Enhanced red blood cell lysis solution, red blood cell DNA extraction kit and method

Abstract

The application discloses an enhanced red blood cell lysis solution, a red blood cell DNA extraction kit and a method. The application provides an enhanced red blood cell lysis solution, which can improve the yield of red blood cell DNA. The application also provides a red blood cell DNA extraction kit and discloses a method for separating or extracting DNA from red blood cells. In addition, the application also discloses applications of the kit and the method for separating or extracting DNA from red blood cells, and the applications include applications in detecting cancer or predicting the risk of suffering from cancer. The enhanced red blood cell lysis solution and the kit provided by the application have important scientific value and application prospect.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to an enhanced red blood cell lysis buffer, a red blood cell DNA extraction kit, and a method thereof. Background Technology

[0002] Blood DNA is a key biomarker in tumor liquid biopsy, playing a crucial role in recurrence monitoring, minimal residual disease assessment, prognosis prediction, and guiding treatment plans. Currently, mainstream methods for blood DNA extraction focus on DNA from whole blood or plasma, but methods for extracting DNA from erythrocytes are relatively limited. Because mature erythrocytes lack a nucleus and have extremely low DNA content, traditional extraction methods result in low DNA yields, failing to meet the demands of high-sensitivity detection and DNA sequencing. Therefore, developing a rapid, convenient, and efficient erythrocyte DNA extraction kit has significant scientific value and application prospects. Summary of the Invention

[0003] To overcome the shortcomings of the prior art, the present invention aims to provide a red blood cell DNA extraction kit and method.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] The first aspect of the present invention provides an enhanced erythrocyte lysis buffer.

[0006] Furthermore, the enhanced erythrocyte lysis buffer includes erythrocyte lysis buffer and NaOH.

[0007] Preferably, the erythrocyte lysis buffer contains NaOH.

[0008] Preferably, the NaOH is a NaOH solution, and the erythrocyte lysis buffer is independent of the NaOH solution.

[0009] Preferably, the erythrocyte lysis buffer is NH4Cl.

[0010] Preferably, the concentration of the NaOH solution is 62.05 mg / ml, and the concentration of the NH4Cl is 8 g / L.

[0011] More preferably, the volume ratio of the NaOH solution to NH4Cl is 0.15:1.

[0012] A second aspect of the present invention provides a red blood cell DNA extraction kit.

[0013] Furthermore, the kit includes the enhanced erythrocyte lysis buffer and DNA extraction buffer described in the first aspect of the present invention.

[0014] Furthermore, the DNA extraction solution includes buffer A and buffer B, wherein buffer A comprises guanidine isothiocyanate, sodium dodecyl sulfate, isopropanol and Tris-HCl solution, and buffer B comprises guanidine isothiocyanate and anhydrous ethanol.

[0015] Preferably, in buffer A, the concentration of guanidine isothiocyanate is 4M, the concentration of sodium dodecyl sulfate is 1%, the concentration of isopropanol is 30%, and the concentration of Tris-HCl solution is 10mM with a pH of 8.0.

[0016] Preferably, in buffer B, the concentration of guanidine isothiocyanate is 1M and the concentration of anhydrous ethanol is 50%.

[0017] Furthermore, the kit also includes reagents commonly used in the DNA extraction process, including but not limited to PBS buffer, RNase, proteinase K, and anhydrous ethanol.

[0018] Furthermore, the kit also includes instructions for use or a label.

[0019] Furthermore, the instruction manual or label indicates that the kit is used for extracting red blood cell DNA.

[0020] In some embodiments of the invention, the kit further comprises one or more sterile containers, such containers being boxes, ampoules, bottles, tubular vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containerizing pharmaceuticals.

[0021] In some embodiments of the present invention, the kit further includes reagents used in the DNA extraction process, including but not limited to CATB extraction solution, anhydrous ethanol, chloroform / isoamyl alcohol, proteinase K, RNase, phenol, TE buffer, etc. The CATB extraction solution (cetyltrimethylammonium bromide) is a commonly used detergent that can effectively lyse cells and release DNA. The anhydrous ethanol is used to precipitate DNA by mixing it with an aqueous DNA solution.

[0022] A third aspect of the present invention provides a method for isolating or extracting DNA from red blood cells.

[0023] Furthermore, the method includes: adding red blood cell lysis buffer to the red blood cell sample, centrifuging to obtain the supernatant mixture; adding NaOH solution to the mixture and incubating for a period of time, and then adding DNA extraction buffer to obtain red blood cell DNA.

