Pretreatment liquid for co-extraction of genome DNA and free DNA and extraction method

Abstract

The invention relates to the technical field of biomedicine, in particular to pretreatment liquid for co-extraction of genome DNA and free DNA and an extraction method, the pretreatment liquid comprises polyhydroxy alcohol, a chelating agent, a preservative, a pH buffer solution and water; the polyhydroxy alcohol comprises high molecular weight polyhydroxy alcohol and low molecular weight polyhydroxy alcohol; the high-molecular-weight polyhydroxy alcohol is selected from at least one of high-molecular-weight polyethylene glycol, and the molecular weight of the high-molecular-weight polyethylene glycol is 1500-40000; the low-molecular-weight polyhydroxy 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 1000 or less. Adding the pretreatment liquid into the sample, and centrifuging to obtain a sample precipitate and a sample supernatant; the sample precipitate is used for extracting genome DNA, and the sample supernate is used for extracting free DNA. According to the extraction method, high-purity extraction of the genome DNA and the free DNA can be realized at the same time.

Description

Technical Field

[0001] The invention relates to a pretreatment solution and an extraction method for co-extracting genomic DNA and free DNA, belonging to the technical field of biomedicine. Background Art

[0002] The technology of co-extraction of genomic DNA and free DNA mainly involves the fields of biology, molecular biology and medicine. This technology is usually used to study DNA in cells and free DNA in liquids (such as blood and 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 that diagnoses diseases by sampling body fluids such as cerebrospinal fluid, saliva, pleural effusion, blood, ascites, and urine. Compared with tissue biopsy, liquid biopsy has the advantages of being non-invasive, enabling early diagnosis, and dynamic monitoring. The DNA in liquid samples can be simply divided into two parts: nucleated cells shed from the human body system, and free DNA that enters the body fluids through the circulatory system. The genomic DNA and free DNA obtained through liquid biopsy can be used in the fields of early disease screening, diagnostic typing, drug concomitant testing, and patient condition monitoring, and has great development potential.

[0004] Currently, both blood and urine can be used as the basis for liquid biopsy technology. Among them, urine is less invasive and more convenient to sample, and its potential as a liquid biopsy sample is gradually being explored. In addition, urine samples have the following advantages over blood samples: (1) They are less contagious to infectious diseases such as HIV; (2) The nucleic acid spectrum is similar to that of blood; (3) The protein content in urine is lower, 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 my country. Many reports have pointed out that the genomic DNA and free DNA of urine exudate cells in patients with bladder cancer can detect a variety of related oncogene mutations and other genetic variations, which indicates that gene mutations in urine samples are associated with disease progression. Similar free DNA in body fluids can not only monitor bladder cancer, but also other non-urinary cancers. For example, 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 the fetus. We can also carry out targeted medication and prognosis prediction based on the mutation sites in the DNA extracted from body fluids.

[0006] Currently, there are few commercial kits on the market that can extract DNA from body fluids, and most of them can only extract genomic DNA or free DNA, and the extraction of free DNA takes a long time. Some products that can extract genomic DNA and free DNA have the problem of difficulty in separating genomic DNA from free DNA, especially 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 free DNA extraction is also developing. Automation technology uses liquid handling workstations and automated instruments to process multiple samples at the same time, reducing manual operation errors and providing high-throughput extraction capabilities. However, the technology of co-extraction of genomic DNA and free DNA through automation technology is also in the process of development, and there is no mature processing technology yet.

[0008] In general, the co-extraction technology of genomic DNA and free DNA is still in the process of development, especially the co-extraction technology of 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 molecular biology research, clinical diagnosis and personalized medicine.

[0009] The co-extraction technology of genomic DNA and free DNA has the following technical difficulties: (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.

[0010] (2) The problem of stable preservation of free DNA: free DNA fragments are small, and the free DNA in urine is smaller than that in blood. In addition, the half-life of free DNA is very short. It is also very important to ensure the stability of free DNA during the period from sampling to DNA extraction.

[0011] (3) The requirement for free genomic DNA contamination in the extracted free DNA: on the one hand, it is necessary to prevent the lysis of detached cells in body fluids; on the other hand, it is necessary to avoid the residue of genomic DNA during the free DNA extraction process.

[0012] Since cell-free DNA extraction takes a long time and has poor stability, there are currently few kits on the market that can extract cell-free DNA, and most can only extract genomic DNA. There are very few that can extract genomic DNA and cell-free DNA, but it is difficult to fully separate the two. The presence of genomic DNA detected in cell-free DNA will affect the purity of the cell-free DNA, thereby affecting the accuracy of downstream experiments. Summary of the invention

[0013] In view of the deficiencies in the prior art, the present invention provides a pretreatment solution and an extraction method for co-extracting genomic DNA and free DNA. The pretreatment solution has the function of preserving exfoliated cells and maintaining the stability of free DNA, so that the extracted free DNA is free of genomic DNA contamination, the purity of the free DNA is guaranteed, and the downstream results are more accurate.

[0014] The technical solution of the present invention to solve the above technical problems is as follows: a pretreatment solution for co-extraction of genomic DNA and free DNA, the pretreatment solution comprising: polyhydroxy alcohol, chelating agent, preservative, pH buffer and water; the pH of the pretreatment solution is 7-9; The polyhydric alcohols include high molecular weight polyhydric alcohols and low molecular weight polyhydric alcohols; The high molecular weight polyhydroxy 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 polyhydroxy 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 less than 1000.

[0015] Furthermore, in the pretreatment liquid, the mass proportion of the high molecular weight polyhydric alcohol is 1%-10%; the mass proportion of the low molecular weight polyhydric alcohol is 10%-65%; the mass proportion of the preservative is 0.5%-5%; and the concentration of the chelating agent in the pretreatment liquid is 10g / L-50g / L.

[0016] Further, the chelating agent is at least one of EDTA-2Na, EDTA-3K, and EDTA-2K; The preservative is at least one of proclin 310, proclin 950, and sodium azide; The pH buffer is a Tris buffer or a citric acid buffer.

[0017] The present invention also discloses a method for co-extracting genomic DNA and free DNA, and the extraction method is: Adding the pretreatment solution of the present invention to the sample, and then centrifuging to obtain a sample precipitate and a sample supernatant; the sample precipitate is used for extracting genomic DNA, and the sample supernatant is used for extracting free DNA; The extraction process of the genomic DNA is as follows: (1) Lysis: Add lysis buffer and protease to the sample precipitate for incubation; (2) Binding: Add binding solution, mix thoroughly by pipetting with a pipette, then add to the DNA binding column, centrifuge and discard the solution; (3) Rinsing: Add rinsing solution I, centrifuge and discard the solution; then add rinsing solution II, centrifuge and discard the solution; (4) Elution: Place the binding column in a centrifuge tube, add elution solution to the binding column, centrifuge, and collect the elution solution after centrifugation, which is the genomic DNA; The extraction process of the free DNA is as follows: (1) Binding: Add an aqueous suspension of silanol nanoparticles or an aqueous suspension of silica to the sample supernatant, mix by inverting, and then centrifuge to discard the supernatant; (2) Rinsing: Add rinse solution I to the precipitate, transfer to the DNA binding column, centrifuge and discard the liquid; then add rinse solution II, centrifuge and discard the liquid; (3) Elution: Place the binding column in a centrifuge tube, add elution solution to the binding column, and centrifuge. The elution solution collected after centrifugation is the free DNA.

[0018] Furthermore, the lysate is a guanidine lysate or an SDS lysate; The guanidine salt lysis solution comprises guanidine salt, TritonX-100, Tris-HCl, Tween-20, EDTA salt and water; the pH of the guanidine salt lysis solution is 8-9; The SDS lysate comprises SDS, phosphate, Tris-HCl, EDTA salt and water; the pH of the SDS lysate is 6-7.

