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

Abstract

The invention discloses an enhanced red blood cell lysis buffer, a red blood cell DNA extraction kit and a method. The invention provides an enhanced red blood cell lysis buffer for the field. The enhanced red blood cell lysis buffer can improve the yield of red blood cell DNA (Deoxyribose Nucleic Acid). The invention 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 invention also discloses application of the kit and a method for separating or extracting DNA from red blood cells, and the application comprises application in detecting cancer or predicting cancer risk. The enhanced red blood cell lysis buffer and the kit provided by the invention have important scientific values and application prospects.

Description

Technical Field

[0001] The present application belongs to the field of biomedical technology, and specifically relates to an enhanced red blood cell lysis solution, a red blood cell DNA extraction kit and a method. Background Art

[0002] Blood DNA is a key biomarker for tumor liquid biopsy and is of great significance in recurrence monitoring, micro-residual lesion assessment, prognosis prediction and treatment guidance. At present, the mainstream methods for blood DNA extraction focus on DNA in whole blood or plasma, but there are relatively few methods for DNA extraction from red blood cells. Since mature red blood cells have no nucleus and the DNA content is extremely low, the DNA yield of traditional extraction methods is low and it is difficult to meet the needs of high-sensitivity detection and DNA sequencing. Therefore, the development of a fast, convenient and efficient red blood cell DNA extraction kit has important scientific value and application prospects. Summary of the invention

[0003] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a red blood cell DNA extraction kit and method.

[0004] In order to achieve the above object, the present invention adopts the following technical solution: A first aspect of the present invention provides an enhanced red blood cell lysing solution.

[0005] Furthermore, the enhanced red blood cell lysing solution includes red blood cell lysing solution and NaOH.

[0006] Preferably, NaOH is mixed in the red blood cell lysing solution.

[0007] Preferably, the NaOH is a NaOH solution, and the red blood cell lysing solution is independent of the NaOH solution.

[0008] Preferably, the red blood cell lysis solution is NH4Cl.

[0009] Preferably, the concentration of the NaOH solution is 62.05 mg / ml, and the concentration of the NH4Cl is 8 g / L.

[0010] More preferably, the volume ratio of the NaOH solution to NH4Cl is 0.15:1.

[0011] A second aspect of the present invention provides a red blood cell DNA extraction kit.

[0012] Furthermore, the kit comprises the enhanced red blood cell lysis solution and DNA extraction solution described in the first aspect of the present invention.

[0013] Furthermore, the DNA extraction solution includes buffer A and buffer B, wherein buffer A includes guanidine isothiocyanate, sodium dodecyl sulfate, isopropanol and Tris-HCl solution, and buffer B includes guanidine isothiocyanate and anhydrous ethanol.

[0014] Preferably, in buffer A, the concentration of guanidine isothiocyanate is 4 M, the concentration of sodium dodecyl sulfate is 1%, the concentration of isopropanol is 30%, the concentration of Tris-HCl solution is 10 mM, and the pH is 8.0.

[0015] Preferably, in buffer B, the concentration of guanidine isothiocyanate is 1 M, and the concentration of anhydrous ethanol is 50%.

[0016] 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.

[0017] Furthermore, the kit also includes instructions for use or a label.

[0018] Furthermore, the instructions or label indicate that the kit is used to extract red blood cell DNA.

[0019] In some embodiments of the present invention, the kit also contains one or more sterile containers, such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for container drugs.

[0020] In some embodiments of the present invention, the kit further comprises reagents used in the DNA extraction process, including but not limited to CATB extract, anhydrous ethanol, chloroform / isoamyl alcohol, proteinase K, RNase, phenol, TE buffer, etc. The CATB extract (hexadecyltrimethylammonium bromide) is a commonly used detergent that can effectively lyse cells and release DNA. The anhydrous ethanol is used to precipitate DNA, and is precipitated by mixing with the DNA aqueous solution.

[0021] A third aspect of the present invention provides a method for separating or extracting DNA from red blood cells.

[0022] Furthermore, the method includes: adding red blood cell lysis solution to the red blood cell sample, centrifuging and obtaining a supernatant mixed solution; adding NaOH solution to the mixed solution and incubating for a period of time, and then adding DNA extraction solution to obtain red blood cell DNA.

[0023] Furthermore, the red blood cell lysis solution is selected from one or a combination of two or more of NH4Cl, KHCO3, EDTA-Na2 or Tris.

[0024] Preferably, the red blood cell lysis solution is NH4Cl.

[0025] Preferably, the volume ratio of the red blood cell sample to NH4Cl is 1:2.

