Pretreatment solution for co-extracting genomic DNA and cell-free DNA and extraction method thereof

Abstract

The present invention relates to the field of biomedical technologies, and particularly to a pretreatment solution and an extraction method for co-extracting genomic DNA and cell-free DNA. The pretreatment solution includes: polyhydric alcohol, chelating agent, preservative, pH buffer and water; the polyhydric alcohol includes high-molecular-weight polyhydric alcohol and low-molecular-weight polyhydric alcohol; the high-molecular-weight polyhydric alcohol is selected from at least one of high-molecular-weight polyethylene glycols, and the molecular weight of the high-molecular-weight polyethylene glycol is 1,500 - 40,000; the low-molecular-weight polyhydric alcohol is selected from at least one of low-molecular-weight polyethylene glycol and glycerol, and the molecular weight of the low-molecular-weight polyethylene glycol is below 1,000. A pretreatment solution is added to a sample, and after centrifugation, a sample precipitate and a sample supernatant are obtained; the sample precipitate is used for the extraction of genomic DNA, and the sample supernatant is used for the extraction of cell-free DNA. The extraction method can simultaneously achieve high-purity extraction of genomic DNA and cell-free DNA.

Description

Technical Field

[0001] The present invention relates to a pretreatment solution and an extraction method for co-extracting genomic DNA and cell-free DNA, and belongs to the field of biomedical technology. Background Art

[0002] The technology for co-extracting genomic DNA and cell-free DNA mainly involves the fields of biology, molecular biology and medicine. This technology is usually used to study the DNA in cells, as well as the cell-free DNA present in liquids (such as blood, urine).

[0003] Since the discovery of circulating tumor cells in 1869, less invasive liquid biopsy technology has become possible. As a branch of in vitro diagnosis, liquid biopsy is a technology for diagnosing diseases by sampling body fluids such as cerebrospinal fluid, saliva, pleural effusion, blood, ascites, urine, etc. Compared with tissue biopsy, liquid biopsy mainly has the advantages of non-invasiveness, early diagnosis, and dynamic monitoring. The DNA in liquid samples can be simply divided into two parts: nucleated cells shed by the human body system, and cell-free DNA that enters body fluids through the circulatory system. The genomic DNA and cell-free DNA obtained by liquid biopsy can be applied to fields such as early disease screening, diagnostic typing, drug companion detection, and patient condition monitoring, and have great development potential.

[0004] Currently, both blood and urine can be used as the basis for liquid biopsy technology. Among them, urine, as a sample with lower invasiveness and more convenient sampling, is gradually being explored for its potential as a liquid biopsy sample. Moreover, urine samples have the following advantages compared to blood samples: (1) lower infectivity to infectious diseases such as HIV; (2) similar nucleic acid profiles to blood; (3) lower protein content in urine, which is more conducive to nucleic acid extraction.

[0005] Urothelial bladder cancer is a malignant tumor that occurs in the bladder mucosa. It is the second most common cancer in the urinary system and has the highest incidence in the urogenital system in China. Multiple reports have pointed out that various relevant carcinogenic gene mutations and other genetic variations can be detected in the genomic DNA and cell-free DNA of urine exudate cells of bladder cancer patients, indicating that there is an association between gene mutations in urine samples and disease progression. Cell-free DNA in similar body fluids can not only monitor bladder cancer, but also monitor other non-urinary system cancers, such as prostate cancer, non-small cell lung cancer, etc.; it can also participate in the diagnosis of infectious diseases (such as tuberculosis), dynamic monitoring after organ transplantation; and non-invasive prenatal diagnosis of fetuses. We can also perform targeted drug use and prognosis prediction based on the mutation sites in the DNA extracted from body fluids.

[0006] Currently, there are relatively few commercial kit products on the market that can extract DNA from body fluids. Moreover, the vast majority of them can only perform single genomic DNA extraction or free DNA extraction, and the extraction of free DNA takes a long time. Some products that can co-extract genomic DNA and free DNA have problems with the difficult separation of genomic DNA and free DNA. In particular, the presence of genomic DNA can be detected in free DNA, which also affects the purity of free DNA.

[0007] The application of automation technology in the extraction of free DNA is also constantly evolving. Automation technology uses liquid handling workstations and automated instruments, which can process multiple samples simultaneously, reduce the error of manual operation, and provide high-throughput extraction capabilities. However, the technology for co-extracting genomic DNA and free DNA through automation technology is also in the development process, and there is currently no mature processing technology.

[0008] Generally speaking, the technology for co-extracting genomic DNA and free DNA is still in the development process, especially the technology for co-extracting genomic DNA and free DNA from urine samples is in its infancy. The continuous development of DNA extraction technology from body fluid samples provides more possibilities for fields such as molecular biology research, clinical diagnosis, and personalized medicine.

[0009] The technology for co-extracting genomic DNA and free DNA has the following technical difficulties:

[0010] (1) The problem of preserving the integrity of exfoliated cells: There are exfoliated cells and free DNA in body fluids; if the exfoliated cells undergo apoptosis and lysis, the released DNA will not only dilute the original free DNA but also interfere with the purity of the extracted free DNA, affecting the reliability of downstream results.

[0011] (2) The problem of stably preserving free DNA: Free DNA fragments are small, and free DNA in urine is even smaller than that in blood; moreover, the half-life of free DNA is very short, so it is also very important to ensure the stability of free DNA during the period from sampling to DNA extraction.

[0012] (3) The requirement of no genomic DNA contamination in the extracted free DNA: On the one hand, it is necessary to prevent the lysis of exfoliated cells in body fluids; on the other hand, it is necessary to avoid the residue of genomic DNA during the extraction of free DNA.

[0013] Since the extraction of cell-free DNA is time-consuming and has poor stability, there are few kits on the market that can extract cell-free DNA. Most of them can only perform single genomic DNA extraction. There are very few that can perform genomic DNA extraction and cell-free DNA extraction, but it is difficult to fully separate the two. The presence of genomic DNA detected in cell-free DNA will affect the purity of cell-free DNA, thereby affecting the accuracy of downstream experiments. Summary of the Invention

[0014] Aiming at the deficiencies of the existing technology, the present invention provides a pretreatment solution and an extraction method for co-extracting genomic DNA and cell-free DNA. The pretreatment solution has the function of both preserving exfoliated cells and maintaining the stability of cell-free DNA, so that the extracted cell-free DNA is free from genomic DNA contamination, ensuring the purity of cell-free DNA and making the downstream results more accurate.

[0015] The technical solution of the present invention to solve the above technical problems is as follows: A pretreatment solution for co-extracting genomic DNA and cell-free DNA, the pretreatment solution includes: polyhydric alcohol, chelating agent, preservative, pH buffer and water; the pH of the pretreatment solution is 7-9;

[0016] The polyhydric alcohol includes high molecular weight polyhydric alcohol and low molecular weight polyhydric alcohol;

[0017] The high molecular weight polyhydric alcohol is selected from at least one of high molecular weight polyethylene glycols, and the number average molecular weight of the high molecular weight polyethylene glycol is 1500-40000;

[0018] The low molecular weight polyhydric alcohol is selected from at least one of low molecular weight polyethylene glycol and glycerol, and the number average molecular weight of the low molecular weight polyethylene glycol is below 1000.

[0019] Further, in the pretreatment solution, the mass ratio of the high molecular weight polyhydric alcohol is 1%-10%; the mass ratio of the low molecular weight polyhydric alcohol is 10%-65%; the mass ratio of the preservative is 0.5%-5%; the concentration of the chelating agent in the pretreatment solution is 10g / L-50g / L.

[0020] Further, the chelating agent is at least one of EDTA-2Na, EDTA-3K, EDTA-2K;

[0021] The preservative is at least one of proclin 310, proclin 950, sodium azide;

[0022] The pH buffer is Tris buffer or citrate buffer.

[0023] The present invention also discloses a method for co-extracting genomic DNA and cell-free DNA. The extraction method is as follows:

[0024] Add the pretreatment solution described in the present invention to the sample, and then centrifuge to obtain a sample precipitate and a sample supernatant; the sample precipitate is used for the extraction of genomic DNA, and the sample supernatant is used for the extraction of cell-free DNA;

[0025] The process for extracting genomic DNA is as follows:

[0026] (1) Lysis: Add a lysis solution and a protease to the sample precipitate and incubate;

[0027] (2) Binding: Add a binding solution, pipette and mix well, then add it to a DNA binding column, and centrifuge to discard the lower liquid;

[0028] (3) Washing: Add Wash Buffer I, centrifuge to discard the lower liquid; then add Wash Buffer II, and centrifuge to discard the lower liquid;

[0029] (4) Elution: Place the binding column into a centrifuge tube, add an elution solution to the binding column, after centrifugation, collect the eluted solution after centrifugation, which is genomic DNA;

[0030] The process for extracting cell-free DNA is as follows:

[0031] (1) Binding: Add an aqueous suspension of silica hydroxyl nanomagnetic beads or an aqueous suspension of silica to the sample supernatant, invert and mix, then centrifuge to discard the supernatant;

[0032] (2) Washing: Add Wash Buffer I to the precipitate, transfer it to a DNA binding column, centrifuge to remove the lower liquid; then add Wash Buffer II, and centrifuge to discard the lower liquid;

[0033] (3) Elution: Place the binding column into a centrifuge tube, add an elution solution to the binding column, after centrifugation, collect the eluted solution after centrifugation, which is cell-free DNA.