[0019] Furthermore, 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; In the SDS lysate, the phosphate is sodium phosphate, and the concentration of the sodium phosphate in the SDS lysate is 0.1M-0.5M; the mass concentration of SDS in the SDS lysate is 0.5%-2%; the concentration of EDTA salt in the SDS lysate is 10mM-50Mm; and the concentration of Tris-HCl in the SDS lysate is 20mM-50mM.

[0020] Furthermore, the binding solution includes guanidine salt, isopropanol and water; 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 the isopropanol in the binding solution is 60%-70%.

[0021] Further, the rinsing liquid I is an aqueous solution of guanidine salt, or the rinsing liquid I includes guanidine salt, ethanol and water; The guanidine salt is guanidine hydrochloride or guanidine isothiocyanate; The concentration of the guanidine salt in the rinse solution I is 4M-5M; the mass concentration of the ethanol in the rinse solution I is 40%-80%; The rinsing solution II includes Tris-HCl, Tris, ethanol and water; the concentration of Tris-HCl in the rinsing solution II is 30-60mM, the concentration of Tris in the rinsing solution II is 0.5M-1M, and the mass concentration of ethanol in the rinsing solution II is 70%-90%; the pH value of the rinsing solution II is 7-8.

[0022] Furthermore, the eluent is at least one of a Tris-HCl aqueous solution, a Tris aqueous solution and an EDTA aqueous solution; and the pH of the eluent is 8-9.

[0023] Furthermore, the inner core of the silicon hydroxyl nano magnetic beads in the aqueous suspension 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; In the aqueous suspension of silicon dioxide, the particle size of silicon dioxide is 50-400 nm.

[0024] The beneficial effects of the present invention are: (1) Pre-treating the sample using the pre-treatment solution of the present invention can achieve fast and efficient sample collection: if the customer directly collects the sample without any treatment, the integrity of the detached cells cannot be guaranteed, which will directly lead to the inability to separate the genomic DNA from the free DNA. If you want to analyze only the free DNA, you must centrifuge the sample to separate the detached cells and only save the supernatant. The addition of the pre-treatment solution in this technology has the effect of preserving the detached cells and maintaining the stability of the free DNA. The advantages are: ① The sample collection process can take place at any time and place without the participation of experimental equipment such as centrifuges; ② After adding the sample pre-treatment solution, the sampling location can be some distance away from the analysis laboratory, and the express delivery in the middle will not affect the subsequent sample analysis.

[0025] (2) The sample is pretreated with the pretreatment solution of the present invention, which can preserve the exfoliated cells and maintain the stability of free DNA, thereby ensuring that there is no mutual interference in the subsequent extraction of genomic DNA and free DNA, and ensuring the purity of each of the genomic DNA and free DNA.

[0026] (3) Pre-treating the sample with the pre-treatment solution of the present invention is beneficial for extracting DNA from difficult samples: adding the pre-treatment solution to treat the sample can change the properties of the sample and increase the solubility of some impurities in the sample, which is beneficial for the subsequent DNA extraction.

[0027] (4) Separation of free DNA by silicon hydroxy nanoparticles or silica is fast and efficient. Under the action of the pretreatment solution, the free DNA is adsorbed onto the silicon hydroxy nanoparticles or silica, and the binding and adsorption of free DNA is completed within 1 minute. Especially when the sample volume is relatively large, there is no need for multiple centrifugation or long-term filtration, thus avoiding the degradation of free DNA caused by long-term operation.

[0028] (5) The extraction method of the present invention can save time and samples. By extracting genomic DNA and free DNA simultaneously, two nucleic acids can be obtained in one sample and one experiment, thereby saving time and samples. This is very valuable for researchers, especially when the number of samples is limited or large-scale analysis is required.

[0029] (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 the detached cells but also maintain the stability of the free DNA, so that the extracted free DNA is free of genomic DNA contamination, thereby ensuring the purity of the free DNA and making the downstream results more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The electrophoresis diagrams show the effect of the isopropanol content in the binding solution on the amount of genomic DNA extracted; Figure 2 The electrophoresis diagrams show the effect of the guanidine salt content in the binding solution on the amount of genomic DNA extracted; Figure 3 The electrophoresis diagrams show the effect of the pH of the eluent on the amount of genomic DNA extracted; Figure 4 The electrophoresis diagrams show the effect of SDS content in the lysate on the amount of genomic DNA extracted; Figure 5 The electrophoresis diagrams are compared to show the effect of guanidine salt content in the lysate on the amount of genomic DNA extracted; Figure 6 The electrophoresis diagrams show the effect of guanidine salt content in the lysate on the amount of free DNA extracted; Figure 7 The electrophoresis diagram of free DNA in Example 1, Example 2, Comparative Example 1 and Comparative Example 2 ( Figure 7 , 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); Figure 8 The electrophoresis diagram of genomic DNA extraction of Example 1, Example 2 and Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6 ( Figure 8 , 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); Fig. 9 The electrophoresis diagram of the urine sample after pretreatment in Example 1 and the urine sample in Comparative Example 7; Fig.10 The electrophoresis diagram of the preservation of genomic DNA in the urine sample after pretreatment in Example 1 at different times ( Fig.10 In the figure, t0, t7, t10, and t30 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, respectively); Fig.11 The electrophoresis diagram of the preservation of free DNA in the urine sample after pretreatment in Example 1 at different times ( Fig.11 In the figure, t0, t4, and t7 represent the free DNA extracted on the day after pretreatment, the 4th day after pretreatment, and the 7th day after pretreatment, respectively).

[0031] Fig.12 The electrophoresis diagrams of free DNA extracted from blood samples of Example 5, Example 6 and Comparative Example 12; Fig.13 The electrophoresis diagrams are of genomic DNA extracted from blood samples of Example 5, Example 6 and Comparative Example 12. DETAILED DESCRIPTION

[0032] The specific implementation of the present invention is described in detail below. The present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used are only for describing specific embodiments and are not intended to limit the present invention.

[0034] A pretreatment solution for co-extraction of genomic DNA and free DNA, the pretreatment solution comprising: polyhydroxy alcohol, chelating agent, preservative, pH buffer and water; the pH of the pretreatment solution is 7-9; The polyhydric alcohols include high molecular weight polyhydric alcohols and low molecular weight polyhydric alcohols; The high molecular weight polyhydroxy 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 polyhydroxy 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 less than 1000.

[0035] More specifically, the number average molecular weight of the high molecular weight polyethylene glycol is applicable in the range of 2000-35000, 2500-30000, 3000-25000, 3500-20000 and 4000-15000; 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; Specifically, in the pretreatment liquid, the mass proportion of the high molecular weight polyhydric alcohol is 1%-10%; the mass proportion of the low molecular weight polyhydric alcohol is 10%-65%; the mass proportion of the preservative is 0.5%-5%; and the concentration of the chelating agent in the pretreatment liquid is 10g / L-50g / L.

[0036] Preferably, in the pretreatment liquid, the mass proportion of the high molecular weight polyhydric alcohol is 2%-8%; the mass proportion of the low molecular weight polyhydric alcohol is 20%-65%.

[0037] More preferably, in the pretreatment liquid, the mass proportion of the high molecular weight polyhydric alcohol is 3%-5%; the mass proportion of the low molecular weight polyhydric alcohol is 20%-40%.

[0038] Specifically, the chelating agent is at least one of EDTA-2Na, EDTA-3K, and EDTA-2K; The preservative is at least one of proclin 310, proclin 950, and sodium azide; The pH buffer is a Tris buffer or a citric acid buffer.

[0039] A method for co-extracting genomic DNA and free DNA, the extraction method comprising: Sample preparation: Add the pretreatment solution to the sample, then centrifuge to obtain a sample precipitate and a sample supernatant; the sample precipitate is used to extract genomic DNA, and the sample supernatant is used to extract free DNA; wherein, the amount of pretreatment solution added is 10%-20% of the sample usage amount. In the specific embodiment, the sample is experimented with a volume of 10mL.