[0024] Furthermore, the erythrocyte lysis buffer is selected from one or more combinations of NH4Cl, KHCO3, EDTA-Na2, or Tris.

[0025] Preferably, the erythrocyte lysis buffer is NH4Cl.

[0026] Preferably, the volume ratio of the red blood cell sample to NH4Cl is 1:2.

[0027] In this invention, the erythrocyte lysis buffer disrupts the erythrocyte cell membrane, releasing DNA from the erythrocytes into the lysis buffer. Simultaneously, the erythrocyte lysis buffer does not disrupt the granulocyte membrane, preserving the morphology of the granulocytes and preventing DNA release into the lysis buffer. Centrifugation is used to discard the precipitate, retaining the lysed components derived from erythrocytes while removing granulocytes and other precipitates.

[0028] In some embodiments, the erythrocyte lysis buffer includes NH4Cl, KHCO3, EDTA-Na2, and Tris; the volume ratio of erythrocytes to the erythrocyte lysis buffer is any ratio between 1:1 and 10, for example, the volume ratio of erythrocytes to erythrocyte lysis buffer can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In a specific embodiment of the present invention, the erythrocyte lysis buffer is NH4Cl, and the volume ratio of the erythrocyte sample to NH4Cl is 1:2.

[0029] In some embodiments, the method for isolating or extracting DNA from cells further includes the steps of collecting peripheral blood from a subject and separating red blood cells. In this application, "subject" means the person being tested. In some specific embodiments, "subject" refers to a human subject. In this application, "peripheral blood" means blood released from hematopoietic organs into the circulatory system and participating in circulation. "Peripheral blood" is different from immature blood cells in hematopoietic organs (such as bone marrow). In this disclosure, peripheral blood can be collected by methods known in the art, such as venous, fingertip, or earlobe sampling. Typically, peripheral blood consists of plasma and blood cells, wherein blood cells further include white blood cells, red blood cells, and platelets. By volume, red blood cells account for approximately 45% of total peripheral blood, plasma accounts for approximately 54.3% of total peripheral blood, and white blood cells account for approximately 0.7% of total peripheral blood.

[0030] In some embodiments, the method further includes the step of adding a erythrocyte stabilizer after peripheral blood collection. The erythrocyte stabilizer is selected from one or more combinations of EDTA, citrate, sodium fluoride, sodium orthovanadate, sodium glycerophosphate, sodium pyrophosphate, tetraimidazole hydrochloride, benzyl sulfonyl fluoride, pepsin inhibitor, EDTA-Na2, formaldehyde polymer, acetaldehyde polymer, propionaldehyde polymer, basic salts, hydrochloric acid, and Tris.

[0031] In other embodiments, methods for separating peripheral blood erythrocytes include natural sedimentation, differential sedimentation, sodium chloride separation, direct centrifugation, and density gradient centrifugation. These methods utilize the density differences between different components of peripheral blood to separate them. For example, density gradient centrifugation can be used to separate different components of peripheral blood.

[0032] Furthermore, the concentration of the NaOH solution is 62.05 mg / ml.

[0033] Preferably, the volume ratio of the NaOH solution to NH4Cl is 0.15:1.

[0034] In this invention, the applicant has experimentally demonstrated for the first time that adding NaOH solution during the extraction of erythrocyte DNA can increase the total amount of erythrocyte DNA obtained. NaOH, due to its alkalinity, can further degrade incompletely broken erythrocyte membranes and hemoglobin, promoting the release and ionization of DNA, offering advantages of low cost and high extraction efficiency. Furthermore, the specific lysis of erythrocytes using ammonium chloride can remove contamination from nucleated cell DNA. The NaOH solution can convert ammonium chloride into ammonia, which can be volatilized by heating, ultimately producing sodium chloride.

[0035] In some embodiments of the present invention, after adding NaOH solution, the mixture is incubated at 45°C to 50°C for 10 min to 20 min; preferably, after adding NaOH solution, the mixture is incubated at 45.5°C for 10 min.

[0036] Furthermore, the DNA extraction solution includes buffer A and buffer B, wherein buffer A comprises guanidine isothiocyanate, sodium dodecyl sulfate, isopropanol and Tris-HCl solution, and buffer B comprises guanidine isothiocyanate and anhydrous ethanol.

[0037] Preferably, during the extraction process, buffer A is added first and reacted for a period of time before anhydrous ethanol is added. Then, the reaction solution is added to the adsorption column, centrifuged, and the waste liquid is discarded. Then, buffer B is added to the adsorption column, centrifuged, washed, and red blood cell DNA is obtained.