[0034] Further, the lysis solution is a guanidine salt lysis solution or an SDS lysis solution;

[0035] The guanidine salt lysis solution includes guanidine salt, Triton X-100, Tris-HCl, Tween-20, EDTA salt and water; the pH of the guanidine salt lysis solution is 8-9;

[0036] The SDS lysis solution includes SDS, phosphate, Tris-HCl, EDTA salt and water; the pH of the SDS lysis solution is 6-7.

[0037] Further, in the guanidine salt lysis solution, the guanidine salt is guanidine hydrochloride or guanidine isothiocyanate, and the concentration of the guanidine salt in the guanidine salt lysis solution is 2M - 8M; the mass concentration of TritonX-100 in the guanidine salt lysis solution is 0.3% - 3%; the mass concentration of Tween-20 in the guanidine salt lysis solution is 3% - 10%; the concentration of EDTA salt in the guanidine salt lysis solution is 10mM - 50mM; the concentration of Tris-HCl in the guanidine salt lysis solution is 20mM - 50mM;

[0038] In the SDS lysis solution, the phosphate is sodium phosphate, and the concentration of the sodium phosphate in the SDS lysis solution is 0.1M - 0.5M; the mass concentration of SDS in the SDS lysis solution is 0.5% - 2%; the concentration of EDTA salt in the SDS lysis solution is 10mM - 50Mm; the concentration of Tris-HCl in the SDS lysis solution is 20mM - 50mM.

[0039] Further, the binding solution includes a guanidine salt, isopropanol, and water;

[0040] The guanidine salt is guanidine hydrochloride or guanidine isothiocyanate, and the concentration of the guanidine salt in the binding solution is 4M - 8M; the mass concentration of isopropanol in the binding solution is 60% - 70%.

[0041] Further, the washing solution I is an aqueous solution of a guanidine salt, or the washing solution I includes a guanidine salt, ethanol, and water;

[0042] The guanidine salt is guanidine hydrochloride or guanidine isothiocyanate;

[0043] The concentration of the guanidine salt in the washing solution I is 4M - 5M; the mass concentration of ethanol in the washing solution I is 40% - 80%;

[0044] The washing solution II includes Tris-HCl, Tris, ethanol, and water; the concentration of Tris-HCl in the washing solution II is 30 - 60mM, the concentration of Tris in the washing solution II is 0.5M - 1M, the mass concentration of ethanol in the washing solution II is 70% - 90%; the pH value of the washing solution II is 7 - 8.

[0045] Further, the eluent is at least one of an aqueous solution of Tris-HCl, an aqueous solution of Tris, and an aqueous solution of EDTA; the pH of the eluent is 8 - 9.

[0046] Further, in the aqueous suspension of the silicon hydroxyl nano-magnetic beads, the core of the silicon hydroxyl nano-magnetic beads is a magnetic substance, and the outer layer is wrapped with SiO2; the diameter of the silicon hydroxyl nano-magnetic beads is 50 - 400nm;

[0047] In the aqueous suspension of silica, the particle size of silica is 50 - 400 nm.

[0048] The beneficial effects of the present invention are as follows:

[0049] (1) Pretreating the sample with the pretreatment solution of the present invention can achieve rapid and efficient sample collection: When customers directly collect samples without any treatment, the integrity of exfoliated cells cannot be guaranteed, which will directly lead to the inability to separate genomic DNA from free DNA. If only free DNA is to be analyzed, then the sample must be centrifuged to separate the exfoliated cells, and only the supernatant is retained. Adding the pretreatment solution in this technology can not only preserve the exfoliated cells but also maintain the stability of free DNA. The advantages are as follows: ① The sample collection process can occur at any time and place without the participation of experimental equipment such as centrifuges; ② After adding the sample pretreatment solution, it can meet the requirement that there is a certain distance between the sampling location and the analysis laboratory, and the subsequent sample analysis will not be affected by the express transportation in the middle.

[0050] (2) Pretreating the sample with the pretreatment solution of the present invention can not only preserve the exfoliated cells but also maintain the stability of free DNA, ensuring that they do not interfere with each other during the subsequent extraction of genomic DNA and free DNA, and guaranteeing the purity of genomic DNA and free DNA respectively.

[0051] (3) Pretreating the sample with the pretreatment solution of the present invention is beneficial for the extraction of DNA from difficult samples: Adding the pretreatment solution to treat the sample can change the properties of the sample, increase the solubility of some impurities in the sample, and is beneficial for the subsequent extraction of DNA.

[0052] (4) Separating free DNA by silica hydroxyl nanomagnetic beads or silica has rapidity and high efficiency. Under the action of the pretreatment solution, free DNA is adsorbed onto the silica hydroxyl nanomagnetic beads or silica, and the binding and adsorption of free DNA are completed within 1 minute. Especially when the sample volume is relatively large, there is no need for multiple centrifugations or long-time filtration, avoiding the degradation of free DNA caused by long-time operations.

[0053] (5) The extraction method of the present invention can save time and samples. By simultaneously extracting genomic DNA and free DNA, two kinds of nucleic acids can be obtained from one sample in one experiment, thus saving time and samples. This is very valuable for researchers, especially when the number of samples is limited or large-scale analysis is required.

[0054] (6) The free DNA extracted by the extraction method of the present invention has high purity. In this technology, the sample is pretreated with a pretreatment solution, which can not only preserve exfoliated cells but also maintain the stability of free DNA, so that the extracted free DNA is free from the contamination of genomic DNA, ensuring the purity of free DNA and making the downstream results more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Comparative electrophoresis diagram of the effect of the isopropanol content in the binding solution on the genomic DNA extraction amount;

[0056] Figure 2 Comparative electrophoresis diagram of the effect of the guanidine salt content in the binding solution on the genomic DNA extraction amount;

[0057] Figure 3 Comparative electrophoresis diagram of the effect of the pH of the eluent on the genomic DNA extraction amount;

[0058] Figure 4 Comparative electrophoresis diagram of the effect of the SDS content in the lysis solution on the genomic DNA extraction amount;

[0059] Figure 5 Comparative electrophoresis diagram of the effect of the guanidine salt content in the lysis solution on the genomic DNA extraction amount;

[0060] Figure 6 Comparative electrophoresis diagram of the effect of the guanidine salt content in the lysis solution on the free DNA extraction amount;

[0061] Figure 7 Electrophoresis diagrams of the free DNA of Example 1, Example 2, Comparative Example 1, and Comparative Example 2 ( Figure 7 In it, lane 1 is the free DNA extracted in Example 1, lane 2 is the free DNA extracted in Example 2, lane 3 is the free DNA extracted in Comparative Example 1, and lane 4 is the free DNA extracted in Comparative Example 2);

[0062] Figure 8 Genomic DNA extraction electrophoresis diagrams of Example 1, Example 2, and Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6 ( Figure 8 In it, lane 1 is the genomic DNA extracted in Example 1, lane 2 is the genomic DNA extracted in Example 2, lane 3 is the genomic DNA extracted in Comparative Example 3, lane 4 is the genomic DNA extracted in Comparative Example 4, lane 5 is the genomic DNA extracted in Comparative Example 5, and lane 6 is the genomic DNA extracted in Comparative Example 6);

[0063] Figure 9 Electrophoresis diagrams of the urine sample after pretreatment in Example 1 and the urine sample in Comparative Example 7;

[0064] Figure 10Electrophoresis diagram of the preservation of genomic DNA in the urine sample pretreated in Example 1 at different times ( Figure 10 In Figure 10 , t0, t7, t10, and t30 respectively represent the genomic DNA extracted on the day after pretreatment, the 7th day after pretreatment, the 10th day after pretreatment, and the 30th day after pretreatment);

[0065] Figure 11 Electrophoresis diagram of the preservation of free DNA in the urine sample pretreated in Example 1 at different times ( Figure 11 In Figure 11 , t0, t4, and t7 respectively represent the free DNA extracted on the day after pretreatment, the 4th day after pretreatment, and the 7th day after pretreatment).

[0066] Figure 12 Electrophoresis diagram of the free DNA extracted from the blood samples of Example 5, Example 6, and Comparative Example 12;

[0067] Figure 13 Electrophoresis diagram of the genomic DNA extracted from the blood samples of Example 5, Example 6, and Comparative Example 12. Detailed Description of the Invention

[0068] The following is a detailed description of the specific embodiments of the present invention. The present invention can be implemented in many other ways different from those described herein. Those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used are only for describing specific embodiments and do not limit the present invention.