[0040] The extraction process of the genomic DNA is as follows: (1) Lysis: Add lysis buffer and protease to the sample precipitate for incubation; the amount of lysis buffer added is 450 μL, and the amount of protease added is 20 μL.

[0041] (2) Binding: Add binding solution, mix well by pipetting, then add to the DNA binding column and centrifuge to discard the solution. The amount of binding solution added is 450 μL.

[0042] (3) Rinse: Add rinsing solution I, centrifuge and discard the solution; then add rinsing solution II, centrifuge and discard the solution; the amount of rinsing solution I added is 500 μL; the amount of rinsing solution II added is 700 μL.

[0043] (4) Elution: Place the binding column in a centrifuge tube, add elution buffer to the binding column, centrifuge, and collect the elution buffer after centrifugation, which is the genomic DNA; the amount of elution buffer added is 30-50 μL.

[0044] The extraction process of the free DNA is as follows: (1) Binding: Add silanol nanoparticles aqueous suspension or silica aqueous suspension to the sample supernatant, mix by inversion, centrifuge and discard the supernatant; the amount of silanol nanoparticles aqueous suspension added is 10-20 μL or the amount of silica aqueous suspension added is 100-200 μL; (2) Rinsing: Add rinsing solution I to the precipitate, transfer it to the DNA binding column, and centrifuge to remove the lower liquid; then add rinsing solution II, centrifuge and discard the lower liquid; the amount of rinsing solution I added is 500 μL; the amount of rinsing solution II added is 700 μL.

[0045] (3) Elution: Place the binding column in a centrifuge tube, add elution solution to the binding column, centrifuge, and collect the elution solution after centrifugation, which is the free DNA. The amount of elution solution added is 30-50 μL.

[0046] Specifically, the specific operation in the sample preparation process is: adding the pretreatment solution to the sample, centrifuging at 18000g for 10 minutes, the sample precipitate is used for genomic DNA extraction, the supernatant is transferred to another new 15mL centrifuge tube, centrifuging at 18000g for 10 minutes, and the sample supernatant is used for free DNA extraction. Multiple centrifugation operations are conducive to extracting high-purity free DNA.

[0047] Specifically, during the extraction of the genomic DNA, proteinase K was added during lysis, and the mixture was incubated at 56° C. for 20 minutes.

[0048] Specifically, during the extraction of free DNA, before the combination operation in step (1), the sample supernatant is centrifuged again at 18,000 g for 10 min and transferred to a new centrifuge tube for free DNA extraction. This is more conducive to purifying high-purity free DNA.

[0049] Specifically, in the process of extracting free DNA, the specific process of step (1) is as follows: adding a silanol nanomagnetic bead aqueous suspension or a silica aqueous suspension to the sample supernatant, inverting the sample 20 times, centrifuging at 1800 g for 5 minutes, and discarding the supernatant.

[0050] In the embodiment of the present invention, the solid content of the silanol nanomagnetic beads in the aqueous suspension of silanol nanomagnetic beads is 50 mg / mL; the solid content of the silicon dioxide in the aqueous suspension of silicon dioxide is 50 mg / mL.

[0051] Specifically, the lysate is a guanidine lysate or an SDS lysate; The guanidine salt lysis solution comprises guanidine salt, TritonX-100, Tris-HCl, Tween-20, EDTA salt and water; the pH of the guanidine salt lysis solution is 8-9; The SDS lysate comprises SDS, phosphate, Tris-HCl, EDTA salt and water; the pH of the SDS lysate is 6-7.

[0052] 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 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; In the SDS lysate, the phosphate is sodium phosphate, and the concentration of the sodium phosphate in the SDS lysate is 0.1M-0.5M; the mass concentration of SDS in the SDS lysate is 0.5%-2%; the concentration of EDTA salt in the SDS lysate is 10mM-50Mm; and the concentration of Tris-HCl in the SDS lysate is 20mM-50mM.

[0053] More specifically, the EDTA salt is at least one of EDTA-2Na, EDTA-3K, and EDTA-2K.

[0054] Specifically, the combination solution includes guanidine salt, isopropanol and water; 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 the isopropanol in the binding solution is 60%-70%.

[0055] Specifically, the rinsing liquid I is an aqueous solution of guanidine salt, or the rinsing liquid I includes guanidine salt, ethanol and water; The guanidine salt is guanidine hydrochloride or guanidine isothiocyanate; The concentration of the guanidine salt in the rinse solution I is 4M-5M; the mass concentration of the ethanol in the rinse solution I is 40%-80%; The rinsing solution II includes Tris-HCl, Tris, ethanol and water; the concentration of Tris-HCl in the rinsing solution II is 30-60mM, the concentration of Tris in the rinsing solution II is 0.5M-1M, and the mass concentration of ethanol in the rinsing solution II is 70%-90%; the pH value of the rinsing solution II is 7-8.

[0056] Specifically, the eluent is at least one of a Tris-HCl aqueous solution, a Tris aqueous solution and an EDTA aqueous solution; and the pH of the eluent is 8-9.

[0057] Specifically, the inner core of the silicon hydroxyl nanomagnetic bead is a magnetic material, and the outer layer is wrapped with SiO2; the diameter of the silicon hydroxyl nanomagnetic bead is 50-400nm.

[0058] Example 1 1. Preparation of pretreatment solution: Add high molecular weight polyhydric alcohol, low molecular weight polyhydric alcohol, chelating agent, preservative, and pH buffer solution into water and mix them evenly to obtain a pretreatment solution; The high molecular weight polyhydroxy alcohol is polyethylene glycol 5000, and the mass concentration in the pretreatment solution is 5%; The low molecular weight polyhydric alcohol is ethylene glycol 200, and the mass concentration in the pretreatment solution is 40%; The chelating agent was EDTA-2Na, and its concentration in the pretreatment solution was 30 g / L; The preservative was proclin 310, and its mass concentration in the pretreatment solution was 2%; The pH buffer is Tris buffer; the pH of the pretreatment solution is 8.

[0059] 2. Co-extraction method of genomic DNA and free DNA: S1. Sample preparation: Add the pretreatment solution to the urine sample (10 mL), centrifuge at 18000g for 10 min, and use the sample precipitate for genomic DNA extraction. Transfer the supernatant to another new 15 mL centrifuge tube, centrifuge at 18000g for 10 min, and use the sample supernatant for free DNA extraction. The amount of pretreatment solution added is 15% of the urine sample mass.

[0060] S2. The extraction process of the genomic DNA is as follows: (1) Lysis: Add lysis buffer and proteinase K to the sample pellet and incubate at 56°C for 20 min. The lysing solution is SDS lysing solution, which specifically consists of: SDS (0.5%), sodium phosphate (0.5M), Tris-HCl (50mM), EDTA-2K (10mM) and water; The amount of lysate added was 450 μL.

[0061] (2) Binding: Add binding solution, mix thoroughly by pipetting with a pipette, then add to the DNA binding column, centrifuge and discard the solution; The binding solution is composed of: guanidine hydrochloride (5M), isopropanol (70%) and water; wherein the amount of the binding solution added is 450 μL.

[0062] (3) Rinsing: Add rinsing solution I, centrifuge and discard the solution; then add rinsing solution II, centrifuge and discard the solution; The rinse solution I is composed of: guanidine hydrochloride (4M), ethanol (60%) and water; wherein the amount of rinse solution I added is 500 μL.

[0063] The composition of the rinsing solution II is: Tris-HCl (40mM), Tris (0.5M), ethanol (70%) and water; the pH value of the rinsing solution II is 7-8, wherein the amount of the rinsing solution II added is 700 μL.

[0064] (4) Elution: Place the binding column in a new 1.5 mL centrifuge tube, add eluent to the binding column, centrifuge for 2 minutes, and collect the eluent after centrifugation, which is the genomic DNA; The eluent is a Tris-HCl aqueous solution (the concentration of Tris-HCl is 15 mM), the pH of the eluent is 8.5, and the amount of the eluent added is 50 μL.