[0038] Preferably, the ratio of the buffer solution A to the total reaction volume is 1:1.

[0039] Preferably, in buffer A, the concentration of guanidine isothiocyanate is 4M, the concentration of sodium dodecyl sulfate is 1%, the concentration of isopropanol is 30%, and the concentration of Tris-HCl solution is 10mM with a pH of 8.0.

[0040] Preferably, in buffer B, the concentration of guanidine isothiocyanate is 1M and the concentration of anhydrous ethanol is 50%.

[0041] In some embodiments of the present invention, after adding buffer A, the reaction is carried out at 45°C to 50°C for 30 to 60 minutes until the solution turns dark brown, then anhydrous ethanol is added, mixed thoroughly, and transferred to the adsorption column. Preferably, after adding buffer A, the reaction is carried out at 56.5°C for 45 minutes, then anhydrous ethanol is added at a ratio of 1:1 with the buffer solution, mixed thoroughly, and transferred to the adsorption column.

[0042] In some embodiments of the present invention, after adding buffer B, anhydrous ethanol may be added for washing, preferably with a concentration of 80%. More preferably, Tris solution is added after washing to dissolve the DNA.

[0043] In this invention, the DNA extraction solution also includes extraction solutions commonly used in the DNA extraction process, including but not limited to CTAB extraction solution, TE buffer, etc.

[0044] Furthermore, the method also includes a step of washing the red blood cells before adding the red blood cell lysis buffer.

[0045] Preferably, the method further includes the step of adding RNase to remove RNA.

[0046] Preferably, the method further includes the step of adding proteinase K to degrade the protein.

[0047] In this invention, the method includes the step of washing the red blood cell sample upon acquisition to remove the influence of plasma DNA. Preferably, washing is performed using PBS buffer.

[0048] In some embodiments of the present invention, the reagent for degrading proteins includes guanidine cyanate, guanidine hydrochloride, sodium iodide, urea, or an enzyme that acts on proteins; preferably, the reagent for degrading proteins is proteinase K, and more preferably, the ratio of the added proteinase K to the total volume is 0.1:1.

[0049] The fourth aspect of the present invention provides for any of the following applications.

[0050] Furthermore, the applications include:

[0051] 1) Application of NaOH solution in the preparation of erythrocyte DNA extraction kit;

[0052] 2) The application of the enhanced erythrocyte lysis buffer described in the first aspect of this invention in the preparation of a erythrocyte DNA extraction kit;

[0053] 3) The application of the kit described in the second aspect of the present invention in detecting cancer or predicting the risk of developing cancer;

[0054] 4) The application of red blood cell DNA extracted by the method described in the third aspect of the present invention in detecting cancer or predicting the risk of developing cancer.

[0055] Furthermore, the concentration of the NaOH solution is 62.05 mg / ml.

[0056] In the occurrence and progression of cancer, genetic information undergoes a series of changes, including DNA mutations, insertions / deletions, chromosomal structural variations, copy number variations, and alterations in epigenetic information. During cancer evolution, DNA sequence variations occur randomly, and only when these variations occur in key growth control genes can they lead to malignant tumors. Most abnormal gene expression stems from epigenetic changes, typically alterations in DNA methylation levels. Studies have shown that changes in gene methylation levels precede gene mutations, and tracking and detecting changes in gene methylation can predict cancer development at an earlier stage. DNA methylation refers to the methylation of the 5th carbon atom of cytosine in CpG dinucleotides. As a stable modified state, it can be inherited by newly formed daughter DNA during DNA replication under the action of DNA methyltransferases, making it an important epigenetic mechanism. During DNA methylation, methylation of the gene promoter region can lead to transcriptional silencing of tumor suppressor genes; therefore, it is closely related to tumorigenesis. Aberrant methylation includes hypermethylation of tumor suppressor genes and DNA repair genes, hypomethylation of repetitive DNA sequences, and loss of imprinting of certain genes, all of which are associated with the development of various tumors. Therefore, the erythrocyte DNA obtained using the kits and methods provided in this application can be used to detect cancer or cancer risk; the cancers include all cancers, especially liver cancer, colorectal cancer, gastric cancer, esophageal cancer, lung cancer, cervical cancer, pancreatic cancer, breast cancer, or head and neck cancer.

