Plasma free DNA extraction method capable of removing genome contamination based on paramagnetic particle method

Abstract

The invention discloses a plasma free DNA extraction method capable of removing genome contamination based on a paramagnetic particle method, and relates to the technical field of free DNA detection.The plasma free DNA extraction method comprises the following specific steps that firstly, a sample is subjected to one-step splitting combination; step 2, cleaning 1: adding a cleaning solution 1 into the centrifugal tube, carrying out vortex oscillation, placing the centrifugal tube on a magnetic frame, standing until magnetic beads are completely adsorbed, and thoroughly removing supernatant; step 3, cleaning 2: adding a cleaning solution 2 into the centrifugal tube, carrying out vortex oscillation and standing until the magnetic beads are completely adsorbed and the supernatant is thoroughly removed, and repeating the step for one time; step 4, elution: adding an eluent, uniformly mixing by vortex and oscillating, placing the centrifuge tube on a magnetic frame for standing, and transferring the supernatant into a new centrifuge tube after the magnetic beads are completely adsorbed; and step 5, genome DNA removal. The plasma free DNA extraction method is easy and convenient to operate, meanwhile, gDNA pollution in the plasma free DNA can be removed, high-purity free DNA is provided for downstream detection, and a paramagnetic particle method is combined for extraction.

Description

Technical Field

[0001] The invention relates to the technical field of free DNA detection, and in particular to a plasma free DNA extraction method capable of removing genome contamination based on a magnetic bead method. Background Art

[0002] Free DNA in blood (cfDNA) is circulating deoxyribonucleotides (DNA), which are partially degraded endogenous DNA in the body that is free from cells in the circulating blood. In healthy individuals, cfDNA mainly comes from bone marrow and lymphocytes, but in certain disease processes or physiological conditions, there may be sources from one or more other tissues. Plasma free DNA usually exists in short fragments of less than 1000 bp, and its fragment length is distributed at 167 bp (the peak corresponding to "nucleosome + connecting histone"), with a periodic fluctuation of 10.4 bp in the range of 100-160 bp. cfDNA is a potential biomarker for certain cancers and disease states, as well as fetal DNA in maternal blood.

[0003] The content of free DNA (cfDNA) in plasma is relatively low, accounting for only a small part of the total DNA, which requires highly sensitive techniques and methods to capture these trace DNA molecules during the extraction process. Currently, there are two main methods for extracting cfDNA: centrifugal column method and magnetic bead method, but in practical applications, there are problems such as low extraction efficiency, genomic contamination, and overly complicated operations. The contamination of genomic DNA may reduce the sensitivity of mutation detection in free DNA, because the increase in background noise makes it more difficult to identify the true mutation signal.

[0004] Among them, the centrifugal column method is more complicated to operate and requires multiple centrifugation steps. At the same time, the reagents include RNA sedimentation aids, which affects the DNA quantification and is not conducive to downstream experiments. The magnetic bead method is an extraction method that has rapidly emerged in recent years. It is simpler to operate than the column method and can be automated. However, at present, most of the sample processing methods in the magnetic bead method extraction of plasma free DNA are mostly lysis first and then binding, which requires two steps to achieve DNA binding and cannot achieve true automated extraction. At the same time, the existing methods basically do not mention how to remove genomic DNA contamination in free DNA. For this reason, we propose a plasma free DNA extraction method based on the magnetic bead method that can remove genomic contamination. Summary of the invention

[0005] 1. Technical issues to be solved

[0006] In view of the deficiencies of the prior art, the present invention provides a method for extracting plasma free DNA based on a magnetic bead method that can remove genomic contamination, thereby solving the technical problem of how to remove genomic DNA contamination in free DNA.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] A method for extracting plasma free DNA that can remove genomic contamination based on a magnetic bead method, the technical solution of the present invention is realized by the following aspects:

[0010] Step 1: One-step sample lysis and binding: Take 2 mL of plasma sample and place it in a 10 mL centrifuge tube. Add 100-500 μL of proteinase K solution, 20-60 μL of magnetic bead suspension 1 and 2-5 mL of lysis and binding solution. Vortex thoroughly to mix, incubate at room temperature for 20 min, perform magnetic separation after incubation, and completely remove the supernatant. Avoid contact with magnetic beads during this period.

[0011] Step 2, Wash 1: Add 1-2 mL of Wash Solution 1 to the centrifuge tube, vortex for 1 min, then transfer all the resuspended magnetic bead solution to a 2 mL centrifuge tube, place it on the magnetic rack, let it stand until the magnetic beads are completely adsorbed, and completely remove the supernatant.