[0065] S3, the extraction process of the free DNA is: The sample supernatant was centrifuged again at 18000g for 10 min and transferred to a new centrifuge tube for extraction of free DNA.

[0066] (1) Binding: Add the aqueous suspension of silanol nanoparticles to the supernatant, invert 20 times, centrifuge at 1800 g for 5 min, and discard the supernatant; The amount of the silicon hydroxy nano-magnetic bead aqueous suspension added was 20 μL.

[0067] (2) Rinsing: Add rinse solution I to the precipitate, transfer to the DNA binding column, centrifuge and discard the liquid; then add rinse solution II, centrifuge and discard the liquid; The rinse solution I is composed of: guanidine hydrochloride (4M), ethanol (60%) and water; wherein the amount of rinse solution I added is 500 μL.

[0068] The composition of the rinsing solution II is: Tris-HCl (40mM), Tris (0.5M), ethanol (70%) and water; the pH value of the rinsing solution II is 7-8, wherein the amount of the rinsing solution II added is 700 μL.

[0069] (3) Elution: Place the binding column in a centrifuge tube, add elution solution to the binding column, and centrifuge. The elution solution collected after centrifugation is the free DNA.

[0070] The eluent is a Tris-HCl aqueous solution (the concentration of Tris-HCl is 15 mM), the pH of the eluent is 8.5, and the amount of the eluent added is 50 μL.

[0071] Example 2 1. Preparation of pretreatment solution: Add high molecular weight polyhydric alcohol, low molecular weight polyhydric alcohol, chelating agent, preservative, and pH buffer solution into water and mix them evenly to obtain a pretreatment solution; The high molecular weight polyhydroxy alcohol is polyethylene glycol 15000, and its mass concentration in the pretreatment solution is 3%; The low molecular weight polyhydric alcohol is ethylene glycol 500, and the mass concentration in the pretreatment solution is 20%; The chelating agent was EDTA-3K, and its concentration in the pretreatment solution was 10 g / L; The preservative was proclin 950, and the mass concentration in the pretreatment solution was 0.5%; The pH buffer is a citric acid buffer; the pH of the pretreatment solution is 7.

[0072] 2. Co-extraction method of genomic DNA and free DNA: S1. Sample preparation: Add the pretreatment solution to the urine sample (10 mL), centrifuge at 18000g for 10 min, and use the sample precipitate for genomic DNA extraction. Transfer the supernatant to another new 15 mL centrifuge tube, centrifuge at 18000g for 10 min, and use the sample supernatant for free DNA extraction. The amount of pretreatment solution added is 15% of the urine sample mass.

[0073] S2. The extraction process of the genomic DNA is as follows: (1) Lysis: Add lysis buffer and proteinase K to the sample pellet and incubate at 56°C for 20 min. The lysis solution is a guanidine salt lysis solution, which specifically comprises: guanidine isothiocyanate (7M), TritonX-100 (0.3%), Tris-HCl (20mM), Tween-20 (3%), EDTA-2Na (10mM) and water; The amount of lysate added was 450 μL.

[0074] (2) Binding: Add binding solution, mix thoroughly by pipetting with a pipette, then add to the DNA binding column, centrifuge and discard the solution; The binding solution is composed of: guanidine hydrochloride (4M), isopropanol (60%) and water; wherein the amount of the binding solution added is 450 μL.

[0075] (3) Rinsing: Add rinsing solution I, centrifuge and discard the solution; then add rinsing solution II, centrifuge and discard the solution; The rinse solution I is composed of: guanidine hydrochloride (5M), ethanol (40%) and water; wherein the amount of rinse solution I added is 500 μL.

[0076] The composition of the rinsing solution II is: Tris-HCl (30mM), Tris (1M), ethanol (90%) and water; the pH value of the rinsing solution II is 7-8, wherein the amount of the rinsing solution II added is 700 μL.

[0077] (4) Elution: Place the binding column in a new 1.5 mL centrifuge tube, add eluent to the binding column, centrifuge for 2 minutes, and collect the eluent after centrifugation, which is the genomic DNA; The eluent is a Tris aqueous solution (the concentration of Tris is 10 mM), and the pH of the eluent is 8. The amount of the eluent added is 30 μL.

[0078] S3, the extraction process of the free DNA is: The sample supernatant was centrifuged again at 18000g for 10 min and transferred to a new centrifuge tube for extraction of free DNA.

[0079] (1) Binding: Add silica aqueous suspension to the supernatant, invert 20 times, centrifuge at 1800 g for 5 min, and discard the supernatant; The amount of silica aqueous suspension added was 200 μL.

[0080] (2) Rinsing: Add rinse solution I to the precipitate, transfer to the DNA binding column, centrifuge and discard the liquid; then add rinse solution II, centrifuge and discard the liquid; The rinse solution I is composed of: guanidine hydrochloride (5M), ethanol (40%) and water; wherein the amount of rinse solution I added is 500 μL.

[0081] The composition of the rinsing solution II is: Tris-HCl (30mM), Tris (1M), ethanol (90%) and water; the pH value of the rinsing solution II is 7-8, wherein the amount of the rinsing solution II added is 700 μL.

[0082] (3) Elution: Place the binding column in a centrifuge tube, add elution solution to the binding column, and centrifuge. The elution solution collected after centrifugation is the free DNA.

[0083] The eluent is a Tris aqueous solution (the concentration of Tris is 10 mM), and the pH of the eluent is 8. The amount of the eluent added is 50 μL.

[0084] Example 3 1. Preparation of pretreatment solution: Add high molecular weight polyhydric alcohol, low molecular weight polyhydric alcohol, chelating agent, preservative, and pH buffer solution into water and mix them evenly to obtain a pretreatment solution; The high molecular weight polyhydroxy alcohol is polyethylene glycol 1500, and the mass concentration in the pretreatment solution is 10%; The low molecular weight polyhydric alcohol is ethylene glycol 400, and the mass concentration in the pretreatment solution is 65%; The chelating agent was EDTA-2K, and its concentration in the pretreatment solution was 50 g / L; The preservative is sodium azide, and the mass concentration in the pretreatment solution is 5%; The pH buffer is a citric acid buffer; the pH of the pretreatment solution is 9.

[0085] 2. Co-extraction method of genomic DNA and free DNA: S1. Sample preparation: Add the pretreatment solution to the urine sample (10 mL), centrifuge at 18000g for 10 min, and use the sample precipitate for genomic DNA extraction. Transfer the supernatant to another new 15 mL centrifuge tube, centrifuge at 18000g for 10 min, and use the sample supernatant for free DNA extraction. The amount of pretreatment solution added is 20% of the urine sample.

[0086] S2. The extraction process of the genomic DNA is as follows: (1) Lysis: Add lysis buffer and proteinase K to the sample pellet and incubate at 56°C for 20 min. The lysis solution is a guanidine salt lysis solution, which specifically comprises: guanidine isothiocyanate (8M), TritonX-100 (3%), Tris-HCl (50mM), Tween-20 (10%), EDTA-2Na (50mM) and water; The amount of lysate added was 450 μL.

[0087] (2) Binding: Add binding solution, mix thoroughly by pipetting with a pipette, then add to the DNA binding column, centrifuge and discard the solution; The binding solution is composed of: guanidine isothiocyanate (8M), isopropanol (70%) and water; wherein the amount of the binding solution added is 450 μL.

[0088] (3) Rinsing: Add rinsing solution I, centrifuge and discard the solution; then add rinsing solution II, centrifuge and discard the solution; The rinse solution I is composed of: guanidine isothiocyanate (4M), ethanol (80%) and water; wherein the amount of rinse solution I added is 500 μL.

[0089] The composition of the rinsing solution II is: Tris-HCl (60mM), Tris (0.5M), ethanol (80%) and water; the pH value of the rinsing solution II is 7-8, wherein the amount of the rinsing solution II added is 700 μL.