[0026] In the present invention, the erythrocyte lysing solution destroys the erythrocyte membrane, so that the DNA in the erythrocyte is released into the lysing solution, and at the same time, the erythrocyte lysing solution does not destroy the granulocyte membrane, protects the integrity of the granulocyte morphology, and prevents the DNA in the granulocyte from being released into the lysing solution. The precipitate is discarded by centrifugation, the lysed components derived from the erythrocyte are retained, and the granulocytes and other precipitates are discarded.

[0027] In some embodiments, the red blood cell lysing solution includes NH4Cl, KHCO3, EDTA-Na2, and Tris; the volume ratio of the red blood cells to the red blood cell lysing solution is any ratio between 1:1-10, for example, the volume ratio of the red blood cells to the red blood cell lysing solution 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 red blood cell lysing solution is NH4Cl, and the volume ratio of the red blood cell sample to NH4Cl is 1:2.

[0028] In some embodiments, the method for separating or extracting DNA from cells also includes the step of collecting peripheral blood from a subject and separating red blood cells. In the present application, "subject" means an object to be tested. In certain specific embodiments, "subject" is a human subject. In the present application, "peripheral blood" means blood released from a hematopoietic organ into the circulatory system and participating in the circulation. "Peripheral blood" is different from immature blood cells in hematopoietic organs (such as bone marrow). In the disclosure herein, peripheral blood can be collected by means known in the art such as blood sampling from a vein, fingertip or earlobe. 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 peripheral whole blood, plasma accounts for approximately 54.3% of peripheral whole blood, and white blood cells account for approximately 0.7% of peripheral whole blood.

[0029] In some embodiments, the method further comprises the step of adding a red blood cell stabilizer after collecting peripheral blood. The red blood cell stabilizer is selected from one or a combination of two or more of EDTA, citrate, sodium fluoride, sodium orthovanadate, sodium glycerophosphate, sodium pyrophosphate, tetraimidazole hydrochloride, phenylmethylsulfonyl fluoride, pepsin inhibitor, EDTA-Na2, formaldehyde polymer, acetaldehyde polymer, propionaldehyde polymer, alkaline salt, hydrochloric acid, and Tris.

[0030] In other embodiments, the separation method of peripheral blood red blood cells includes natural sedimentation method, differential sedimentation method, sodium chloride separation method, direct centrifugation and density gradient centrifugation method, etc. The density difference between different components of peripheral blood can be used to separate different components of peripheral blood. For example, different components of peripheral blood can be separated by density gradient centrifugation.

[0031] Furthermore, the concentration of the NaOH solution is 62.05 mg / ml.

[0032] Preferably, the volume ratio of the NaOH solution to NH4Cl is 0.15:1.

[0033] In the present invention, the applicant has experimentally demonstrated for the first time that the addition of NaOH solution during the extraction of red blood cell DNA can increase the total amount of red blood cell DNA obtained. NaOH can utilize its alkalinity to further degrade the incompletely broken red blood cell membrane and hemoglobin, promote the release and freeing of DNA, and has the advantages of low cost and high extraction efficiency. In addition, the use of ammonium chloride to specifically lyse red blood cells can remove the contamination of nucleated cell DNA. NaOH solution can convert ammonium chloride into ammonia, and ammonia can be volatilized by heating to eventually generate sodium chloride.

[0034] In some embodiments of the present invention, after adding the NaOH solution, incubation is carried out at 45° C. to 50° C. for 10 min to 20 min; preferably, after adding the NaOH solution, incubation is carried out at 45.5° C. for 10 min.

[0035] Furthermore, the DNA extraction solution includes buffer A and buffer B, wherein buffer A includes guanidine isothiocyanate, sodium dodecyl sulfate, isopropanol and Tris-HCl solution, and buffer B includes guanidine isothiocyanate and anhydrous ethanol.

[0036] Preferably, during the extraction process, buffer A is first added to react for a period of time and then anhydrous ethanol is added, after which the reaction solution is added to an adsorption column and centrifuged to discard the waste liquid; buffer B is then added to the adsorption column and centrifuged, and red blood cell DNA is obtained after washing.

[0037] Preferably, the ratio of buffer A to the total reaction volume is 1:1.

[0038] Preferably, in buffer A, the concentration of guanidine isothiocyanate is 4 M, the concentration of sodium dodecyl sulfate is 1%, the concentration of isopropanol is 30%, the concentration of Tris-HCl solution is 10 mM, and the pH is 8.0.

[0039] Preferably, in buffer B, the concentration of guanidine isothiocyanate is 1 M, and the concentration of anhydrous ethanol is 50%.