[0012] Step 3, Wash 2: Add 0.5-1 mL of Wash Solution 2 to the centrifuge tube, vortex for 1 min, place the centrifuge tube on a magnetic rack, let it stand until the magnetic beads are completely adsorbed, and completely remove the supernatant. Repeat this step once.

[0013] Step 4. Elution: Add 20-100 μL of elution buffer, vortex and shake for 5 minutes, place the centrifuge tube on the magnetic rack and let it stand for 1 minute. After the magnetic beads are completely adsorbed, transfer the supernatant to a new centrifuge tube to obtain free DNA.

[0014] Step 5: Genomic DNA Removal

[0015] Preferably, the specific steps of step five are as follows: ① add a certain proportion of magnetic bead suspension 2 to the above-mentioned free DNA eluate, vortex thoroughly to mix, incubate at room temperature for 5 minutes, and then perform magnetic separation, and transfer all the supernatant to a clean 1.5mL centrifuge tube.

[0016] ②Add a certain proportion of magnetic bead suspension 2 to the supernatant, vortex thoroughly to mix, and incubate at room temperature for 5 minutes. Then perform magnetic separation and completely remove the supernatant, avoiding contact with the magnetic beads.

[0017] ③Keep the centrifuge tube on the magnetic stand and add 80% ethanol to wash the magnetic beads twice.

[0018] ④ Place the centrifuge tube on the magnetic rack, place it in a fume hood, and air-dry for 2 minutes.

[0019] ⑤ Add 20-60 μL of elution buffer, vortex and mix for 5 minutes, place the centrifuge tube on the magnetic rack and let it stand for 1 minute. After the magnetic beads are completely adsorbed, transfer the supernatant to a new centrifuge tube to obtain free DNA without genomic DNA contamination.

[0020] This gDNA removal method is not only applicable to the cfDNA extracted by this patent, but also to the removal of gDNA from cfDNA extracted by other methods.

[0021] Preferably, the lysis and binding solution is 50-500mmol / L sodium chloride, 5-20mmol / L disodium ethylenediaminetetraacetic acid, 2-5mol / L guanidine thiocyanate, 0.5-2% sodium dodecyl sulfate, 1-10% polyethylene glycol p-isooctylphenyl ether, 1-10% polyoxyethylene ether, 10-100mmol / L Tris-Hcl (pH 8.0), and 20-50% isopropanol. Guanidine thiocyanate is a denaturant that can interfere with the hydrogen bonds and intramolecular interactions of macromolecules, dissolve proteins, and eliminate the secondary structure of proteins, resulting in degradation of cell structures and rapid separation of nucleoproteins from nucleic acids.

[0022] Preferably, sodium dodecyl sulfate is an anionic detergent that can destroy the electrostatic attraction or coordination bond between proteins and nucleic acids in cells, causing chromosome segregation, protein denaturation, and nucleic acid release.

[0023] Preferably, polyethylene glycol 4-isooctylphenyl ether is a non-ionic surfactant, which can act synergistically with sodium dodecyl sulfate to improve the ability to lyse the surface membrane structure of cells or enveloped viruses and promote the release of nucleic acids. Polyoxyethylene ether, as a non-ionic surfactant, can destroy the lipid bilayer, dissolve and destroy most of the proteins adsorbed and bound between molecules, and help release nucleic acids.

[0024] Preferably, the proteinase K solution is 20-50 mg / mL proteinase K. Proteinase K is a strong proteinase that dissolves proteins and mainly acts to hydrolyze histones bound to nucleic acids, thereby freeing DNA in the solution.

[0025] Preferably, the magnetic bead suspension 1 is a superparamagnetic silicon hydroxyl nanomagnetic microsphere with a particle size of 50-500nm and a concentration of 50-200mg / mL. The surface of the silicon hydroxyl magnetic beads is modified with a large number of silicon hydroxyl groups, which can specifically bind to nucleic acids in the solution under high salt and low pH conditions through hydrophobic effects, hydrogen bonding effects and electrostatic effects, but not bind to other impurities (such as proteins), and can quickly separate nucleic acids from biological samples.

[0026] Preferably, the cleaning solution 1 is 2-5 mol / L guanidine thiocyanate, 1-10% polyethylene glycol p-isooctylphenyl ether, 1-10% polyoxyethylene ether, 10-100 mmol / L Tris-Hcl (pH 8.0), and 20-50% isopropanol. Guanidine thiocyanate, polyethylene glycol p-isooctylphenyl ether, and polyoxyethylene ether are used as protein denaturants to further clean impurities such as proteins adhering to the magnetic beads.