[0090] (4) Elution: Place the binding column in a new 1.5 mL centrifuge tube, add eluent to the binding column, centrifuge for 2 minutes, and collect the eluent after centrifugation, which is the genomic DNA; The eluent is an EDTA aqueous solution (the concentration of EDTA is 1 mM), and the pH of the eluent is 9. The amount of the eluent added is 40 μL.

[0091] S3, the extraction process of the free DNA is: The sample supernatant was centrifuged again at 18000g for 10 min and transferred to a new centrifuge tube for extraction of free DNA.

[0092] (1) Binding: Add the aqueous suspension of silanol nanoparticles to the supernatant, invert 20 times, centrifuge at 1800 g for 5 min, and discard the supernatant; The amount of the silicon hydroxy nano-magnetic bead aqueous suspension added was 20 μL.

[0093] (2) Rinsing: Add rinse solution I to the precipitate, transfer to the DNA binding column, centrifuge and discard the liquid; then add rinse solution II, centrifuge and discard the liquid; The rinse solution I is composed of: guanidine isothiocyanate (4M), ethanol (80%) and water; wherein the amount of rinse solution I added is 500 μL.

[0094] The composition of the rinsing solution II is: Tris-HCl (60mM), Tris (0.5M), ethanol (80%) and water; the pH value of the rinsing solution II is 7-8, wherein the amount of the rinsing solution II added is 700 μL.

[0095] (3) Elution: Place the binding column in a centrifuge tube, add elution solution to the binding column, and centrifuge. The elution solution collected after centrifugation is the free DNA.

[0096] The eluent is an EDTA aqueous solution (the concentration of EDTA is 1 mM), and the pH of the eluent is 9. The amount of the eluent added is 50 μL.

[0097] Example 4 1. Preparation of pretreatment solution: Add high molecular weight polyhydric alcohol, low molecular weight polyhydric alcohol, chelating agent, preservative, and pH buffer solution into water and mix them evenly to obtain a pretreatment solution; The high molecular weight polyhydroxy alcohol is polyethylene glycol 5000, and the mass concentration in the pretreatment solution is 1%; The low molecular weight polyhydric alcohol is ethylene glycol 200, and the mass concentration in the pretreatment solution is 10%; The chelating agent was EDTA-2K, and its concentration in the pretreatment solution was 20 g / L; The preservative was proclin 310, and its mass concentration in the pretreatment solution was 1%; The pH buffer is Tris buffer; the pH of the pretreatment solution is 8.5.

[0098] 2. Co-extraction method of genomic DNA and free DNA: S1. Sample preparation: Add the pretreatment solution to the urine sample (10 mL), centrifuge at 18000g for 10 min, and use the sample precipitate for genomic DNA extraction. Transfer the supernatant to another new 15 mL centrifuge tube, centrifuge at 18000g for 10 min, and use the sample supernatant for free DNA extraction. The amount of pretreatment solution added is 10% of the urine sample.

[0099] S2. The extraction process of the genomic DNA is as follows: (1) Lysis: Add lysis buffer and proteinase K to the sample pellet and incubate at 56°C for 20 min. The lysis solution is a guanidine salt lysis solution, which specifically comprises: guanidine hydrochloride (2M), TritonX-100 (2%), Tris-HCl (30mM), Tween-20 (6%), EDTA-2K (30mM) and water; The amount of lysate added was 450 μL.

[0100] (2) Binding: Add binding solution, mix thoroughly by pipetting with a pipette, then add to the DNA binding column, centrifuge and discard the solution; The binding solution is composed of: guanidine isothiocyanate (4M), isopropanol (60%) and water; wherein the amount of the binding solution added is 450 μL.

[0101] (3) Rinsing: Add rinsing solution I, centrifuge and discard the solution; then add rinsing solution II, centrifuge and discard the solution; The rinse solution I is composed of: guanidine isothiocyanate (4M), ethanol (60%) and water; wherein the amount of rinse solution I added is 500 μL.

[0102] The composition of the rinsing solution II is: Tris-HCl (50mM), Tris (0.8M), ethanol (70%) and water; the pH value of the rinsing solution II is 7-8, wherein the amount of the rinsing solution II added is 700 μL.

[0103] (4) Elution: Place the binding column in a new 1.5 mL centrifuge tube, add eluent to the binding column, centrifuge for 2 minutes, and collect the eluent after centrifugation, which is the genomic DNA; The eluent is a Tris-HCl aqueous solution (the concentration of Tris-HCl is 10 mM), the pH of the eluent is 8.5, and the amount of the eluent added is 50 μL.

[0104] S3, the extraction process of the free DNA is: The sample supernatant was centrifuged again at 18000g for 10 min and transferred to a new centrifuge tube for extraction of free DNA.

[0105] (1) Binding: Add the aqueous suspension of silanol nanoparticles to the supernatant, invert 20 times, centrifuge at 1800 g for 5 min, and discard the supernatant; The amount of the silicon hydroxy nano-magnetic bead aqueous suspension added was 20 μL.

[0106] (2) Rinsing: Add rinse solution I to the precipitate, transfer to the DNA binding column, centrifuge and discard the liquid; then add rinse solution II, centrifuge and discard the liquid; The rinse solution I is composed of: guanidine isothiocyanate (4M), ethanol (60%) and water; wherein the amount of rinse solution I added is 500 μL.

[0107] The composition of the rinsing solution II is: Tris-HCl (50mM), Tris (0.8M), ethanol (70%) and water; the pH value of the rinsing solution II is 7-8, wherein the amount of the rinsing solution II added is 700 μL.

[0108] (3) Elution: Place the binding column in a centrifuge tube, add elution solution to the binding column, and centrifuge. The elution solution collected after centrifugation is the free DNA.

[0109] The eluent is a Tris-HCl aqueous solution (the concentration of Tris-HCl is 10 mM), the pH of the eluent is 8.5, and the amount of the eluent added is 50 μL.

[0110] Example 5 1. Preparation of pretreatment solution: Add high molecular weight polyhydric alcohol, low molecular weight polyhydric alcohol, chelating agent, preservative, and pH buffer solution into water and mix them evenly to obtain a pretreatment solution; The high molecular weight polyhydroxy alcohol is polyethylene glycol 5000, and the mass concentration in the pretreatment solution is 1%; The low molecular weight polyhydric alcohol is ethylene glycol 200, and the mass concentration in the pretreatment solution is 10%; The chelating agent was EDTA-2K, and its concentration in the pretreatment solution was 20 g / L; The preservative was proclin 310, and its mass concentration in the pretreatment solution was 1%; The pH buffer is Tris buffer; the pH of the pretreatment solution is 8.5.

[0111] 2. Co-extraction method of genomic DNA and free DNA: S1. Sample preparation: Add the pretreatment solution to the blood sample (1 mL), centrifuge at 18000g for 10 min, use the middle layer of the sample for genomic DNA extraction, carefully transfer the supernatant to another new 2 mL centrifuge tube, centrifuge at 18000g for 10 min, and use the sample supernatant for free DNA extraction. The amount of pretreatment solution added is 15% of the blood sample.

[0112] S2. The extraction process of the genomic DNA is as follows: (1) Lysis: Add lysis buffer and proteinase K to the sample middle layer and incubate at 56°C for 20 min. The lysis solution is a guanidine salt lysis solution, which specifically comprises: guanidine hydrochloride (5M), TritonX-100 (1.5%), Tris-HCl (30mM), Tween-20 (5%), EDTA-2Na (20mM) and water; The amount of lysate added was 450 μL.

[0113] (2) Binding: Add binding solution, mix thoroughly by pipetting with a pipette, then add to the DNA binding column, centrifuge and discard the solution; The binding solution is composed of: guanidine isothiocyanate (4M), isopropanol (70%) and water; wherein the amount of the binding solution added is 450 μL.