[0070] A pretreatment solution for co-extracting genomic DNA and free DNA, the pretreatment solution includes: polyhydric alcohol, chelating agent, preservative, pH buffer, and water; the pH of the pretreatment solution is 7-9;

[0071] The polyhydric alcohol includes high molecular weight polyhydric alcohol and low molecular weight polyhydric alcohol;

[0072] The high molecular weight polyhydric alcohol is selected from at least one of high molecular weight polyethylene glycols, and the number average molecular weight of the high molecular weight polyethylene glycol is 1500-40000;

[0073] The low molecular weight polyhydric alcohol is selected from at least one of low molecular weight polyethylene glycol and glycerol, and the number average molecular weight of the low molecular weight polyethylene glycol is below 1000.

[0074] More specifically, the applicable range of the number-average molecular weight of the high-molecular-weight polyethylene glycol is 2000-35000, 2500-30000, 3000-25000, 3500-20000, and 4000-15000;

[0075] More preferably, the number-average molecular weight of the high-molecular-weight polyethylene glycol is 5000-15000; the number-average molecular weight of the low-molecular-weight polyethylene glycol is 200-500;

[0076] Specifically, in the pretreatment solution, the mass ratio of the high-molecular-weight polyhydric alcohol is 1%-10%; the mass ratio of the low-molecular-weight polyhydric alcohol is 10%-65%; the mass ratio of the preservative is 0.5%-5%; the concentration of the chelating agent in the pretreatment solution is 10 g / L-50 g / L.

[0077] Preferably, in the pretreatment solution, the mass ratio of the high-molecular-weight polyhydric alcohol is 2%-8%; the mass ratio of the low-molecular-weight polyhydric alcohol is 20%-65%.

[0078] More preferably, in the pretreatment solution, the mass ratio of the high-molecular-weight polyhydric alcohol is 3%-5%; the mass ratio of the low-molecular-weight polyhydric alcohol is 20%-40%.

[0079] Specifically, the chelating agent is at least one of EDTA-2Na, EDTA-3K, and EDTA-2K;

[0080] The preservative is at least one of proclin 310, proclin 950, and sodium azide;

[0081] The pH buffer is Tris buffer or citrate buffer.

[0082] A method for co-extracting genomic DNA and free DNA, and the extraction method is as follows:

[0083] Sample preparation: Add the pretreatment solution to the sample, and then centrifuge to obtain a sample precipitate and a sample supernatant; the sample precipitate is used for the extraction of genomic DNA, and the sample supernatant is used for the extraction of free DNA; among them, the addition amount of the pretreatment solution is 10%-20% of the sample usage amount. In specific examples, the sample is all experimented with a volume of 10 mL.

[0084] The extraction process of the genomic DNA is as follows:

[0085] (1) Lysis: Add a lysis solution and a protease to the sample precipitate for incubation; among them, the addition amount of the lysis solution is 450 μL, and the addition amount of the protease is 20 μL.

[0086] (2) Binding: Add the binding solution, pipette and mix well with a pipette tip, then add it to the DNA binding column and centrifuge to discard the lower liquid; among them, the addition amount of the binding solution is 450 μL.

[0087] (3) Rinsing: Add Wash Solution I, centrifuge to discard the lower liquid; then add Wash Solution II and centrifuge to discard the lower liquid; the addition amount of Wash Solution I is 500 μL; the addition amount of Wash Solution II is 700 μL.

[0088] (4) Elution: Place the binding column into a centrifuge tube, add the elution buffer to the binding column, after centrifugation, collect the eluted solution after centrifugation, which is genomic DNA; the addition amount of the elution buffer is 30 - 50 μL.

[0089] The extraction process of the free DNA is as follows:

[0090] (1) Binding: Add the aqueous suspension of silica hydroxyl nanomagnetic beads or the aqueous suspension of silica to the sample supernatant, mix by inverting up and down, then centrifuge to discard the supernatant; the addition amount of the aqueous suspension of silica hydroxyl nanomagnetic beads is 10 - 20 μL or the addition amount of the aqueous suspension of silica is 100 - 200 μL;

[0091] (2) Rinsing: Add Wash Solution I to the precipitate, transfer it to the DNA binding column, centrifuge to remove the lower liquid; then add Wash Solution II and centrifuge to discard the lower liquid; the addition amount of Wash Solution I is 500 μL; the addition amount of Wash Solution II is 700 μL.

[0092] (3) Elution: Place the binding column into a centrifuge tube, add the elution buffer to the binding column, after centrifugation, collect the eluted solution after centrifugation, which is free DNA. The addition amount of the elution buffer is 30 - 50 μL.

[0093] Specifically, the specific operation in the sample preparation process is: Add the pretreatment solution to the sample, centrifuge at 18000g for 10 min, the sample precipitate is used for the extraction of genomic DNA, the supernatant is transferred to another new 15 mL centrifuge tube, centrifuge at 18000g for 10 min, and the sample supernatant is used for the extraction of free DNA. Multiple centrifugation operations are beneficial to achieve the extraction of high - purity free DNA.

[0094] Specifically, during the extraction process of the genomic DNA, during lysis, after adding proteinase K, incubate at 56 °C for 20 min.

[0095] Specifically, during the extraction process of the free DNA, before the binding operation in step (1), centrifuge the sample supernatant again at 18000g for 10 min, and transfer it to a new centrifuge tube for the extraction of free DNA. It is more beneficial to purify high - purity free DNA.

[0096] Specifically, in the process of extracting the free DNA, the specific process of step (1) of binding is as follows: Add an aqueous suspension of silicon hydroxyl nanomagnetic beads or an aqueous suspension of silica to the sample supernatant, invert 20 times, centrifuge at 1800 g for 5 min, and discard the supernatant.

[0097] In the embodiment of the present invention, the solid content of the silicon hydroxyl nanomagnetic beads in the aqueous suspension of silicon hydroxyl nanomagnetic beads is 50 mg / mL; the solid content of silica in the aqueous suspension of silica is 50 mg / mL.

[0098] Specifically, the lysis solution is a guanidine salt lysis solution or an SDS lysis solution;

[0099] The guanidine salt lysis solution includes guanidine salt, Triton X-100, Tris-HCl, Tween-20, EDTA salt and water; the pH of the guanidine salt lysis solution is 8-9;

[0100] The SDS lysis solution includes SDS, phosphate, Tris-HCl, EDTA salt and water; the pH of the SDS lysis solution is 6-7.

[0101] More specifically, in the guanidine salt lysis solution, the guanidine salt is guanidine hydrochloride or guanidine isothiocyanate, and the concentration of the guanidine salt in the guanidine salt lysis solution is 2M-8M; the mass concentration of Triton X-100 in the guanidine salt lysis solution is 0.3%-3%; the mass concentration of Tween-20 in the guanidine salt lysis solution is 3%-10%; the concentration of EDTA salt in the guanidine salt lysis solution is 10 mM-50 mM; the concentration of Tris-HCl in the guanidine salt lysis solution is 20 mM-50 mM;

[0102] In the SDS lysis solution, the phosphate is sodium phosphate, and the concentration of the sodium phosphate in the SDS lysis solution is 0.1M-0.5M; the mass concentration of SDS in the SDS lysis solution is 0.5%-2%; the concentration of EDTA salt in the SDS lysis solution is 10 mM-50 mM; the concentration of Tris-HCl in the SDS lysis solution is 20 mM-50 mM.

[0103] More specifically, the EDTA salt is at least one of EDTA-2Na, EDTA-3K, and EDTA-2K.

[0104] Specifically, the binding solution includes guanidine salt, isopropanol and water;

[0105] The guanidine salt is guanidine hydrochloride or guanidine isothiocyanate, and the concentration of the guanidine salt in the binding solution is 4M-8M; the mass concentration of isopropanol in the binding solution is 60%-70%.

[0106] Specifically, the washing solution I is an aqueous solution of guanidine salt, or the washing solution I comprises guanidine salt, ethanol and water;

[0107] The guanidine salt is guanidine hydrochloride or guanidine isothiocyanate;

[0108] The concentration of the guanidine salt in the washing solution I is 4M - 5M; the mass concentration of the ethanol in the washing solution I is 40% - 80%;

[0109] The washing solution II comprises Tris-HCl, Tris, ethanol and water; the concentration of Tris-HCl in the washing solution II is 30 - 60 mM, the concentration of Tris in the washing solution II is 0.5M - 1M, the mass concentration of the ethanol in the washing solution II is 70% - 90%; the pH value of the washing solution II is 7 - 8.

[0110] Specifically, the eluent is at least one of an aqueous solution of Tris-HCl, an aqueous solution of Tris and an aqueous solution of EDTA; the pH of the eluent is 8 - 9.

[0111] Specifically, the core of the silica hydroxyl group nanomagnetic beads is a magnetic substance, and the outer layer is coated with SiO2; the diameter of the silica hydroxyl group nanomagnetic beads is 50 - 400 nm.

[0112] Example 1

[0113] I. Preparation of the pretreatment solution:

[0114] Add high molecular weight polyhydric alcohol, low molecular weight polyhydric alcohol, chelating agent, preservative, and pH buffer mixture into water and mix evenly to obtain the pretreatment solution;

[0115] The high molecular weight polyhydric alcohol is polyethylene glycol 5000, and its mass concentration in the pretreatment solution is 5%;

[0116] The low molecular weight polyhydric alcohol is ethylene glycol 200, and its mass concentration in the pretreatment solution is 40%;

[0117] The chelating agent is EDTA-2Na, and its concentration in the pretreatment solution is 30 g / L;

[0118] The preservative is proclin 310, and its mass concentration in the pretreatment solution is 2%;

[0119] The pH buffer is Tris buffer; the pH of the pretreatment solution is 8.