[0040] In some embodiments of the present invention, after adding buffer A, the mixture is placed at 45°C to 50°C for 30min to 60min until the solution turns dark brown, and then anhydrous ethanol is added, mixed and transferred to the adsorption column. Preferably, after adding buffer A, the mixture is reacted at 56.5°C for 45min, and then anhydrous ethanol is added in a ratio of 1:1 with the buffer, mixed and transferred to the adsorption column.

[0041] In some embodiments of the present invention, anhydrous ethanol may be added for washing after adding buffer B, preferably, the concentration of anhydrous ethanol is 80%. More preferably, Tris solution is added after washing to dissolve DNA.

[0042] In the present invention, the DNA extracting solution also includes the extracting solution conventionally used in the DNA extraction process, including but not limited to CTAB extracting solution, TE buffer solution and the like.

[0043] Furthermore, the method also includes the step of washing the red blood cells before adding the red blood cell lysing solution.

[0044] Preferably, the method further comprises the step of adding RNase to remove RNA.

[0045] Preferably, the method further comprises the step of adding proteinase K to degrade proteins.

[0046] In the present invention, the method includes the step of washing the red blood cell sample to remove the influence of plasma DNA when obtaining the red blood cell sample. Preferably, PBS buffer is used for washing.

[0047] In some embodiments of the present invention, the reagent for degrading proteins includes guanidine cyanate, guanidine hydrochloride, sodium iodide, urea or an enzyme acting 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.

[0048] A fourth aspect of the present invention provides any of the following applications.

[0049] Furthermore, the application includes: 1) Application of NaOH solution in the preparation of red blood cell DNA extraction kit; 2) Use of the enhanced red blood cell lysis solution described in the first aspect of the present invention in the preparation of a red blood cell DNA extraction kit; 3) Use of the kit according to the second aspect of the present invention in detecting cancer or predicting the risk of cancer; 4) Use 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 cancer.

[0050] Furthermore, the concentration of the NaOH solution is 62.05 mg / ml.

[0051] During the occurrence and progression of cancer, genetic information will undergo a series of changes, including DNA mutations, insertions / deletions, chromosome structural variations, copy number variations, and changes in epigenetic information. During the evolution of cancer, DNA sequence variations occur randomly, and only when variations occur in key growth control genes can they lead to the occurrence of malignant tumors. Most gene expression abnormalities are caused by epigenetic changes, usually changes 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 the occurrence of cancer earlier. DNA methylation refers to the methylation process of the 5th carbon atom on cytosine in CpG dinucleotides. As a stable modification state, it can be inherited to new offspring DNA during the DNA replication process under the action of DNA methyltransferase. It is an important epigenetic mechanism. When DNA is methylated, methylation of the gene promoter region can lead to transcriptional silencing of tumor suppressor genes, so it is closely related to the occurrence of tumors. Abnormal methylation includes hypermethylation of tumor suppressor genes and DNA repair genes, hypomethylation of repetitive sequence DNA, and loss of imprinting of certain genes, which are related to the occurrence of various tumors. Therefore, the red blood cell DNA obtained by the kit and method provided in this application can be used to detect cancer or cancer risk; the cancer includes all cancers, especially liver cancer, colorectal cancer, gastric cancer, esophageal cancer, lung cancer, cervical cancer, pancreatic cancer, breast cancer or head and neck cancer.

[0052] Advantages and beneficial effects of the present invention: The present invention innovatively discovers 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 solution for the art, and providing a fast, convenient and efficient red blood cell DNA extraction kit, which has important scientific value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The results of the determination of the DNA concentration extracted from the examples of the present invention and the comparative examples are shown. DETAILED DESCRIPTION

[0054] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solution and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0055] The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified. Generally, the terms and techniques related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry and hybridization described herein are well-known and commonly used terms in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods known in the art.