[0114] (3) Rinsing: Add rinsing solution I, centrifuge and discard the solution; then add rinsing solution II, centrifuge and discard the solution; The rinse solution I is composed of: guanidine hydrochloride (5M), ethanol (60%) and water; wherein the amount of rinse solution I added is 500 μL.

[0115] The composition of the rinsing solution II is: Tris-HCl (50mM), Tris (0.5M), ethanol (70%) and water; the pH value of the rinsing solution II is 7-8, wherein the amount of the rinsing solution II added is 700 μL.

[0116] (4) Elution: Place the binding column in a new 1.5 mL centrifuge tube, add eluent to the binding column, centrifuge for 2 minutes, and collect the eluent after centrifugation, which is the genomic DNA; The eluent is a Tris-HCl aqueous solution (the concentration of Tris-HCl is 10 mM), the pH of the eluent is 8.5, and the amount of the eluent added is 50 μL.

[0117] S3, the extraction process of the free DNA is: The sample supernatant was centrifuged again at 18000g for 10 min and transferred to a new centrifuge tube for extraction of free DNA.

[0118] (1) Binding: Add the aqueous suspension of silanol nanoparticles to the supernatant, invert 20 times, centrifuge at 1800 g for 5 min, and discard the supernatant; The amount of the silicon hydroxy nano-magnetic bead aqueous suspension added was 20 μL.

[0119] (2) Rinsing: Add rinse solution I to the precipitate, transfer to the DNA binding column, centrifuge and discard the liquid; then add rinse solution II, centrifuge and discard the liquid; The rinse solution I is composed of: guanidine hydrochloride (5M), ethanol (60%) and water; wherein the amount of rinse solution I added is 500 μL.

[0120] The composition of the rinsing solution II is: Tris-HCl (50mM), Tris (0.5M), ethanol (70%) and water; the pH value of the rinsing solution II is 7-8, wherein the amount of the rinsing solution II added is 700 μL.

[0121] (3) Elution: Place the binding column in a centrifuge tube, add elution solution to the binding column, and centrifuge. The elution solution collected after centrifugation is the free DNA.

[0122] The eluent is a Tris-HCl aqueous solution (the concentration of Tris-HCl is 10 mM), the pH of the eluent is 8.5, and the amount of the eluent added is 50 μL.

[0123] Example 6 1. Preparation of pretreatment solution: Add high molecular weight polyhydric alcohol, low molecular weight polyhydric alcohol, chelating agent, preservative, and pH buffer solution into water and mix them evenly to obtain a pretreatment solution; The high molecular weight polyhydroxy alcohol is polyethylene glycol 5000, and the mass concentration in the pretreatment solution is 5%; The low molecular weight polyhydric alcohol is ethylene glycol 200, and the mass concentration in the pretreatment solution is 40%; The chelating agent was EDTA-2Na, and its concentration in the pretreatment solution was 30 g / L; The preservative was proclin 310, and its mass concentration in the pretreatment solution was 2%; The pH buffer is Tris buffer; the pH of the pretreatment solution is 8.

[0124] 2. Co-extraction method of genomic DNA and free DNA: S1. Sample preparation: Add the pretreatment solution to the blood sample (1 mL), centrifuge at 18000g for 10 min, use the middle layer of the sample for genomic DNA extraction, carefully transfer the supernatant to another new 2 mL centrifuge tube, centrifuge at 18000g for 10 min, and use the sample supernatant for free DNA extraction. The amount of pretreatment solution added is 15% of the blood sample.

[0125] S2. The extraction process of the genomic DNA is as follows: (1) Lysis: Add lysis buffer and proteinase K to the sample middle layer and incubate at 56°C for 20 min. The lysing solution is SDS lysing solution, which specifically comprises: SDS (2%), sodium phosphate (0.1 M), Tris-HCl (20 mM), EDTA-2K (50 mM) and water; The amount of lysate added was 450 μL.

[0126] (2) Binding: Add binding solution, mix thoroughly by pipetting with a pipette, then add to the DNA binding column, centrifuge and discard the solution; The binding solution is composed of: guanidine hydrochloride (8M), isopropanol (60%) and water; wherein the amount of the binding solution added is 450 μL.

[0127] (3) Rinsing: Add rinsing solution I, centrifuge and discard the solution; then add rinsing solution II, centrifuge and discard the solution; The rinse solution I is composed of: guanidine hydrochloride (4M), ethanol (50%) and water; wherein the amount of rinse solution I added is 500 μL.

[0128] The composition of the rinsing solution II is: Tris-HCl (40mM), Tris (0.5M), ethanol (90%) and water; the pH value of the rinsing solution II is 7-8, wherein the amount of the rinsing solution II added is 500 μL.

[0129] (4) Elution: Place the binding column in a new 1.5 mL centrifuge tube, add eluent to the binding column, centrifuge for 2 minutes, and collect the eluent after centrifugation, which is the genomic DNA; The eluent is a Tris-HCl aqueous solution (the concentration of Tris-HCl is 10 mM), the pH of the eluent is 8.5, and the amount of the eluent added is 50 μL.

[0130] S3, the extraction process of the free DNA is: The sample supernatant was centrifuged again at 18000g for 10 min and transferred to a new centrifuge tube for extraction of free DNA.

[0131] (1) Binding: Add silica aqueous suspension to the supernatant, invert 20 times, centrifuge at 1800 g for 5 min, and discard the supernatant; The amount of silica aqueous suspension added was 200 μL.

[0132] (2) Rinsing: Add rinse solution I to the precipitate, transfer to the DNA binding column, centrifuge and discard the liquid; then add rinse solution II, centrifuge and discard the liquid; The rinse solution I is composed of: guanidine hydrochloride (4M), ethanol (50%) and water; wherein the amount of rinse solution I added is 500 μL.

[0133] The composition of the rinsing solution II is: Tris-HCl (40mM), Tris (0.5M), ethanol (90%) and water; the pH value of the rinsing solution II is 7-8, wherein the amount of the rinsing solution II added is 700 μL.

[0134] (3) Elution: Place the binding column in a centrifuge tube, add elution solution to the binding column, and centrifuge. The elution solution collected after centrifugation is the free DNA.

[0135] The eluent is a Tris-HCl aqueous solution (the concentration of Tris-HCl is 10 mM), the pH of the eluent is 8.5, and the amount of the eluent added is 30-50 μL.

[0136] Example 7 The same sample was pretreated using the same pretreatment solution as in Example 1, and then the genomic DNA and free DNA were co-extracted using the same method as in Example 1, except that the mass concentration of isopropanol in the binding solution of Example 7 was 60%.

[0137] The optimal concentration of isopropanol in the binding solution was verified by the purification results of Example 1 and Example 7. In order to make the experimental data more convincing, three samples were purified using the methods of Example 1 and Example 7, respectively. The electrophoresis diagram of the purified genomic DNA is shown in FIG. Figure 1 As shown. Figure 1 It can be seen that when the isopropanol concentration in the binding solution is 70%, the purification effect is better.

[0138] Example 8 The same sample was pretreated with the same pretreatment solution as in Example 1, and then the genomic DNA and free DNA were co-extracted with the same method as in Example 1, except that the concentration of guanidine salt in the binding solution of Example 8 was 6M.

[0139] Example 9 The same sample was pretreated with the same pretreatment solution as in Example 1, and then the genomic DNA and free DNA were co-extracted with the same method as in Example 1, except that the concentration of guanidine salt in the binding solution of Example 9 was 8M.

[0140] The optimal concentration of guanidine salt in the binding solution was verified by the purification of Example 1 and Example 8-9. In order to make the experimental data more convincing, two samples were purified by the methods of Example 1 and Example 8-9, respectively. The electrophoresis diagram of the purified genomic DNA is shown in FIG. Figure 2 As shown. Figure 2 It can be seen that the yield of genomic DNA is not much different. Considering the cost, 5M guanidine salt concentration can be selected.