[0120] II. Method for co-extracting genomic DNA and free DNA:

[0121] S1. Sample Preparation: Add the pretreatment solution to the urine sample (10 mL), centrifuge at 18,000 g for 10 min. The sample precipitate is used for genomic DNA extraction, and the supernatant is transferred to another new 15-mL centrifuge tube and centrifuged at 18,000 g for 10 min. The sample supernatant is used for free DNA extraction. Among them, the addition amount of the pretreatment solution is 15% of the mass of the urine sample.

[0122] S2. The process of extracting the genomic DNA is as follows:

[0123] (1) Lysis: Add the lysis solution and proteinase K to the sample precipitate, and incubate at 56 °C for 20 min;

[0124] The lysis solution is SDS lysis solution, and the specific composition is: SDS (0.5%), sodium phosphate (0.5 M), Tris-HCl (50 mM), EDTA-2K (10 mM) and water;

[0125] Among them, the addition amount of the lysis solution is 450 μL.

[0126] (2) Binding: Add the binding solution, mix well with a pipette tip and then add it to the DNA binding column, and centrifuge to discard the lower liquid;

[0127] The composition of the binding solution is: guanidine hydrochloride (5 M), isopropanol (70%) and water; among them, the addition amount of the binding solution is 450 μL.

[0128] (3) Rinsing: Add rinsing solution I, centrifuge to discard the lower liquid; then add rinsing solution II, and centrifuge to discard the lower liquid;

[0129] The composition of the rinsing solution I is: guanidine hydrochloride (4 M), ethanol (60%) and water; among them, the addition amount of the rinsing solution I is 500 μL.

[0130] The composition of the rinsing solution II is: Tris-HCl (40 mM), Tris (0.5 M), ethanol (70%) and water; the pH value of the rinsing solution II is 7 - 8, and among them, the addition amount of the rinsing solution II is 700 μL.

[0131] (4) Elution: Place the binding column in a new 1.5-mL centrifuge tube, add the elution solution to the binding column, and after centrifuging for 2 min, collect the eluted solution after centrifugation as the genomic DNA;

[0132] The elution solution is an aqueous solution of Tris-HCl (the concentration of Tris-HCl is 15 mM), and the pH of the elution solution is 8.5. The addition amount of the elution solution is 50 μL.

[0133] S3. The process of extracting the free DNA is as follows:

[0134] Centrifuge the sample supernatant again at 18,000 g for 10 min, and transfer it to a new centrifuge tube for extraction of free DNA.

[0135] (1) Binding: Add an aqueous suspension of silica hydroxyl nanomagnetic beads to the supernatant, invert 20 times, centrifuge at 1,800 g for 5 min, and discard the supernatant;

[0136] The addition amount of the aqueous suspension of silica hydroxyl nanomagnetic beads is 20 μL.

[0137] (2) Rinsing: Add Wash Buffer I to the precipitate, transfer it to a DNA binding column, centrifuge to remove the liquid; then add Wash Buffer II and centrifuge to discard the liquid;

[0138] The composition of Wash Buffer I is: guanidine hydrochloride (4 M), ethanol (60%) and water; the addition amount of Wash Buffer I is 500 μL.

[0139] The composition of Wash Buffer II is: Tris-HCl (40 mM), Tris (0.5 M), ethanol (70%) and water; the pH value of Wash Buffer II is 7 - 8, and the addition amount of Wash Buffer II is 700 μL.

[0140] (3) Elution: Place the binding column in a centrifuge tube, add eluent to the binding column, after centrifugation, collect the eluent after centrifugation as free DNA.

[0141] The eluent is an aqueous solution of Tris-HCl (the concentration of Tris-HCl is 15 mM), and the pH of the eluent is 8.5. The addition amount of the eluent is 50 μL.

[0142] Example 2

[0143] I. Preparation of the pretreatment solution:

[0144] Add high molecular weight polyhydric alcohol, low molecular weight polyhydric alcohol, chelating agent, preservative, and pH buffer mixture to water and mix evenly to obtain the pretreatment solution;

[0145] The high molecular weight polyhydric alcohol is polyethylene glycol 15000, and its mass concentration in the pretreatment solution is 3%;

[0146] The low molecular weight polyhydric alcohol is ethylene glycol 500, and its mass concentration in the pretreatment solution is 20%;

[0147] The chelating agent is EDTA-3K, and its concentration in the pretreatment solution is 10 g / L;

[0148] The preservative is proclin 950, and its mass concentration in the pretreatment solution is 0.5%;

[0149] The pH buffer is a citrate buffer; the pH of the pretreatment solution is 7.

[0150] II. Method for co-extracting genomic DNA and free DNA:

[0151] S1. Sample preparation: Add the pretreatment solution to the urine sample (10 mL), centrifuge at 18,000 g for 10 min. The sample precipitate is used for the extraction of genomic DNA, and the supernatant is transferred to another new 15 mL centrifuge tube and centrifuged at 18,000 g for 10 min. The sample supernatant is used for the extraction of free DNA. Among them, the addition amount of the pretreatment solution is 15% of the mass of the urine sample.

[0152] S2. The extraction process of the genomic DNA is as follows:

[0153] (1) Lysis: Add lysis buffer and proteinase K to the sample precipitate, and incubate at 56 °C for 20 min;

[0154] The lysis buffer is a guanidine salt lysis buffer, and the specific composition is: guanidine isothiocyanate (7 M), Triton X-100 (0.3%), Tris-HCl (20 mM), Tween-20 (3%), EDTA-2Na (10 mM) and water;

[0155] Among them, the addition amount of the lysis buffer is 450 μL.

[0156] (2) Binding: Add the binding solution, pipette and mix well, then add it to the DNA binding column, and centrifuge to discard the lower liquid;

[0157] The composition of the binding solution is: guanidine hydrochloride (4 M), isopropanol (60%) and water; among them, the addition amount of the binding solution is 450 μL.

[0158] (3) Rinsing: Add wash buffer I, centrifuge to discard the lower liquid; then add wash buffer II, and centrifuge to discard the lower liquid;

[0159] The composition of the wash buffer I is: guanidine hydrochloride (5 M), ethanol (40%) and water; among them, the addition amount of the wash buffer I is 500 μL.

[0160] The composition of the wash buffer II is: Tris-HCl (30 mM), Tris (1 M), ethanol (90%) and water; the pH value of the wash buffer II is 7-8, and among them, the addition amount of the wash buffer II is 700 μL.

[0161] (4) Elution: Place the binding column into a new 1.5 mL centrifuge tube, add elution buffer to the binding column, after centrifuging for 2 min, collect the eluate after centrifugation, which is the genomic DNA;

[0162] The eluent is an aqueous Tris solution (the concentration of Tris is 10 mM), the pH of the eluent is 8, and the addition amount of the eluent is 30 μL.

[0163] S3. The extraction process of the free DNA is as follows:

[0164] Centrifuge the sample supernatant again at 18,000 g for 10 min and transfer it to a new centrifuge tube for the extraction of free DNA.

[0165] (1) Binding: Add a silica aqueous suspension to the supernatant, invert it 20 times, centrifuge at 1,800 g for 5 min, and discard the supernatant;

[0166] Among them, the addition amount of the silica aqueous suspension is 200 μL.

[0167] (2) Rinsing: Add Wash Buffer I to the precipitate, transfer it to a DNA binding column, and centrifuge to discard the lower liquid; then add Wash Buffer II and centrifuge to discard the lower liquid;

[0168] The composition of Wash Buffer I is: guanidine hydrochloride (5 M), ethanol (40%), and water; the addition amount of Wash Buffer I is 500 μL.

[0169] The composition of Wash Buffer II is: Tris-HCl (30 mM), Tris (1 M), ethanol (90%), and water; the pH value of Wash Buffer II is 7 - 8, and the addition amount of Wash Buffer II is 700 μL.

[0170] (3) Elution: Place the binding column in a centrifuge tube, add the eluent to the binding column, and after centrifugation, collect the eluent after centrifugation as the free DNA.

[0171] The eluent is an aqueous Tris solution (the concentration of Tris is 10 mM), the pH of the eluent is 8, and the addition amount of the eluent is 50 μL.

[0172] Example 3

[0173] I. Preparation of the pretreatment solution:

[0174] Add high molecular weight polyhydric alcohol, low molecular weight polyhydric alcohol, chelating agent, preservative, and pH buffer mixture to water and mix evenly to obtain the pretreatment solution;

[0175] The high molecular weight polyhydric alcohol is polyethylene glycol 1500, and its mass concentration in the pretreatment solution is 10%;

[0176] The low molecular weight polyhydric alcohol is ethylene glycol 400, and its mass concentration in the pretreatment solution is 65%;

[0177] The chelating agent is EDTA-2K, and its concentration in the pretreatment solution is 50 g / L;

[0178] The preservative is sodium azide, and its mass concentration in the pretreatment solution is 5%;

[0179] The pH buffer is a citrate buffer; the pH of the pretreatment solution is 9.