[0027] Preferably, the cleaning solution 2 is 5-50mmol / L Tris-Hcl, pH 8.0, 80% ethanol. DNA is insoluble in 80% ethanol, but salt ions are soluble, so the residual salt impurities can be effectively removed.

[0028] Preferably, the eluent is 5-10 mmol / L Tris-Hcl, pH 8.0.

[0029] Preferably, the magnetic bead suspension 2 is 1-3 mol / L sodium chloride, 5-50% polyethylene glycol 8000, and 1-5% superparamagnetic carboxyl magnetic microspheres. Polyethylene glycol and salt can cause the DNA molecules to change from a curled conformation to a spherical conformation. First, a relatively low concentration of polyethylene glycol is selected to promote the carboxyl magnetic beads to adsorb large fragments of DNA, and the large fragment contamination is removed by discarding the magnetic beads. Then, a high concentration of polyethylene glycol is used to allow the magnetic beads to adsorb the remaining small fragments of DNA, thereby achieving the separation of large and small fragments of DNA.

[0030] Preferably, the preferred magnetic bead suspension 2 is 2-2.5 mol / L sodium chloride, 25-30% polyethylene glycol 8000, and 1.5-2% superparamagnetic carboxyl magnetic microspheres.

[0031] (III) Beneficial effects

[0032] A simple method for extracting plasma free DNA has been invented, which can remove gDNA contamination and provide a high-purity free DNA for downstream testing. The extraction is combined with the magnetic bead method to realize an automated process. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings.

[0034] Figure 1 This is an overall effect diagram of a plasma free DNA extraction method based on a magnetic bead method capable of removing genomic contamination in Example 1 of the present invention;

[0035] Figure 2This is a detection diagram of cfDNA Agilent 2100 sample 1 extracted by two methods in Example 2 of the present invention;

[0036] Figure 3 This is a detection diagram of cfDNA Agilent 2100 sample 2 extracted by two methods in Example 2 of the present invention;

[0037] Figure 4 This is a detection diagram of cfDNA Agilent 2100 sample 3 extracted by two methods in Example 2 of the present invention. DETAILED DESCRIPTION

[0038] The embodiments of the present application provide a method for extracting free DNA that is easy to operate, thereby solving the technical problem of how to remove genomic DNA contamination in free DNA in the prior art. The method for rapid separation and purification of DNA based on the magnetic bead method has the advantage of being automated and easy to operate. At the same time, the high-purity free DNA obtained has important guiding significance for downstream liquid biopsy methods such as cancer diagnosis, medication guidance, monitoring and prognosis evaluation.

[0039] Example 1

[0040] The technical solution in the embodiment of the present application is to solve the technical problem of how to remove genomic DNA contamination in free DNA. The overall idea is as follows:

[0041] In view of the shortcomings of the current extraction methods, the purpose of the present invention is to provide a method for extracting free DNA that is easy to operate, and also includes a method for removing gDNA contamination in free DNA;

[0042] The technical solution of the present invention is achieved by the following aspects:

[0043] The specific steps for routine plasma free DNA extraction are as follows:

[0044] Step 1: One-step lysis and binding of samples: Take 2 mL of plasma sample and place it in a 10 mL centrifuge tube, add 200 μL of proteinase K solution, 20 μL of magnetic bead suspension 1 and 3 mL of lysis and binding solution, vortex thoroughly to mix, incubate at room temperature for 20 min, perform magnetic separation after the incubation, and completely remove the supernatant, avoiding contact with magnetic beads during the process;

[0045] Step 2, Wash 1: Add 1 mL of Wash Solution 1 to the centrifuge tube, vortex for 1 min, then transfer all the resuspended magnetic bead solution to a 2 mL centrifuge tube, place it on a magnetic rack, let it stand until the magnetic beads are completely adsorbed, and completely remove the supernatant;

[0046] Step 3, washing 2: add 1 mL of washing solution 2 to the centrifuge tube, vortex and oscillate for 1 min, place the centrifuge tube on a magnetic rack, let it stand until the magnetic beads are completely adsorbed, and completely remove the supernatant. Repeat this washing step once;

[0047] Step 4: Elution: Add 40 μL of elution buffer, vortex and mix for 5 minutes, place the centrifuge tube on the magnetic rack and let it stand for 1 minute. After the magnetic beads are completely adsorbed, transfer the supernatant to a new centrifuge tube to obtain free DNA.