[0056] Example Method for Extracting Red Blood Cell DNA 1. Experimental Methods 1. The method for extracting red blood cell DNA in the embodiment of the present invention is as follows: (1) Collect 500 µL of red blood cells, place in a 1.5 mL enzyme-free centrifuge tube, add 1 mL of PBS, invert to mix, centrifuge at 400 g for 5 minutes, and recover the red blood cell pellet; (2) Repeat step (1) once to remove residual plasma DNA; (3) Add 1 mL of ammonium chloride lysis solution in a ratio of 1:2, wait for 5 to 10 minutes, invert and mix several times, and observe the properties of the solution; (4) After the solution is clarified, centrifuge at 400g for 5 minutes. A white precipitate and a red supernatant are visible. Under a light microscope, the white precipitate is red blood cell fragments and a small amount of nucleated cells. Carefully aspirate the red supernatant and place it in a new 1.5 mL enzyme-free centrifuge tube. (5) Add 10 µL of 100 mg / ml RNase, invert to mix, and let stand at room temperature for 5 minutes to remove RNA; (6) Add 150 µl of NaOH solution at a ratio of 1:0.15 to the red lysate and mix thoroughly by inverting; (7) Place the centrifuge tube in a 45.5°C metal bath, open the lid, and react for 10 minutes; (8) Add 150 µl of 20 mg / mL proteinase K at a ratio of 1:0.1 to the total volume in a 5 mL enzyme-free centrifuge tube at room temperature and mix by inversion. (9) Add approximately 1.6 mL of buffer A at a ratio of 1:1 to the total volume and mix by inversion. (10) Place the centrifuge tube in a 56.5°C water bath and react for 45 minutes until the solution turns dark brown. (11) Add about 1.6 mL of anhydrous ethanol in a ratio of 1:1 to buffer A and mix by inversion. The total system is about 4.5-4.8 mL. (12) Pipette 600 µl of the solution and add it dropwise to a silica gel adsorption column. Centrifuge at 12,000 g for 1 min and discard the waste liquid. (13) Repeat step (12) 6 to 7 times until all the solution in the system is completely absorbed; (14) Add 600 µl of buffer B to the silica gel adsorption column, centrifuge at 12,000 g for 1 min, and discard the waste liquid; (15) Add 600 µl of 80% anhydrous ethanol to the silica gel adsorption column, centrifuge at 12,000 g for 1 min, and discard the waste liquid; (16) Repeat step (15) twice; (17) Centrifuge the empty silica gel adsorption column at 12000g for 2 minutes, discard the waste liquid, and leave it open at room temperature for 5 minutes to dry; (18) Add 60-80 µL of 10 mM Tris solution (pH 8.0) to the silica gel adsorption column, let stand for 5 min, centrifuge at 12,000 g for 2 min, and collect the filtered liquid as the red blood cell DNA solution; (19) Red blood cell DNA solution can be stored at -80℃ or -20℃.

[0057] 2. The comparative extraction method is as follows: (1) Collect 500 µL of red blood cells, place in a 1.5 mL enzyme-free centrifuge tube, add 1 mL of PBS, invert to mix, centrifuge at 400 g for 5 minutes, and recover the red blood cell pellet; (2) Repeat step (1) once to remove residual plasma DNA; (3) Add 1 mL of ammonium chloride lysis solution in a ratio of 1:2, wait for 5 to 10 minutes, invert and mix several times, and observe the properties of the solution; (4) After the solution is clarified, centrifuge at 400g for 5 minutes. A white precipitate and a red supernatant are visible. Under a light microscope, the white precipitate is red blood cell fragments and a small amount of nucleated cells. Carefully aspirate the red supernatant and place it in a new 1.5mL enzyme-free centrifuge tube; (5) Add 10 µL of 100 mg / ml RNase, invert to mix, and let stand at room temperature for 5 minutes to remove RNA; (6) Add 150 µl of 20 mg / mL proteinase K at a ratio of 1:0.1 to the total volume in a 5 mL enzyme-free centrifuge tube at room temperature and mix by inversion. (7) Add approximately 1.6 mL of buffer A at a ratio of 1:1 to the total volume and mix by inversion. (8) Place the centrifuge tube in a 56.5°C water bath and react for 45 minutes until the solution turns dark brown. (9) Add about 1.6 mL of anhydrous ethanol in a ratio of 1:1 to buffer A and mix by inversion. The total system is about 4.5-4.8 mL. (10) Pipette 600 µl of the solution and add it dropwise to a silica gel adsorption column. Centrifuge at 12,000 g for 1 min and discard the waste liquid. (11) Repeat step (10) 6 to 7 times until all the solution in the system is completely absorbed; (12) Add 600 µl of buffer B to the silica gel adsorption column, centrifuge at 12,000 g for 1 min, and discard the waste liquid; (13) Add 600 µl of 80% anhydrous ethanol to the silica gel adsorption column, centrifuge at 12,000 g for 1 min, and discard the waste liquid; (14) Repeat step (13) twice; (15) Centrifuge the empty silica gel adsorption column at 12000g for 2 minutes, discard the waste liquid, and leave it open at room temperature for 5 minutes to dry; (16) Add 60-80 µL of 10 mM Tris solution (pH 8.0) to the silica gel adsorption column, let it stand for 5 minutes, centrifuge at 12,000 g for 2 minutes, and collect the filtered liquid as the red blood cell DNA solution.