[0180] II. Method for co-extracting genomic DNA and free DNA:

[0181] S1. Sample preparation: Add the pretreatment solution to the urine sample (10 mL), centrifuge at 18000 g for 10 min. The sample precipitate is used for the extraction of genomic DNA, and the supernatant is transferred to another new 15 mL centrifuge tube and centrifuged at 18000 g for 10 min. The sample supernatant is used for the extraction of free DNA. Among them, the addition amount of the pretreatment solution is 20% of the urine sample.

[0182] S2. The extraction process of the genomic DNA is as follows:

[0183] (1) Lysis: Add the lysis solution and proteinase K to the sample precipitate, and incubate at 56 °C for 20 min;

[0184] The lysis solution is a guanidine salt lysis solution, and its specific composition is: guanidine isothiocyanate (8M), TritonX-100 (3%), Tris-HCl (50 mM), Tween-20 (10%), EDTA-2Na (50 mM) and water;

[0185] Among them, the addition amount of the lysis solution is 450 μL.

[0186] (2) Binding: Add the binding solution, pipette and mix well, then add it to the DNA binding column, and centrifuge to discard the lower liquid;

[0187] The composition of the binding solution is: guanidine isothiocyanate (8M), isopropanol (70%) and water; among them, the addition amount of the binding solution is 450 μL.

[0188] (3) Rinsing: Add the rinsing solution I, centrifuge to discard the lower liquid; then add the rinsing solution II, and centrifuge to discard the lower liquid;

[0189] The composition of the rinsing solution I is: guanidine isothiocyanate (4M), ethanol (80%) and water; among them, the addition amount of the rinsing solution I is 500 μL.

[0190] The composition of the rinsing solution II is: Tris-HCl (60 mM), Tris (0.5M), ethanol (80%) and water; the pH value of the rinsing solution II is 7-8, and among them, the addition amount of the rinsing solution II is 700 μL.

[0191] (4)Elution: Place the binding column into a new 1.5 mL centrifuge tube, add the elution buffer to the binding column, and after centrifugation for 2 min, collect the eluate after centrifugation, which is the genomic DNA;

[0192] The elution buffer is an aqueous solution of EDTA (the concentration of EDTA is 1 mM), and the pH of the elution buffer is 9. The addition amount of the elution buffer is 40 μL.

[0193] S3. The extraction process of the free DNA is as follows:

[0194] Centrifuge the sample supernatant again at 18,000 g for 10 min, and transfer it to a new centrifuge tube for the extraction of free DNA.

[0195] (1)Binding: Add the aqueous suspension of silica hydroxyl nanomagnetic beads to the supernatant, invert it up and down 20 times, centrifuge at 1,800 g for 5 min, and discard the supernatant;

[0196] The addition amount of the aqueous suspension of silica hydroxyl nanomagnetic beads is 20 μL.

[0197] (2)Rinsing: Add the rinsing buffer I to the precipitate, transfer it to the DNA binding column, and centrifuge to remove the lower liquid; then add the rinsing buffer II and centrifuge to discard the lower liquid;

[0198] The composition of the rinsing buffer I is: guanidine isothiocyanate (4 M), ethanol (80%) and water; the addition amount of the rinsing buffer I is 500 μL.

[0199] The composition of the rinsing buffer II is: Tris-HCl (60 mM), Tris (0.5 M), ethanol (80%) and water; the pH value of the rinsing buffer II is 7 - 8, and the addition amount of the rinsing buffer II is 700 μL.

[0200] (3)Elution: Place the binding column into a centrifuge tube, add the elution buffer to the binding column, and after centrifugation, collect the eluate after centrifugation, which is the free DNA.

[0201] The elution buffer is an aqueous solution of EDTA (the concentration of EDTA is 1 mM), and the pH of the elution buffer is 9. The addition amount of the elution buffer is 50 μL.

[0202] Example 4

[0203] I. Preparation of the pretreatment solution:

[0204] Add high molecular weight polyhydric alcohol, low molecular weight polyhydric alcohol, chelating agent, preservative, and pH buffer mixture to water and mix evenly to obtain the pretreatment solution;

[0205] The high molecular weight polyhydric alcohol is polyethylene glycol 5000, and its mass concentration in the pretreatment solution is 1%;

[0206] The low-molecular-weight polyhydric alcohol is ethylene glycol 200, and its mass concentration in the pretreatment solution is 10%;

[0207] The chelating agent is EDTA-2K, and its concentration in the pretreatment solution is 20 g / L;

[0208] The preservative is proclin 310, and its mass concentration in the pretreatment solution is 1%;

[0209] The pH buffer is Tris buffer; the pH of the pretreatment solution is 8.5.

[0210] II. Method for co-extracting genomic DNA and free DNA:

[0211] S1. Sample preparation: Add the above-mentioned pretreatment solution to the urine sample (10 mL), centrifuge at 18000 g for 10 min, use the sample precipitate for the extraction of genomic DNA, transfer the supernatant to another new 15-mL centrifuge tube, centrifuge at 18000 g for 10 min, and use the sample supernatant for the extraction of free DNA. Among them, the addition amount of the pretreatment solution is 10% of the urine sample.

[0212] S2. The extraction process of the genomic DNA is as follows:

[0213] (1) Lysis: Add the lysis solution and proteinase K to the sample precipitate, and incubate at 56 °C for 20 min;

[0214] The lysis solution is a guanidine salt lysis solution, and its specific composition is: guanidine hydrochloride (2 M), TritonX-100 (2%), Tris-HCl (30 mM), Tween-20 (6%), EDTA-2K (30 mM) and water;

[0215] Among them, the addition amount of the lysis solution is 450 μL.

[0216] (2) Binding: Add the binding solution, mix well with a pipette tip and then add it to the DNA binding column, and centrifuge to discard the lower liquid;

[0217] The composition of the binding solution is: guanidine isothiocyanate (4 M), isopropanol (60%) and water; among them, the addition amount of the binding solution is 450 μL.

[0218] (3) Rinsing: Add the rinsing solution I, centrifuge to discard the lower liquid; then add the rinsing solution II, and centrifuge to discard the lower liquid;

[0219] The composition of the rinsing solution I is: guanidine isothiocyanate (4 M), ethanol (60%) and water; among them, the addition amount of the rinsing solution I is 500 μL.

[0220] The composition of the washing solution II is: Tris-HCl (50 mM), Tris (0.8 M), ethanol (70%), and water; the pH value of the washing solution II is 7-8, and the addition amount of the washing solution II is 700 μL.

[0221] (4) Elution: Place the binding column into a new 1.5 mL centrifuge tube, add the eluent to the binding column, after centrifuging for 2 min, collect the centrifuged eluent, which is the genomic DNA;

[0222] The eluent is an aqueous Tris-HCl solution (the concentration of Tris-HCl is 10 mM), and the pH of the eluent is 8.5. The addition amount of the eluent is 50 μL.

[0223] S3. The extraction process of the free DNA is as follows:

[0224] Centrifuge the sample supernatant again at 18000 g for 10 min, and transfer it to a new centrifuge tube for the extraction of free DNA.

[0225] (1) Binding: Add the aqueous suspension of silica hydroxyl nanomagnetic beads to the supernatant, invert it 20 times up and down, centrifuge at 1800 g for 5 min, and discard the supernatant;

[0226] The addition amount of the aqueous suspension of silica hydroxyl nanomagnetic beads is 20 μL.

[0227] (2) Washing: Add the washing solution I to the precipitate, transfer it to the DNA binding column, centrifuge to remove the lower liquid; then add the washing solution II, and centrifuge to discard the lower liquid;

[0228] The composition of the washing solution I is: guanidine isothiocyanate (4 M), ethanol (60%), and water; the addition amount of the washing solution I is 500 μL.

[0229] The composition of the washing solution II is: Tris-HCl (50 mM), Tris (0.8 M), ethanol (70%), and water; the pH value of the washing solution II is 7-8, and the addition amount of the washing solution II is 700 μL.

[0230] (3) Elution: Place the binding column into a centrifuge tube, add the eluent to the binding column, after centrifuging, collect the centrifuged eluent, which is the free DNA.

[0231] The eluent is an aqueous Tris-HCl solution (the concentration of Tris-HCl is 10 mM), and the pH of the eluent is 8.5. The addition amount of the eluent is 50 μL.

[0232] Example 5

[0233] I. Preparation of the pretreatment solution:

[0234] Add a high molecular weight polyhydric alcohol, a low molecular weight polyhydric alcohol, a chelating agent, a preservative, and a pH buffer mixture to water and mix evenly to obtain a pretreatment solution;

[0235] The high molecular weight polyhydric alcohol is polyethylene glycol 5000, and its mass concentration in the pretreatment solution is 1%;

[0236] The low molecular weight polyhydric alcohol is ethylene glycol 200, and its mass concentration in the pretreatment solution is 10%;

[0237] The chelating agent is EDTA-2K, and its concentration in the pretreatment solution is 20 g / L;

[0238] The preservative is proclin 310, and its mass concentration in the pretreatment solution is 1%;

[0239] The pH buffer is Tris buffer; the pH of the pretreatment solution is 8.5.