[0048] The steps for removing genomic gDNA contamination are as follows:

[0049] ① Take 30 μL of the above free DNA elution solution, add 9 μL of magnetic bead suspension 2, vortex thoroughly to mix, incubate at room temperature for 5 minutes, then perform magnetic separation, and transfer all the supernatant to a clean 1.5 mL centrifuge tube;

[0050] ②Add 75 μL of magnetic bead suspension 2 to the supernatant, vortex thoroughly to mix, and incubate at room temperature for 5 minutes. Then perform magnetic separation and completely remove the supernatant, avoiding contact with the magnetic beads;

[0051] ③ Keep the centrifuge tube on the magnetic stand and add 80% ethanol to wash the magnetic beads twice;

[0052] ④ Place the centrifuge tube on a magnetic rack in a fume hood and air-dry for 2 minutes;

[0053] ⑤ Add 30 μL of elution buffer, vortex and mix for 5 minutes, place the centrifuge tube on the magnetic rack and let it stand for 1 minute. After the magnetic beads are completely adsorbed, transfer the supernatant to a new centrifuge tube to obtain free DNA without genomic DNA contamination.

[0054] like Figure 1 As shown in the figure (Note: the blue line represents the free DNA sample before gDNA removal, and the red line represents the free DNA sample after gDNA removal). After the extraction, the difference before and after gDNA removal in the eluate was measured using the Agilent 2100 Bioanalyzer (the results are shown in Figure 1 ). According to the results in the figure below, it can be seen from the figure that the gDNA contamination in the plasma free DNA extracted by the present invention can be basically completely removed to obtain high-purity cfDNA.

[0055] Example 2

[0056] Comparison of the extraction effect of the present invention with that of the existing magnetic bead-based free DNA extraction kit

[0057] Three different plasma samples were selected, and two 2 mL portions of each sample were taken, and extracted using the present method and an existing magnetic bead-based free DNA extraction kit (manufacturer C), respectively. The present method was performed with reference to Example 1, and the existing kit extraction method was performed with reference to the instructions of the purchased kit.

[0058] After extraction, the free DNA extracted by the above two methods was measured by a fluorescence photometer (Qubit 4.0) for the content of cfDNA in the eluate (the results are shown in Table 1), and the cfDNA extracted by the above two methods was analyzed by Agilent 2100 Bioanalyzer for fragment size analysis (the results are shown in Figure 2-4 ).

[0059]

[0060]

[0061] Table 1 Qubit assay for cfDNA content in the eluate of Example 2

[0062] comprehensive Figure 2-4 As shown (Note: the blue line represents cfDNA extracted by the magnetic bead free DNA extraction kit of manufacturer C, and the red line represents cfDNA extracted by the patent method), according to the following results, it can be seen that the amount of cfDNA extracted by the present invention is not much different from that of the existing magnetic bead kit; however, through the analysis of the Agilent 2100 results, it was found that the residual gDNA in the cfDNA extracted by the present invention was significantly lower than that of the existing magnetic bead kit, indicating that the present invention can effectively remove gDNA contamination during cfDNA extraction, and provide an efficient and reliable extraction method for subsequent liquid biopsy research. This gDNA removal method is not only applicable to cfDNA extracted by this patent, but also to the removal of gDNA in cfDNA extracted by other methods.

[0063] The lysis and binding solution is 50-500mmol / L sodium chloride, 5-20mmol / L disodium ethylenediaminetetraacetate, 2-5mol / L guanidine thiocyanate, 0.5-2% sodium dodecyl sulfate, 1-10% polyethylene glycol p-isooctylphenyl ether, 1-10% polyoxyethylene ether, 10-100mmol / L Tris-Hcl (pH 8.0), and 20-50% isopropanol. Guanidine thiocyanate is a denaturant that can interfere with the hydrogen bonds and intramolecular interactions of macromolecules, dissolve proteins, and make the secondary structure of proteins disappear, resulting in degradation of cell structures and rapid separation of nucleoproteins from nucleic acids.

[0064] Sodium dodecyl sulfate is an anionic detergent that can destroy the electrostatic attraction or coordination bond between proteins and nucleic acids in cells, causing chromosome segregation, protein denaturation, and release of nucleic acids.

[0065] Polyethylene glycol 4-isooctylphenyl ether is a non-ionic surfactant that can work synergistically with sodium dodecyl sulfate to improve the ability to lyse the surface membrane structure of cells or enveloped viruses and promote the release of nucleic acids.