[0058] 2. Experimental Results The results of the DNA concentrations extracted from the embodiments and comparative examples are shown in Tables 1 and Figure 1 As shown, by comparing the examples and comparative examples, it can be seen that the addition of NaOH solution can increase the total amount of red blood cell DNA obtained. NaOH can utilize its alkalinity to further degrade the incompletely broken red blood cell membranes and hemoglobin, promote the release and freeness of DNA, and has the advantages of low cost and high extraction efficiency. In addition, the use of ammonium chloride to specifically lyse red blood cells can remove the contamination of nucleated cell DNA. NaOH solution can convert ammonium chloride into ammonia, and ammonia can promote its volatilization by heating, and finally generate sodium chloride. The extracted red blood cell DNA can be used directly for DNA-related experiments, or it can be used for DNA sequencing after further purification.

[0059] Table 1 Determination results of DNA concentration extracted from Examples and Comparative Examples

[0060] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. An enhanced red blood cell lysis solution, characterized in that: The enhanced red blood cell lysing solution comprises red blood cell lysing solution and NaOH; Preferably, the red blood cell lysate is mixed with NaOH; Preferably, the NaOH is a NaOH solution, and the red blood cell lysing solution is independent of the NaOH solution; Preferably, the red blood cell lysis solution is NH4Cl; Preferably, the concentration of the NaOH solution is 62.05 mg / ml, and the concentration of the NH4Cl is 8 g / L; More preferably, the volume ratio of the NaOH solution to NH4Cl is 0.15:

1.

2. A red blood cell DNA extraction kit, characterized in that: The kit comprises the enhanced red blood cell lysis solution and DNA extraction solution according to claim 1.

3. The kit according to claim 2, characterized in that The DNA extraction solution comprises 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; Preferably, in buffer A, the concentration of guanidine isothiocyanate is 4 M, the concentration of sodium dodecyl sulfate is 1%, the concentration of isopropanol is 30%, the concentration of Tris-HCl solution is 10 mM, and the pH is 8.0; Preferably, in buffer B, the concentration of guanidine isothiocyanate is 1 M, and the concentration of anhydrous ethanol is 50%.

4. The kit according to claim 1, characterized in that 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.

5. A method for separating or extracting DNA from red blood cells, characterized in that: The method comprises: adding red blood cell lysis solution to a red blood cell sample, centrifuging to obtain a supernatant mixed solution; adding a NaOH solution to the mixed solution for incubation for a period of time, and then adding a DNA extraction solution to obtain red blood cell DNA.

6. The method according to claim 5, characterized in that The red blood cell lysis solution is selected from one or a combination of two or more of NH4Cl, KHCO3, EDTA-Na2 or Tris; Preferably, the red blood cell lysis solution is NH4Cl; Preferably, the volume ratio of the red blood cell sample to NH4Cl is 1:

2.

7. The method according to claim 5 or claim 6, characterized in that: The concentration of the NaOH solution is 62.05 mg / ml; Preferably, the volume ratio of the NaOH solution to NH4Cl is 0.15:

1.

8. The method according to claim 5, characterized in that The DNA extraction solution comprises 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; Preferably, during the extraction process, buffer A is first added to react 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 buffer B is added to the adsorption column and centrifuged, and red blood cell DNA is obtained after washing; Preferably, the ratio of buffer A to the total reaction volume is 1:1; Preferably, in buffer A, the concentration of guanidine isothiocyanate is 4 M, the concentration of sodium dodecyl sulfate is 1%, the concentration of isopropanol is 30%, the concentration of Tris-HCl solution is 10 mM, and the pH is 8.0; Preferably, in buffer B, the concentration of guanidine isothiocyanate is 1 M, and the concentration of anhydrous ethanol is 50%.

9. The method according to claim 5, characterized in that The method further comprises the step of washing the red blood cells before adding the red blood cell lysing solution; Preferably, the method further comprises the step of adding RNase to remove RNA; Preferably, the method further comprises the step of adding proteinase K to degrade proteins.

10. Any one of the following applications, characterized in that: The applications include: 1) Application of NaOH solution in the preparation of red blood cell DNA extraction kit; 2) Use of the enhanced red blood cell lysis solution according to claim 1 in the preparation of a red blood cell DNA extraction kit; 3) Use of the kit according to any one of claims 2 to 4 in detecting cancer or predicting the risk of cancer; 4) Use of red blood cell DNA extracted by the method according to any one of claims 5 to 9 in detecting cancer or predicting the risk of cancer; Preferably, the concentration of the NaOH solution is 62.05 mg / ml.

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