[0240] II. Method for co-extracting genomic DNA and free DNA:

[0241] S1. Sample preparation: Add the above pretreatment solution to a blood sample (1 mL), centrifuge at 18000 g for 10 min. The middle layer of the sample is used for the extraction of genomic DNA. Carefully aspirate the supernatant and transfer it to another new 2 mL centrifuge tube, and centrifuge at 18000 g for 10 min. The supernatant of the sample is used for the extraction of free DNA. Among them, the addition amount of the pretreatment solution is 15% of the blood sample.

[0242] S2. The extraction process of the genomic DNA is as follows:

[0243] (1) Lysis: Add a lysis solution and proteinase K to the middle layer of the sample, and incubate at 56 °C for 20 min;

[0244] The lysis solution is a guanidine salt lysis solution, and its specific composition is: guanidine hydrochloride (5 M), TritonX-100 (1.5%), Tris-HCl (30 mM), Tween-20 (5%), EDTA-2Na (20 mM) and water;

[0245] Among them, the addition amount of the lysis solution is 450 μL.

[0246] (2) Binding: Add a binding solution, mix well with a pipette tip and then add it to a DNA binding column, and centrifuge to discard the lower liquid;

[0247] The composition of the binding solution is: guanidine isothiocyanate (4 M), isopropanol (70%) and water; among them, the addition amount of the binding solution is 450 μL.

[0248] (3)Rinsing: Add washing solution I, centrifuge and discard the supernatant; then add washing solution II, centrifuge and discard the supernatant.

[0249] The composition of the washing solution I is: guanidine hydrochloride (5M), ethanol (60%) and water; the addition amount of the washing solution I is 500 μL.

[0250] The composition of the washing solution II is: Tris-HCl (50 mM), Tris (0.5M), ethanol (70%) and water; the pH value of the washing solution II is 7 - 8, and the addition amount of the washing solution II is 700 μL.

[0251] (4)Elution: Place the binding column into a new 1.5 mL centrifuge tube, add the elution solution to the binding column, centrifuge for 2 min, and collect the eluted solution after centrifugation, which is the genomic DNA.

[0252] The elution solution is an aqueous solution of Tris-HCl (the concentration of Tris-HCl is 10 mM), and the pH of the elution solution is 8.5. The addition amount of the elution solution is 50 μL.

[0253] S3. The extraction process of the free DNA is as follows:

[0254] Centrifuge the sample supernatant again at 18000 g for 10 min, and transfer it to a new centrifuge tube for the extraction of free DNA.

[0255] (1)Binding: Add the aqueous suspension of silicon hydroxyl nanomagnetic beads to the supernatant, invert 20 times, centrifuge at 1800 g for 5 min, and discard the supernatant.

[0256] The addition amount of the aqueous suspension of silicon hydroxyl nanomagnetic beads is 20 μL.

[0257] (2)Rinsing: Add washing solution I to the precipitate, transfer it to the DNA binding column, centrifuge and discard the supernatant; then add washing solution II, centrifuge and discard the supernatant.

[0258] The composition of the washing solution I is: guanidine hydrochloride (5M), ethanol (60%) and water; the addition amount of the washing solution I is 500 μL.

[0259] The composition of the washing solution II is: Tris-HCl (50 mM), Tris (0.5M), ethanol (70%) and water; the pH value of the washing solution II is 7 - 8, and the addition amount of the washing solution II is 700 μL.

[0260] (3)Elution: Place the binding column into a centrifuge tube, add the elution solution to the binding column, centrifuge, and collect the eluted solution after centrifugation, which is the free DNA.

[0261] The eluent is an aqueous Tris-HCl solution (the concentration of Tris-HCl is 10 mM), and the pH of the eluent is 8.5. The addition amount of the eluent is 50 μL.

[0262] Example 6

[0263] I. Preparation of the pretreatment solution:

[0264] Add high molecular weight polyhydric alcohol, low molecular weight polyhydric alcohol, chelating agent, preservative, and pH buffer mixture into water and mix evenly to obtain the pretreatment solution;

[0265] The high molecular weight polyhydric alcohol is polyethylene glycol 5000, and its mass concentration in the pretreatment solution is 5%;

[0266] The low molecular weight polyhydric alcohol is ethylene glycol 200, and its mass concentration in the pretreatment solution is 40%;

[0267] The chelating agent is EDTA-2Na, and its concentration in the pretreatment solution is 30 g / L;

[0268] The preservative is proclin 310, and its mass concentration in the pretreatment solution is 2%;

[0269] The pH buffer is Tris buffer; the pH of the pretreatment solution is 8.

[0270] II. Method for co-extracting genomic DNA and free DNA:

[0271] S1. Sample preparation: Add the above-mentioned pretreatment solution to the blood sample (1 mL), centrifuge at 18000 g for 10 min, the middle layer of the sample is used for the extraction of genomic DNA, carefully transfer the supernatant to another new 2 mL centrifuge tube, and centrifuge at 18000 g for 10 min. The supernatant of the sample is used for the extraction of free DNA. Among them, the addition amount of the pretreatment solution is 15% of the blood sample.

[0272] S2. The extraction process of the genomic DNA is as follows:

[0273] (1) Lysis: Add lysis buffer and proteinase K to the middle layer of the sample, and incubate at 56 °C for 20 min;

[0274] The lysis buffer is SDS lysis buffer, and its specific composition is: SDS (2%), sodium phosphate (0.1 M), Tris-HCl (20 mM), EDTA-2K (50 mM) and water;

[0275] Among them, the addition amount of the lysis buffer is 450 μL.

[0276] (2) Binding: Add the binding solution, pipette and mix evenly, then add it to the DNA binding column, and centrifuge to discard the lower liquid;

[0277] The composition of the binding solution is: guanidine hydrochloride (8M), isopropanol (60%) and water; the addition amount of the binding solution is 450 μL.

[0278] (3) Rinsing: Add rinsing solution I, centrifuge and discard the lower liquid; then add rinsing solution II, centrifuge and discard the lower liquid;

[0279] The composition of the rinsing solution I is: guanidine hydrochloride (4M), ethanol (50%) and water; the addition amount of the rinsing solution I is 500 μL.

[0280] The composition of the rinsing solution II is: Tris-HCl (40 mM), Tris (0.5M), ethanol (90%) and water; the pH value of the rinsing solution II is 7 - 8, and the addition amount of the rinsing solution II is 500 μL.

[0281] (4) Elution: Place the binding column into a new 1.5 mL centrifuge tube, add the elution solution to the binding column, after centrifuging for 2 min, collect the eluted solution after centrifugation, which is genomic DNA;

[0282] The elution solution is an aqueous Tris-HCl solution (the concentration of Tris-HCl is 10 mM), and the pH of the elution solution is 8.5. The addition amount of the elution solution is 50 μL.

[0283] S3. The extraction process of the free DNA is as follows:

[0284] Centrifuge the sample supernatant again at 18000g for 10 min, and transfer it to a new centrifuge tube for the extraction of free DNA.

[0285] (1) Binding: Add the silica aqueous suspension to the supernatant, invert it 20 times, centrifuge at 1800g for 5 min, and discard the supernatant;

[0286] Among them, the addition amount of the silica aqueous suspension is 200 μL.

[0287] (2) Rinsing: Add the rinsing solution I to the precipitate, transfer it to the DNA binding column, centrifuge and discard the lower liquid; then add the rinsing solution II, centrifuge and discard the lower liquid;

[0288] The composition of the rinsing solution I is: guanidine hydrochloride (4M), ethanol (50%) and water; the addition amount of the rinsing solution I is 500 μL.

[0289] The composition of the rinsing solution II is: Tris-HCl (40 mM), Tris (0.5M), ethanol (90%) and water; the pH value of the rinsing solution II is 7 - 8, and the addition amount of the rinsing solution II is 700 μL.

[0290] (3) Elution: Place the binding column into a centrifuge tube, add eluent to the binding column, after centrifugation, collect the eluent after centrifugation, which is the free DNA.

[0291] The eluent is an aqueous Tris-HCl solution (the concentration of Tris-HCl is 10 mM), and the pH of the eluent is 8.5. The addition amount of the eluent is 30 - 50 μL.

[0292] Example 7

[0293] Pretreat the same sample with the same pretreatment solution as in Example 1, and then co-extract genomic DNA and free DNA using the same method as in Example 1. The difference is that in the binding solution of this Example 7, the mass concentration of isopropanol is 60%.

[0294] Verify the optimal concentration of isopropanol in the binding solution through the purification conditions of Example 1 and Example 7. To make the experimental data more persuasive, the purification methods of Example 1 and Example 7 were respectively used to purify three samples. The electrophoresis diagrams of the purified genomic DNA are as Figure 1 shown. From Figure 1 it can be seen that when the concentration of isopropanol in the binding solution is 70%, the purification effect is better.