[0057] Advantages and benefits of the present invention: The innovative discovery of the present invention is that adding NaOH solution during the red blood cell DNA extraction process can increase the total amount of red blood cell DNA obtained, thereby providing an enhanced red blood cell lysis buffer for the field, and providing a rapid, convenient and efficient red blood cell DNA extraction kit, which has important scientific value and application prospects. Attached Figure Description

[0058] Figure 1 This shows the measurement results of the extracted DNA concentration in the embodiments and comparative examples of the present invention. Detailed Implementation

[0059] The present invention will be further described below with reference to specific embodiments. However, the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

[0060] Unless otherwise specified, the materials, reagents, etc., used in the following examples are commercially available. Generally, the terms and techniques related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally performed according to conventional methods known in the art.

[0061] Example: Method for extracting DNA from red blood cells

[0062] I. Experimental Methods

[0063] 1. The method for extracting red blood cell DNA in this embodiment of the invention is as follows:

[0064] (1) Collect 500µL of red blood cells, place them in a 1.5mL enzyme-free centrifuge tube, add 1mL of PBS, invert to mix, centrifuge at 400g for 5 minutes, and recover the red blood cell pellet.

[0065] (2) Repeat step (1) once to remove residual plasma DNA;

[0066] (3) Add 1 mL of ammonium chloride red solution at a ratio of 1:2, wait for 5 to 10 minutes, invert and mix several times during this period, and observe the properties of the solution;

[0067] (4) After the solution is clear, centrifuge at 400g for 5 minutes. A white precipitate and a red supernatant will be visible. Under a light microscope, the white precipitate is observed to be red blood cell fragments and a small number of nucleated cells. Carefully aspirate the red supernatant and place it in a new 1.5mL enzyme-free centrifuge tube.

[0068] (5) Add 10µL of 100mg / ml RNase, invert and mix well, and let stand at room temperature for 5 minutes to remove RNA;

[0069] (6) Add 150µl of NaOH solution at a ratio of 1:0.15 with the red lysate, and mix by inverting.

[0070] (7) Place the centrifuge tube in a 45.5℃ metal bath, open the lid, and react for 10 minutes;

[0071] (8) Add 150µl of 20mg / mL proteinase K at room temperature in a ratio of 1:0.1 to the total volume, place it in a 5mL enzyme-free centrifuge tube, and mix by inverting.

[0072] (9) Add approximately 1.6 mL of buffer A at a ratio of 1:1 to the total volume, and mix by inverting.

[0073] (10) Place the centrifuge tube in a water bath at 56.5℃ and react for 45 minutes. The solution will turn dark brown.

[0074] (11) Add about 1.6 mL of anhydrous ethanol at a ratio of 1:1 with buffer A, invert and mix well, the total volume of the system is about 4.5~4.8 mL;

[0075] (12) Take 600µl of solution and add it dropwise to the silica gel adsorption column. Centrifuge at 12000g for 1 minute and discard the waste liquid.

[0076] (13) Repeat step (12) 6 to 7 times until all the solution in the system is absorbed;

[0077] (14) Add 600µl of buffer B to the silica gel adsorption column, centrifuge at 12000g for 1 minute, and discard the waste liquid;

[0078] (15) Add 600µl of 80% anhydrous ethanol to the silica gel adsorption column, centrifuge at 12000g for 1 minute, and discard the waste liquid;

[0079] (16) Repeat step (15) twice;

[0080] (17) Centrifuge the empty silica gel adsorption column at 12000g for 2 minutes, remove the waste liquid, and let it stand at room temperature for 5 minutes to dry.

[0081] (18) Add 60~80µL of 10mM Tris solution with pH 8.0 to the silica adsorption column, let stand for 5 minutes, centrifuge at 12000g for 2 minutes, and collect the filtered liquid as the red blood cell DNA solution.

[0082] (19) Red blood cell DNA solution can be stored at -80°C or -20°C.

[0083] 2. The method for extracting the proportion is as follows:

[0084] (1) Collect 500µL of red blood cells, place them in a 1.5mL enzyme-free centrifuge tube, add 1mL of PBS, invert to mix, centrifuge at 400g for 5 minutes, and recover the red blood cell pellet.

[0085] (2) Repeat step (1) once to remove residual plasma DNA;

[0086] (3) Add 1 mL of ammonium chloride red solution at a ratio of 1:2, wait for 5 to 10 minutes, invert and mix several times during this period, and observe the properties of the solution;

[0087] (4) After the solution becomes clear, centrifuge at 400g for 5 minutes. A white precipitate and a red supernatant will be visible. Under a light microscope, the white precipitate is observed to be fragments of red blood cells and a small number of nucleated cells. Carefully aspirate the red supernatant and place it in a new 1.5mL enzyme-free centrifuge tube;

[0088] (5) Add 10µL of 100mg / ml RNase, invert and mix well, and let stand at room temperature for 5 minutes to remove RNA;

[0089] (6) Add 150µl of 20mg / mL proteinase K at room temperature in a ratio of 1:0.1 to the total volume, place it in a 5mL enzyme-free centrifuge tube, and mix by inverting.