[0141] Example 10 The same sample was pretreated with the same pretreatment solution as in Example 1, and then the genomic DNA and free DNA were co-extracted with the same method as in Example 1, except that the pH of the eluent in this Example 10 was 8.

[0142] Embodiment 11 The same sample was pretreated using the same pretreatment solution as in Example 1, 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.

[0143] The optimal pH of the eluent was verified by the purification results of Example 1 and Examples 10-11. The electrophoresis diagram of the purified genomic DNA is shown in Figure 3 As shown. Figure 3 It can be seen that the best pH is 8.5.

[0144] Example 12 The same sample was pretreated using the same pretreatment solution as in Example 1, and then genomic DNA and free DNA were co-extracted using the same method as in Example 1, except that the content of SDS in the lysate in this Example 12 was 1%.

[0145] Embodiment 13 The same sample was pretreated using the same pretreatment solution as in Example 1, and then genomic DNA and free DNA were co-extracted using the same method as in Example 1, except that the SDS content in the lysate in this Example 13 was 2%.

[0146] The optimal concentration of SDS in the lysate was verified by the purification results of Example 1 and Example 12-13. In order to make the experimental data more convincing, two samples were purified by the methods of Example 1 and Example 12-13, respectively. The electrophoresis diagram of the purified genomic DNA is shown in FIG. Figure 4 As shown. Figure 4 It can be seen that the yield of genomic DNA is not much different. Considering the cost, the concentration of SDS in the lysis buffer can be selected as 0.5%.

[0147] Example 14-Example 17 The same sample was pretreated using the same pretreatment solution as in Example 2, 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 salt in the lysate in Examples 14-17 were 2M, 4M, 6M, and 8M, respectively.

[0148] The optimal concentration of guanidine salt in the lysate was verified by the purification results of Example 1 and Examples 14-17. 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, the optimal concentration of guanidine salt in the lysate is 7M.

[0149] Comparative Example 1 The same method as in Example 1 was used to extract free DNA and genomic DNA, except that the aqueous suspension of silicon hydroxyl nanoparticles in Example 1 was replaced with commercially available product Zymo, Clearing Beads, Cat No. D3061-2-1.

[0150] Comparative Example 2 The same method as in Example 1 was used to extract free DNA and genomic DNA, except that the aqueous suspension of silicon hydroxyl nanomagnetic beads in Example 1 was replaced with commercially available product Magen, MagPure Particles F, Cat No. MPF-100.

[0151] The electrophoresis diagrams of free DNA extraction of Example 1, Example 2 and Comparative Example 1 and Comparative Example 2 are as follows: Figure 7 As shown, it can be seen that there is genomic DNA contamination on the gel image of lane 3 of comparative example 1, which shows that the use of the silicon hydroxy nano magnetic beads or silica of the present invention is more conducive to achieving high-purity extraction of free DNA.

[0152] Comparative Example 3 The same method as in Example 1 was used to extract free DNA and genomic DNA, except that the lysis buffer in Example 1 was replaced with commercially available product Zymo, Genomic Lysis Buffer, Cat No. D3004-1-50.

[0153] Comparative Example 4 The same method as in Example 1 was used to extract free DNA and genomic DNA, except that the lysis solution in Example 1 was replaced with a commercially available product Norgen, Urine DNA Isolation Kit (Slurry Format), Cat No. 48800.

[0154] Comparative Example 5 The same method as in Example 1 was used to extract free DNA and genomic DNA, except that the lysis buffer in Example 1 was replaced with a commercially available Qiagen, QIAamp® DNA Micro Kit, Cat No. 56304.

[0155] Comparative Example 6 The same method as in Example 1 was used to extract free DNA and genomic DNA, except that the lysis solution in Example 1 was replaced with a commercially available product, Magen, HiPure Circulating DNA Midi Kit G, Cat No. D3182.

[0156] The electrophoresis diagrams of genomic DNA extraction of Example 1, Example 2 and Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6 are as follows: Figure 8 As shown, it can be seen that the lysis solution defined in the present invention is more conducive to the extraction of genomic DNA.

[0157] Comparative Example 7 The same method as in Example 1 was used to extract free DNA and genomic DNA, except that the samples were not pretreated with the pretreatment solution.

[0158] The electrophoresis diagrams of the sample after pretreatment in Example 1 and the sample in Comparative Example 7 are as follows: Fig. 9 As shown, from Fig. 9 It can be seen that if no pretreatment solution is added to the sample, there is no inhibitory effect on the growth of bacteria in the sample. The bacterial proliferation leads to a significant increase in the yield of genomic DNA, which will dilute the DNA of the detached cells in the sample.

[0159] Fig.10 and Fig.11 The electrophoretic diagrams of the storage conditions of genomic DNA and free DNA in the urine sample pretreated in Example 1 at different times are shown in FIG. Fig.10 , Fig.11 It can be seen that the genomic DNA and free DNA in the urine sample pretreated with the pretreatment solution of the present invention can be stored more stably.

[0160] Comparative Example 8 The same method as in Example 1 was used to extract free DNA and genomic DNA, except that no high molecular weight polyhydroxy alcohol was added to the pretreatment solution of this comparative example 8.

[0161] Comparative Example 9 The same method as in Example 1 was used to extract free DNA and genomic DNA, except that no low molecular weight polyhydroxy alcohol was added to the pretreatment solution of this comparative example 9.

[0162] Comparative Example 10 The same method as in Example 1 was used to extract free DNA and genomic DNA, except that Tween-20 was not added to the lysate of Comparative Example 10.

[0163] Comparative Example 11 The free DNA and genomic DNA were extracted by the same method as in Example 1, except that the amount of Tween-20 added to the lysate in Comparative Example 10 was increased, and the mass concentration of Tween-20 in the lysate in Comparative Example 11 was 12%.

[0164] Comparative Example 12 The same method as in Example 5 was used to extract free DNA and genomic DNA, except that the lysis solution in Example 1 was replaced with a commercially available product, Magen, HiPure Circulating DNA Midi Kit G, Cat No. D3182.

[0165] The electrophoresis diagrams of free DNA and genomic DNA extraction of Example 5, Example 6 and Comparative Example 12 are as follows: Fig.12 and Fig.13 As shown, it can be seen that the lysis solution defined in Example 5 and Example 6 is more conducive to the extraction of free DNA.

[0166] The purification conditions of the above embodiments and comparative examples were tested, and the extraction conditions are shown in Table 1. The detection methods involved are Invitrogen™ Qubit™ 4 Fluorometer, Qubit™ 1X dsDNA HS Assay Kits; ThermoScientific NanoDrop One Spectrophotometer. The specific test results are shown in Table 1. Among them, the determination method of the genomic DNA preservation stability (column 5 of Table 1) and the free DNA preservation stability (column 6 of Table 1) is: after the sample is pretreated with a pretreatment solution, it is placed at room temperature for 30 days, and then the genomic DNA and free DNA are extracted, and the yield data of the test is (comparative example 7 does not add a pretreatment solution, and the sample is directly placed at room temperature for 30 days, and then the genomic DNA and free DNA are extracted for yield data testing). The smaller the difference between the data in columns 2 and 5 in Table 1, the smaller the difference between the data in columns 4 and 6, indicating that the preservation stability is better. Among them, the data in columns 2 to 4 in Table 1 are all experimentally measured data completed within 6 hours after the sample is obtained.

[0167] Table 1 Extraction conditions in samples of embodiments and comparative examples

[0168] It can be seen from the above table data and the accompanying drawings that Examples 1-17 use the extraction method of the present invention to extract genomic DNA and free DNA, and can achieve the extraction of high-purity genomic DNA and free DNA from one sample at the same time. The pretreatment solution used has the function of preserving the detached cells and maintaining the stability of the free DNA, so that the extracted free DNA is free of genomic DNA contamination, ensuring the purity of the free DNA, and making the downstream results more accurate.