[0295] Example 8

[0296] Pretreat the same sample with the same pretreatment solution as in Example 1, and then co-extract genomic DNA and free DNA using the same method as in Example 1. The difference is that in the binding solution of this Example 8, the concentration of guanidine salt is 6M.

[0297] Example 9

[0298] Pretreat the same sample with the same pretreatment solution as in Example 1, and then co-extract genomic DNA and free DNA using the same method as in Example 1. The difference is that in the binding solution of this Example 9, the concentration of guanidine salt is 8M.

[0299] Verify the optimal concentration of guanidine salt in the binding solution through the purification conditions of Example 1 and Examples 8 - 9. To make the experimental data more persuasive, the purification methods of Example 1 and Examples 8 - 9 were respectively used to purify two samples. The electrophoresis diagrams of the purified genomic DNA are as Figure 2 shown. From Figure 2 it can be seen that the yields of genomic DNA are not very different. Considering the cost, a guanidine salt concentration of 5M is sufficient.

[0300] Example 10

[0301] The same pretreatment solution as in Example 1 was used to pretreat the same samples, and then the genomic DNA and free DNA were co-extracted using the same method as in Example 1, except that: the pH of the eluent in this Example 10 was 8.

[0302] Example 11

[0303] The same pretreatment solution as in Example 1 was used to pretreat the same samples, and then the genomic DNA and free DNA were co-extracted using the same method as in Example 1, except that: the pH of the eluent in this Example 11 was 9.

[0304] The purification conditions of the eluent were verified by the purification results of Example 1 and Examples 10 - 11. The electrophoresis pattern of the purified genomic DNA is as Figure 3 shown. It can be seen from Figure 3 that: the best pH is 8.5.

[0305] Example 12

[0306] The same pretreatment solution as in Example 1 was used to pretreat the same samples, and then the genomic DNA and free DNA were co-extracted using the same method as in Example 1, except that: the content of SDS in the lysis buffer in this Example 12 was 1%.

[0307] Example 13

[0308] The same pretreatment solution as in Example 1 was used to pretreat the same samples, and then the genomic DNA and free DNA were co-extracted using the same method as in Example 1, except that: the content of SDS in the lysis buffer in this Example 13 was 2%.

[0309] The optimal concentration of SDS in the lysis buffer was verified by the purification results of Example 1 and Examples 12 - 13. To make the experimental data more persuasive, the purification methods of Example 1 and Examples 12 - 13 were respectively used to purify two samples. The electrophoresis pattern of the purified genomic DNA is as Figure 4 shown. It can be seen from Figure 4 that: the yields of genomic DNA are not very different. Considering the cost, a concentration of 0.5% of SDS in the lysis buffer is sufficient.

[0310] Examples 14 - 17

[0311] The same pretreatment solution as in Example 2 was used to pretreat the same samples, and then the genomic DNA and free DNA were co-extracted using the same method as in Example 2, except that: the concentrations of guanidine salts in the lysis buffer in these Examples 14 - 17 were 2M, 4M, 6M, and 8M respectively.

[0312] Verify the optimal concentration of guanidine salt in the lysis solution through the purification conditions of Example 1 and Examples 14 - 17. The specific results are as Figure 5 and Figure 6 . From Figure 5 and Figure 6 it can be seen that: Considering the extraction of genomic DNA and free DNA comprehensively, the concentration of guanidine salt in the lysis solution is preferably 7M.

[0313] Comparative Example 1

[0314] Extract free DNA and genomic DNA using the same method as in Example 1, with the difference that: Replace the aqueous suspension of silica hydroxyl nanomagnetic beads in Example 1 with the commercially available product Zymo, Clearing Beads, Cat No. D3061 - 2 - 1.

[0315] Comparative Example 2

[0316] Extract free DNA and genomic DNA using the same method as in Example 1, with the difference that: Replace the aqueous suspension of silica hydroxyl nanomagnetic beads in Example 1 with the commercially available product Magen, MagPure Particles F, Cat No.MPF - 100.

[0317] The electrophoresis diagrams of free DNA extraction in Example 1, Example 2, Comparative Example 1, and Comparative Example 2 are as Figure 7 shown. It can be seen that: There is contamination of genomic DNA on the gel diagram of lane 3 in Comparative Example 1. Thus, it can be shown that using the silica hydroxyl nanomagnetic beads or silica dioxide described in the present invention is more conducive to achieving high - purity extraction of free DNA.

[0318] Comparative Example 3

[0319] Extract free DNA and genomic DNA using the same method as in Example 1, with the difference that: Replace the lysis solution in Example 1 with the commercially available product Zymo, Genomic Lysis Buffer, Cat No. D3004 - 1 - 50.

[0320] Comparative Example 4

[0321] Extract free DNA and genomic DNA using the same method as in Example 1, with the difference that: Replace the lysis solution in Example 1 with the commercially available product Norgen, Urine DNA Isolation Kit (Slurry Format), Cat No.48800.

[0322] Comparative Example 5

[0323] Extract free DNA and genomic DNA using the same method as in Example 1, except that: replace the lysis solution in Example 1 with a commercially available product Qiagen, QIAamp® DNA Micro Kit, Cat No.56304.

[0324] Comparative Example 6

[0325] Extract free DNA and genomic DNA using the same method as in Example 1, except that: replace the lysis solution in Example 1 with a commercially available product Magen, HiPure Circulating DNA Midi Kit G, Cat No. D3182.

[0326] The genomic DNA extraction electrophoresis diagrams of Example 1, Example 2, and Comparative Examples 3, 4, 5, and 6 are as Figure 8 shown. It can be seen that: using the lysis solution defined in the present invention is more conducive to the extraction of genomic DNA.

[0327] Comparative Example 7

[0328] Extract free DNA and genomic DNA using the same method as in Example 1, except that: the sample is not pretreated with the pretreatment solution.

[0329] The electrophoresis diagrams of the sample after pretreatment in Example 1 and the sample in Comparative Example 7 are as Figure 9 shown. From Figure 9 it can be seen that: if the pretreatment solution is not added to the sample, it has no inhibitory effect on the growth of bacteria in the sample. The proliferation of bacteria leads to a significant increase in the yield of genomic DNA, and instead, it will dilute the DNA of exfoliated cells in the sample.

[0330] Figure 10 and Figure 11 are respectively the electrophoresis diagrams of the genomic DNA and free DNA in the urine sample after pretreatment in Example 1 at different times. From Figure 10 and Figure 11 it can be seen that: the genomic DNA and free DNA in the urine sample pretreated with the pretreatment solution described in the present invention can be more stably preserved.

[0331] Comparative Example 8

[0332] Extract free DNA and genomic DNA using the same method as in Example 1, except that: the high molecular weight polyhydric alcohol is not added to the pretreatment solution of this Comparative Example 8.

[0333] Comparative Example 9

[0334] Extract free DNA and genomic DNA using the same method as in Example 1, except that: no low molecular weight polyhydric alcohol was added to the pretreatment solution of Comparative Example 9.

[0335] Comparative Example 10

[0336] Extract free DNA and genomic DNA using the same method as in Example 1, except that: Tween-20 was not added to the lysis solution of Comparative Example 10.

[0337] Comparative Example 11

[0338] Extract free DNA and genomic DNA using the same method as in Example 1, except that: the amount of Tween-20 added to the lysis solution of Comparative Example 10 was increased, and the mass concentration of Tween-20 in the lysis solution of Comparative Example 11 was 12%.

[0339] Comparative Example 12

[0340] Extract free DNA and genomic DNA using the same method as in Example 5, except that: the lysis solution of Example 1 was replaced with a commercially available product, Magen, HiPure Circulating DNA Midi Kit G, Cat No. D3182.

[0341] The electrophoresis diagrams of the extraction of free DNA and genomic DNA in Example 5, Example 6 and Comparative Example 12 are as Figure 12 and Figure 13 shown, and it can be seen that: the lysis solutions defined in Example 5 and Example 6 of the present invention are more conducive to the extraction of free DNA.

[0342] The purification of the above examples and comparative examples was detected, and the extraction results are shown in Table 1. The detection methods involved were Invitrogen™ Qubit™ 4 fluorometer and Qubit™ 1X dsDNA HS Assay Kits; Thermo Scientific NanoDrop One spectrophotometer. The specific detection results are shown in Table 1 below. Among them, the measurement methods for the stability of genomic DNA preservation (the 5th column in Table 1) and the stability of free DNA preservation (the 6th column in Table 1) are as follows: After the sample is pretreated with the pretreatment solution and placed at room temperature for 30 days, genomic DNA and free DNA are extracted, and the obtained yield data is detected (in Comparative Example 7, the pretreatment solution was not added, and the sample was directly placed at room temperature for 30 days, and then genomic DNA and free DNA were extracted for yield data testing). The smaller the difference between the data in the 2nd column and the 5th column in Table 1, and the smaller the difference between the data in the 4th column and the 6th column, the better the preservation stability. Among them, in Table 1, the data in the 2nd to 4th columns are all the data measured within 6 hours after the sample was obtained.