[0066] As a non-ionic surfactant, polyoxyethylene ether can destroy the lipid bilayer, dissolve and destroy most of the proteins adsorbed and bound between molecules, and help release nucleic acids.

[0067] The proteinase K solution is 20-50 mg / mL proteinase K. Proteinase K is a strong proteinase that dissolves proteins bound to nucleic acids, making DNA free in the solution.

[0068] Magnetic bead suspension 1 is superparamagnetic silicon hydroxy nano-magnetic microspheres with a particle size of 50-500nm and a concentration of 50-200mg / mL. The surface of silicon hydroxy magnetic beads is modified with a large number of silicon hydroxy groups, which can specifically bind to nucleic acids in solution under high salt and low pH conditions through hydrophobic effects, hydrogen bonding effects, and electrostatic effects, but not bind to other impurities (such as proteins), and can quickly separate nucleic acids from biological samples.

[0069] The cleaning solution 1 is 2-5 mol / L guanidine thiocyanate, 1-10% polyethylene glycol p-isooctylphenyl ether, 1-10% polyoxyethylene ether, 10-100 mmol / L Tris-Hcl (pH 8.0), and 20-50% isopropanol. Guanidine thiocyanate, polyethylene glycol p-isooctylphenyl ether, and polyoxyethylene ether are used as protein denaturants to further clean impurities such as proteins adhering to the magnetic beads.

[0070] Cleaning solution 2 is 5-50mmol / L Tris-Hcl, pH 8.0, 80% ethanol. DNA is insoluble in 80% ethanol, but salt ions are soluble, so residual salt impurities can be effectively removed.

[0071] The eluent was 5-10 mmol / L Tris-Hcl, pH 8.0.

[0072] Magnetic bead suspension 2 is 1-3 mol / L sodium chloride, 5-50% polyethylene glycol 8000, and 1-5% superparamagnetic carboxyl magnetic microspheres. Polyethylene glycol and salt can cause DNA molecules to change from a curled conformation to a spherical conformation. First, a lower concentration of polyethylene glycol is used to promote the carboxyl magnetic beads to adsorb large fragments of DNA, and the large fragment contamination is removed by discarding the magnetic beads. Then, a high concentration of polyethylene glycol is used to allow the magnetic beads to adsorb the remaining small fragments of DNA, thereby achieving the separation of large and small fragments of DNA.

[0073] The preferred magnetic bead suspension 2 is 2-2.5 mol / L sodium chloride, 25-30% polyethylene glycol 8000, and 1.5-2% superparamagnetic carboxyl magnetic microspheres.

[0074] The technical problem to be solved by the present invention is that no method for extracting free DNA without genomic contamination has been reported so far. The present method is a method for rapid separation and purification of DNA based on the magnetic bead method, which has the advantages of being automated and easy to operate; the high-purity free DNA obtained at the same time has important guiding significance for downstream liquid biopsy methods such as cancer diagnosis, medication guidance, monitoring and prognosis evaluation.

[0075] Finally, it should be noted that: Obviously, the above embodiments are only examples for clearly explaining the present invention, and are not intended to limit the implementation methods. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from this are still within the scope of protection of the present invention.

Claims

1. A method for extracting plasma free DNA based on magnetic beads to remove genomic contamination, characterized in that: The specific steps are as follows Step 1: One-step lysis and binding of samples: Take 2 mL of plasma sample in a 10 mL centrifuge tube, add 100-500 μL of proteinase K solution, 20-60 μL of magnetic bead suspension 1 and 2-5 mL of lysis and binding solution, vortex thoroughly to mix, incubate at room temperature for 20 min, perform magnetic separation after the incubation, and completely remove the supernatant, avoiding contact with magnetic beads during the process; Step 2, Wash 1: Add 1-2 mL of Wash Solution 1 to the centrifuge tube, vortex for 1 min, then transfer all the resuspended magnetic bead solution to a 2 mL centrifuge tube, place it on a magnetic rack, let it stand until the magnetic beads are completely adsorbed, and completely remove the supernatant; Step 3, washing 2: add 0.5-1mL washing solution 2 to the centrifuge tube, vortex and oscillate for 1min, place the centrifuge tube on the magnetic rack, let it stand until the magnetic beads are completely adsorbed, and completely remove the supernatant. Repeat this washing step once; Step 4: Elution: Add 20-100 μL of elution buffer, vortex and mix for 5 minutes, place the centrifuge tube on the magnetic rack and let it stand for 1 minute. After the magnetic beads are completely adsorbed, transfer the supernatant to a new centrifuge tube to obtain free DNA. Step 5: Removal of genomic DNA.

