for 4 minutes; S14 Transfer: Without disturbing the lower layer of precipitate, transfer the supernatant to a new 2ml sample tube and discard the sample tube containing the precipitate. S15 detection, purification, and storage: The obtained supernatant can be directly used for loading onto a bone DNA rapid detection instrument; it can also be further purified using DNA purification methods, such as magnetic bead method, silica bead method, Chelex method, organic method, DNA purification kit, etc.; the supernatant can also be stored at -20℃ for later use.
4. The method of using the lysis buffer as described in claim 3, characterized in that, The DNA purification method involves transferring the supernatant to a 2 ml spiral sample tube, adding an equal volume of binding buffer (5.0 M guanidine isothiocyanate (GuSCN), 60% isopropanol), mixing well, adding 8 µl of magnetic beads, and binding DNA at room temperature in the dark for 90 min. After DNA binding, the sample is washed once each with 1.0 ml of 75%, 75%, and 100% ethanol, and finally eluted with 30 µl of 0.5×TE. The sample is stored at -20°C in a sealed, light-protected environment.