[0343] Table 1 Extraction of samples in examples and comparative examples

[0344]

[0345] It can be seen from the above table data and the attached drawings that: In Examples 1-17, the extraction method described in the present invention was used to extract genomic DNA and free DNA, and it was possible to simultaneously extract high-purity genomic DNA and free DNA from one sample. The pretreatment solution used can not only preserve exfoliated cells but also maintain the stability of free DNA, so that the extracted free DNA is free from genomic DNA contamination, ensuring the purity of free DNA and making the downstream results more accurate.

[0346] From the comparison of the experimental results of Example 1, Example 2, Comparative Example 1, and Comparative Example 2, it can be seen that using the silicon hydroxyl nano magnetic beads or silica defined in the present invention is more conducive to achieving the high-purity extraction of free DNA.

[0347] From the comparison of the experimental results of Example 1, Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, and the comparison of the experimental results of Example 5, Example 6, and Comparative Example 12, it can be seen that using the lysis solution defined in the present invention is more conducive to achieving the extraction of genomic DNA.

[0348] From the comparison of the experimental results of Example 1 and Comparative Example 7, it can be seen that: if the sample is not pretreated with the pretreatment solution, it is very difficult to co-extract genomic DNA and cell-free DNA. Moreover, since the pretreatment solution is not added to the sample, there is no inhibitory effect on the growth of bacteria in the sample. Bacterial proliferation leads to a significant increase in the yield of genomic DNA, and a large amount of bacterial genomic DNA will be extracted, diluting the genomic DNA of exfoliated cells in the sample. At the same time, cell-free DNA cannot be extracted, and the required genomic DNA and cell-free DNA cannot be obtained. The pretreatment solution of the present invention can increase the dissolution of some impurities in the sample, which is beneficial to the subsequent DNA extraction, and is more conducive to the stable storage of genomic DNA and cell-free DNA.

[0349] From the comparison of the experimental results of Example 1 and Comparative Example 8 and Comparative Example 9, it can be seen that the pretreatment solution prepared when high-molecular-weight polyhydric alcohols and low-molecular-weight polyhydric alcohols coexist is more conducive to the co-extraction of genomic DNA and cell-free DNA.

[0350] From the comparison of the experimental results of Example 1 and Comparative Example 10 and Comparative Example 11, it can be seen that when the concentration of Tween-20 in the lysis solution is within the range defined by the present invention, it is more conducive to the extraction of genomic DNA.

[0351] In summary, the extraction method of the present invention has the following advantages:

[0352] (1) Saving time and samples: By co-extracting genomic DNA and cell-free DNA, two kinds of nucleic acids can be obtained from one sample in one experiment, thus saving time and samples. This is very valuable for researchers, especially when the number of samples is limited or large-scale analysis is required.

[0353] (2) Maintaining the stability of exfoliated cells and cell-free DNA in the sample, which is also a prerequisite for ensuring the purity of cell-free DNA: The components of the sample pretreatment solution included in this technology can ensure that exfoliated cells do not apoptose or lyse within 7 days and cell-free DNA does not degrade within 30 days when exfoliated cells and cell-free DNA coexist.

[0354] (3) No contamination of genomic DNA in the extracted cell-free DNA: With the help of the sample pretreatment solution, when extracting cell-free DNA, the contamination of genomic DNA can be completely removed only by centrifugation, ensuring the purity of cell-free DNA.

[0355] The pretreatment solution can not only increase the solubility of some impurities in the sample, which is beneficial to the subsequent DNA extraction, but also preserve the sample, which is beneficial to transportation. The preservation effect on the sample mainly includes preserving exfoliated cells and maintaining the stability of cell-free DNA.

[0356] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, all possible combinations of the technical features in the above-described embodiments are not exhaustively listed. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0357] For those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. The protection scope of the present invention shall be subject to the appended claims.

Claims

1. A method for co-extracting genomic DNA and cell-free DNA, characterized in that, The extraction method is as follows: Add a pretreatment solution to the sample, and then centrifuge to obtain a sample precipitate and a sample supernatant; the sample precipitate is used for the extraction of genomic DNA, and the sample supernatant is used for the extraction of cell-free DNA; the sample is urine or blood; The process for extracting genomic DNA is as follows: (1) Lysis: Add a lysis solution and a protease to the sample precipitate and incubate; (2) Binding: Add a binding solution, pipette and mix well, then add it to a DNA binding column, and centrifuge to discard the lower liquid; (3) Washing: Add Wash Buffer I, and centrifuge to discard the lower liquid; Then add Wash Buffer II, and centrifuge to discard the lower liquid; (4) Elution: Place the binding column in a centrifuge tube, add an elution solution to the binding column, and after centrifugation, collect the eluted solution after centrifugation, which is genomic DNA; The process for extracting cell-free DNA is as follows: (1) Binding: Add an aqueous suspension of silica hydroxyl nanomagnetic beads or an aqueous suspension of silica to the sample supernatant, invert and mix, and then centrifuge to discard the supernatant; in the aqueous suspension of silica, the particle size of silica is 50 - 400 nm; (2) Washing: Add Wash Buffer I to the precipitate, transfer it to a DNA binding column, and centrifuge to remove the lower liquid; then add Wash Buffer II, and centrifuge to discard the lower liquid; (3) Elution: Place the binding column in a centrifuge tube, add an elution solution to the binding column, and after centrifugation, collect the eluted solution after centrifugation, which is cell-free DNA; The lysis solution is a guanidine salt lysis solution or an SDS lysis solution; The guanidine salt lysis solution includes guanidine salt, Triton X-100, Tris-HCl, Tween-20, EDTA salt, and water; the pH of the guanidine salt lysis solution is 8 - 9; The SDS lysis solution includes SDS, phosphate, Tris-HCl, EDTA salt, and water; the pH of the SDS lysis solution is 6 - 7; In the guanidine salt lysis solution, the concentration of guanidine salt in the guanidine salt lysis solution is 2M - 8M; the mass concentration of Triton X-100 in the guanidine salt lysis solution is 0.3% - 3%; the mass concentration of Tween-20 in the guanidine salt lysis solution is 3% - 10%; the concentration of EDTA salt in the guanidine salt lysis solution is 10 mM - 50 mM; the concentration of Tris-HCl in the guanidine salt lysis solution is 20 mM - 50 mM; In the SDS lysis solution, the phosphate is sodium phosphate, and the concentration of sodium phosphate in the SDS lysis solution is 0.1M - 0.5M; the mass concentration of SDS in the SDS lysis solution is 0.5% - 2%; the concentration of EDTA salt in the SDS lysis solution is 10 mM - 50 mM; the concentration of Tris-HCl in the SDS lysis solution is 20 mM - 50 mM; The binding solution includes guanidine salt, isopropanol, and water; the concentration of guanidine salt in the binding solution is 4M - 8M; the mass concentration of isopropanol in the binding solution is 60% - 70%; The Wash Buffer I is an aqueous solution of guanidine salt, or the Wash Buffer I includes guanidine salt, ethanol, and water; the concentration of guanidine salt in the Wash Buffer I is 4M - 5M; the mass concentration of ethanol in the Wash Buffer I is 40% - 80%; The guanidine salt is guanidine hydrochloride or guanidine isothiocyanate; The washing solution II includes Tris-HCl, Tris, ethanol and water; the concentration of Tris-HCl in the washing solution II is 30-60 mM, the concentration of Tris in the washing solution II is 0.5 M-1 M, and the mass concentration of ethanol in the washing solution II is 70%-90%; the pH value of the washing solution II is 7-8; The pretreatment solution is composed of the following components: polyhydric alcohol, chelating agent, preservative, pH buffer and water; the pH of the pretreatment solution is 7-9; The polyhydric alcohol is high molecular weight polyhydric alcohol and low molecular weight polyhydric alcohol; The high molecular weight polyhydric alcohol is selected from at least one of high molecular weight polyethylene glycols, and the number average molecular weight of the high molecular weight polyethylene glycol is 1500-40000; The low molecular weight polyhydric alcohol is selected from at least one of low molecular weight polyethylene glycols, and the number average molecular weight of the low molecular weight polyethylene glycol is below 1000; The chelating agent is at least one of EDTA-2Na, EDTA-3K, EDTA-2K; In the pretreatment solution, the mass ratio of the high molecular weight polyhydric alcohol is 1%-10%; the mass ratio of the low molecular weight polyhydric alcohol is 10%-65%; the mass ratio of the preservative is 0.5%-5%; the concentration of the chelating agent in the pretreatment solution is 10 g / L-50 g / L.

2. The method for co-extracting genomic DNA and cell-free DNA according to claim 1, wherein The preservative is at least one of proclin 310, proclin 950, sodium azide; The pH buffer is Tris buffer or citrate buffer.

3. The method for co-extracting genomic DNA and cell-free DNA according to claim 1, wherein The eluent is at least one of Tris-HCl aqueous solution, Tris aqueous solution and EDTA aqueous solution; the pH of the eluent is 8-9.

4. The method for co-extracting genomic DNA and cell-free DNA according to claim 1, wherein, In the aqueous suspension of the silicon hydroxyl nano magnetic beads, the core of the silicon hydroxyl nano magnetic beads is a magnetic substance, and the outer layer is wrapped with SiO2; the diameter of the silicon hydroxyl nano magnetic beads is 50-400 nm.

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