2. A method for extracting plasma free DNA based on a magnetic bead method for removing genomic contamination as claimed in claim 1, characterized in that: The specific steps of step five are as follows ① Add a certain proportion of magnetic bead suspension 2 to the above free DNA eluate, vortex thoroughly to mix, incubate at room temperature for 5 minutes, then perform magnetic separation, and transfer all the supernatant to a clean 1.5 mL centrifuge tube; ② Add a certain proportion of magnetic bead suspension 2 to the supernatant, vortex thoroughly to mix, incubate at room temperature for 5 minutes, and then perform magnetic separation to completely remove the supernatant, avoiding contact with the magnetic beads during the process; ③ Keep the centrifuge tube on the magnetic stand and add 80% ethanol to wash the magnetic beads twice; ④ Place the centrifuge tube on a magnetic rack in a fume hood and air-dry for 2 minutes; ⑤ Add 20-60 μL of elution buffer, vortex and mix for 5 minutes, place the centrifuge tube on the magnetic rack and let it stand for 1 minute. After the magnetic beads are completely adsorbed, transfer the supernatant to a new centrifuge tube to obtain free DNA without genomic DNA contamination.

3. The method for extracting plasma free DNA based on magnetic beads and capable of removing genomic contamination as claimed in claim 1, characterized in that: The lysis and binding solution is 50-500mmol / L sodium chloride, 5-20mmol / L disodium ethylenediaminetetraacetate, 2-5mol / L guanidine thiocyanate, 0.5-2% sodium dodecyl sulfate, 1-10% polyethylene glycol p-isooctylphenyl ether, 1-10% polyoxyethylene ether, 10-100mmol / L Tris-Hcl (pH 8.0), and 20-50% isopropanol.

4. The method for extracting plasma free DNA based on magnetic beads and capable of removing genomic contamination as claimed in claim 1, characterized in that: The proteinase K solution contains 20-50 mg / mL proteinase K.

5. The method for extracting plasma free DNA based on magnetic beads and capable of removing genomic contamination as claimed in claim 1, characterized in that: The magnetic bead suspension 1 is superparamagnetic silicon hydroxy nano magnetic microspheres with a particle size of 50-500nm and a concentration of 50-200mg / mL.

6. The method for extracting plasma free DNA based on magnetic beads and capable of removing genomic contamination as claimed in claim 1, characterized in that: The cleaning solution 1 is 2-5 mol / L guanidine thiocyanate, 1-10% polyethylene glycol p-isooctylphenyl ether, 1-10% polyoxyethylene ether, 10-100 mmol / L Tris-Hcl (pH 8.0), and 20-50% isopropanol.

7. The method for extracting plasma free DNA based on magnetic beads and capable of removing genomic contamination as claimed in claim 1, characterized in that: The cleaning solution 2 is 5-50mmol / L Tris-Hcl, pH 8.0, 80% ethanol. DNA is insoluble in 80% ethanol, but salt ions are soluble, so the residual salt impurities can be effectively removed.

8. The method for extracting plasma free DNA based on magnetic beads and capable of removing genomic contamination as claimed in claim 1, characterized in that: The eluent is 5-10 mmol / L Tris-Hcl, pH 8.

0.

9. The method for extracting plasma free DNA based on magnetic beads and capable of removing genomic contamination as claimed in claim 2, characterized in that: The magnetic bead suspension 2 is 1-3 mol / L sodium chloride, 5-50% polyethylene glycol 8000, and 1-5% superparamagnetic carboxyl magnetic microspheres. Polyethylene glycol and salt can cause the DNA molecules to change from a curled conformation to a spherical conformation. First, a relatively low concentration of polyethylene glycol is selected to promote the carboxyl magnetic beads to adsorb large fragments of DNA, and the large fragment contamination is removed by discarding the magnetic beads. Then, a high concentration of polyethylene glycol is used to allow the magnetic beads to adsorb the remaining small fragments of DNA.

10. The method for extracting plasma free DNA based on magnetic beads and capable of removing genomic contamination as claimed in claim 2, characterized in that: The magnetic bead suspension 2 is 2-2.5 mol / L sodium chloride, 25-30% polyethylene glycol 8000, and 1.5-2% superparamagnetic carboxyl magnetic microspheres.

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