[0169] From the comparison of the experimental results of Example 1, Example 2 and Comparative Example 1, Comparative Example 2, it can be seen that the use of the silicon hydroxy nano-magnetic beads or silicon dioxide defined in the present invention is more conducive to achieving high-purity extraction of free DNA.

[0170] Comparison of the experimental results of Example 1, Example 2 and Comparative Example 3, Comparative Example 4, Comparative Example 5, and Comparative Example 6, as well as comparison of the experimental results of Example 5, Example 6 and Comparative Example 12 shows that the use of the lysis solution defined in the present invention is more conducive to the extraction of genomic DNA.

[0171] Comparison of the experimental results of Example 1 and Comparative Example 7 shows that if the sample is not pretreated with a pretreatment solution, it is difficult to achieve co-extraction of genomic DNA and free DNA, and since no pretreatment solution is 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 genomic DNA yield, and a lot of bacterial genomic DNA is extracted, which dilutes the genomic DNA of the exfoliated cells in the sample, and at the same time, the free DNA cannot be extracted, and the required genomic DNA and 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 extraction of DNA, and is more conducive to the stable storage of genomic DNA and free DNA.

[0172] Comparison of the experimental results of Example 1 and Comparative Examples 8 and 9 shows that the pretreatment solution prepared when high molecular weight polyhydroxy alcohol and low molecular weight polyhydroxy alcohol co-exist is more conducive to the co-extraction of genomic DNA and free DNA.

[0173] From the comparison of the experimental results of Example 1 and Comparative Examples 10 and 11, it can be seen that the concentration of Tween-20 in the lysate is within the range defined by the present invention, which is more conducive to the extraction of genomic DNA.

[0174] In summary, the extraction method of the present invention has the following advantages: (1) Save time and samples: By extracting genomic DNA and free DNA simultaneously, two nucleic acids can be obtained in one sample and 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.

[0175] (2) The stability of the exfoliated cells and free DNA in the sample is maintained, which is also the prerequisite for ensuring the purity of the free DNA: the components of the sample pretreatment solution included in this technology can ensure that the exfoliated cells do not undergo apoptosis or lysis within 7 days and the free DNA does not degrade within 30 days when the exfoliated cells and free DNA coexist.

[0176] (3) There is no genomic DNA contamination in the extracted free DNA: With the help of sample pretreatment solution, when extracting free DNA, the genomic DNA contamination can be completely removed by centrifugation alone, thus ensuring the purity of the free DNA.

[0177] The pretreatment solution can not only increase the solubility of some impurities in the sample, which is beneficial for the subsequent DNA extraction, but also preserve the sample, which is beneficial for transportation. The preservation of the sample mainly includes preserving the detached cells and maintaining the stability of free DNA.

[0178] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are exhaustively listed. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0179] For those skilled in the art, several modifications and improvements may be made without departing from the concept of the present invention, all of which belong to the protection scope of the present invention. The protection scope of the present invention shall be based on the attached claims.

Claims

1. A pretreatment solution for co-extraction of genomic DNA and free DNA, characterized in that: The pretreatment solution comprises: polyhydric alcohol, chelating agent, preservative, pH buffer and water; the pH of the pretreatment solution is 7-9; The polyhydric alcohols include high molecular weight polyhydric alcohols and low molecular weight polyhydric alcohols; The high molecular weight polyhydroxy 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 polyhydroxy 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 less than 1000.

2. A pretreatment solution for co-extraction of genomic DNA and free DNA according to claim 1, characterized in that: In the pretreatment liquid, the mass proportion of the high molecular weight polyhydric alcohol is 1%-10%; the mass proportion of the low molecular weight polyhydric alcohol is 10%-65%; the mass proportion of the preservative is 0.5%-5%; and the concentration of the chelating agent in the pretreatment liquid is 10g / L-50g / L.

3. A pretreatment solution for co-extraction of genomic DNA and free DNA according to claim 1, characterized in that: The chelating agent is at least one of EDTA-2Na, EDTA-3K, and EDTA-2K; The preservative is at least one of proclin 310, proclin 950, and sodium azide; The pH buffer is a Tris buffer or a citric acid buffer.

4. A method for co-extraction of genomic DNA and free DNA, characterized in that: The extraction method is: Adding the pretreatment solution of any one of claims 1 to 3 to the sample, and then centrifuging to obtain a sample precipitate and a sample supernatant; the sample precipitate is used for extracting genomic DNA, and the sample supernatant is used for extracting free DNA; The extraction process of the genomic DNA is as follows: (1) Lysis: Add lysis buffer and protease to the sample precipitate for incubation; (2) Binding: Add binding solution, mix thoroughly by pipetting with a pipette, then add to the DNA binding column, centrifuge and discard the solution; (3) Rinsing: Add rinsing solution I, centrifuge and discard the solution; Then add rinse solution II, centrifuge and discard the solution; (4) Elution: Place the binding column in a centrifuge tube, add elution solution to the binding column, centrifuge, and collect the elution solution after centrifugation, which is the genomic DNA; The extraction process of the free DNA is as follows: (1) Binding: Add an aqueous suspension of silanol nanoparticles or an aqueous suspension of silica to the sample supernatant, mix by inverting, and then centrifuge to discard the supernatant; (2) Rinsing: Add rinsing solution I to the precipitate, transfer to the DNA binding column, and centrifuge to remove the lower liquid; Then add rinse solution II, centrifuge and discard the solution; (3) Elution: Place the binding column in a centrifuge tube, add elution solution to the binding column, and centrifuge. The elution solution collected after centrifugation is the free DNA.

5. A method for co-extracting genomic DNA and free DNA according to claim 4, characterized in that: The lysate is a guanidine lysate or an SDS lysate; The guanidine salt lysis solution comprises guanidine salt, TritonX-100, Tris-HCl, Tween-20, EDTA salt and water; the pH of the guanidine salt lysis solution is 8-9; The SDS lysate comprises SDS, phosphate, Tris-HCl, EDTA salt and water; the pH of the SDS lysate is 6-7.

6. A method for co-extraction of genomic DNA and free DNA according to claim 5, characterized in that: 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; In the SDS lysate, the phosphate is sodium phosphate, and the concentration of the sodium phosphate in the SDS lysate is 0.1M-0.5M; the mass concentration of SDS in the SDS lysate is 0.5%-2%; the concentration of EDTA salt in the SDS lysate is 10mM-50Mm; and the concentration of Tris-HCl in the SDS lysate is 20mM-50mM.

7. A method for co-extracting genomic DNA and free DNA according to claim 4, characterized in that: The binding solution includes guanidine salt, isopropanol and water; 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 the isopropanol in the binding solution is 60%-70%.

8. A method for co-extracting genomic DNA and free DNA according to claim 4, characterized in that: The rinsing solution I is an aqueous solution of guanidine salt, or the rinsing solution I includes guanidine salt, ethanol and water; The guanidine salt is guanidine hydrochloride or guanidine isothiocyanate; The concentration of the guanidine salt in the rinse solution I is 4M-5M; the mass concentration of the ethanol in the rinse solution I is 40%-80%; The rinsing solution II includes Tris-HCl, Tris, ethanol and water; the concentration of Tris-HCl in the rinsing solution II is 30-60mM, the concentration of Tris in the rinsing solution II is 0.5M-1M, and the mass concentration of ethanol in the rinsing solution II is 70%-90%; the pH value of the rinsing solution II is 7-8.

9. A method for co-extracting genomic DNA and free DNA according to claim 4, characterized in that: The eluent is at least one of a Tris-HCl aqueous solution, a Tris aqueous solution and an EDTA aqueous solution; the pH of the eluent is 8-9.

10. A method for co-extraction of genomic DNA and free DNA according to claim 4, characterized in that: The inner core of the silicon hydroxyl nano magnetic beads in the aqueous suspension 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; In the aqueous suspension of silicon dioxide, the particle size of silicon dioxide is 50-400 nm.

Images