[0090] (7) Add approximately 1.6 mL of buffer A at a ratio of 1:1 to the total volume, and mix by inverting.

[0091] (8) Place the centrifuge tube in a 56.5℃ water bath and react for 45 minutes. The solution will turn dark brown.

[0092] (9) Add about 1.6 mL of anhydrous ethanol at a ratio of 1:1 with buffer A, invert and mix well, the total volume of the system is about 4.5~4.8 mL;

[0093] (10) Take 600µl of solution and add it dropwise to the silica gel adsorption column. Centrifuge at 12000g for 1 minute and discard the waste liquid.

[0094] (11) Repeat step (10) 6-7 times until all the solution in the system is absorbed;

[0095] (12) Add 600µl of buffer B to the silica gel adsorption column, centrifuge at 12000g for 1 minute, and discard the waste liquid;

[0096] (13) Add 600µl of 80% anhydrous ethanol to the silica gel adsorption column, centrifuge at 12000g for 1 minute, and discard the waste liquid;

[0097] (14) Repeat step (13) twice;

[0098] (15) Centrifuge the empty silica gel adsorption column at 12000g for 2 minutes, remove the waste liquid, and let it stand at room temperature for 5 minutes to dry.

[0099] (16) Add 60~80µL of 10mM Tris solution with pH 8.0 to the silica adsorption column, let stand for 5 minutes, centrifuge at 12000g for 2 minutes, and collect the filtered liquid as the red blood cell DNA solution.

[0100] II. Experimental Results

[0101] The results of DNA concentration determination in the examples and comparative examples are shown in Table 1 and... Figure 1As shown in the comparative examples and comparative cases, adding NaOH solution can increase the total amount of erythrocyte DNA obtained. NaOH, due to its alkalinity, can further degrade incompletely broken erythrocyte membranes and hemoglobin, promoting DNA release and ionization, offering advantages such as low cost and high extraction efficiency. Furthermore, using ammonium chloride to specifically lyse erythrocytes can remove contamination from nucleated cell DNA. NaOH solution can convert ammonium chloride to ammonia, which can be volatilized by heating, ultimately producing sodium chloride. The extracted erythrocyte DNA can be used directly for DNA-related experiments or, after further purification, for DNA sequencing.

[0102] Table 1. Results of DNA concentration determination in the examples and comparative examples.

[0103]

[0104] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A method for isolating or extracting DNA from red blood cells, characterized in that, The method comprises: adding an NH4Cl lysis solution to a red blood cell sample, centrifuging to obtain a supernatant mixture; adding a NaOH solution to the mixture and incubating for a period of time, and then adding a DNA extraction solution to obtain red blood cell DNA; The concentration of NH4Cl is 8 g / L, the volume ratio of the red blood cell sample to NH4Cl is 1:2, the concentration of the NaOH solution is 62.05 mg / ml, and the volume ratio of the NaOH solution to NH4Cl is 0.15:1; The DNA extraction solution comprises buffer A and buffer B, the buffer A comprises guanidine isothiocyanate, sodium dodecyl sulfate, isopropanol and Tris-HCl solution, and the buffer B comprises guanidine isothiocyanate and anhydrous ethanol; During the extraction process, the buffer A is first added and reacted for a period of time, and then anhydrous ethanol is added, and then the reaction solution is added to an adsorption column and centrifuged to discard the waste liquid; then the buffer B is added to the adsorption column and centrifuged to obtain red blood cell DNA, the ratio of the buffer A to the total reaction volume is 1:1, and the volume ratio of the anhydrous ethanol to the buffer A is 1:1; In the buffer A, the concentration of guanidine isothiocyanate is 4M, the concentration of sodium dodecyl sulfate is 1%, the concentration of isopropanol is 30%, and the concentration of the Tris-HCl solution is 10 mM and the pH is 8.0; In the buffer B, the concentration of guanidine isothiocyanate is 1M and the concentration of anhydrous ethanol is 50%.

2. The method of claim 1, wherein, The method further comprises a step of washing the red blood cells before adding the red blood cell lysis solution.

3. The method of claim 1, wherein, The method further comprises a step of adding RNase to remove RNA.

4. The method of claim 1, wherein, The method further comprises a step of adding proteinase K to degrade proteins. The method further comprises a step of washing the red blood cells before adding the red blood cell lysis solution.

Images