Rapid free DNA enrichment method based on paramagnetic particle method

Abstract

The invention discloses a rapid free DNA enrichment method based on a paramagnetic particle method, which comprises the following steps: (1) taking plasma, adding a protease K solution, fully and uniformly mixing, and incubating; (2) adding the magnetic beads and the binding liquid, oscillating, instantaneously centrifuging and standing until the magnetic beads are completely adhered to the wall; (3) absorbing and abandoning all supernatant; (4) adding an eluent, oscillating, instantaneously centrifuging, continuously placing on the magnetic frame, and standing for 5 minutes until the magnetic beads are completely adhered to the wall; and (5) taking the supernatant, and transferring the supernatant into a sample storage tube for enrichment. According to the method, the DNA purity is high, a QSep result shows that no impurity peak interference exists, the feasibility of the method is proved, and the corresponding magnetic beads carrying positive charges can be used for extraction of the method, so that the method has relatively high practicability. A high-quality template can be provided for downstream molecule detection and analysis (including NGS sequencing, digital PCR and the like), and particularly, important values are shown in clinical application scenes such as liquid biopsy and antenatal diagnosis.

Description

Technical Field

[0001] The present invention relates to a DNA enrichment method, and in particular to a rapid free DNA enrichment method based on a magnetic bead method and an application thereof. Background Art

[0002] The development of body fluid DNA enrichment technology is the result of medical needs, technological breakthroughs and the expansion of application scenarios. From the early crude extraction methods to today's targeted and automated technologies, its core goal has always been to improve sensitivity and increase operational convenience to meet the urgent needs of precision medicine and instant diagnosis. Current body fluid DNA enrichment methods are mainly based on the separation and purification of DNA based on physical, chemical or biological properties. Common methods may include centrifugation, magnetic bead method, column membrane method, chemical precipitation method, and some emerging technologies such as microfluidics. At present, the magnetic bead method is a commonly used method for enriching DNA on the market. It is widely used in the market due to its low toxicity, simple operation, and suitability for high-throughput automated extraction. The silicon-based or functionalized groups (such as carboxyl groups) coated on the surface of the magnetic beads can specifically adsorb DNA and reduce contamination by proteins, RNA or other impurities. However, the common magnetic beads on the market, such as carboxyl magnetic beads and silicon-based magnetic beads, form hydrogen bonds through the groups coated with the magnetic beads, thereby adsorbing DNA and achieving the purpose of enriching DNA. However, this method has a low adsorption efficiency for short-fragment DNA (<200bp) and is prone to cause the loss of short-fragment DNA (<200bp). Therefore, this type of magnetic beads has a low yield when used to enrich free DNA (cfDNA), which is easy to affect downstream detection. Summary of the Invention

[0003] Purpose of the invention: The purpose of the present invention is to provide a rapid free DNA enrichment method based on magnetic beads.

[0004] This method is used to quickly enrich free DNA in large volumes of body fluids. Magnetic beads coated with positive charges can be used to adsorb negatively charged DNA, and DNA enrichment can be performed quickly and efficiently through electrostatic adsorption. The following will clearly and completely describe the concept of the present invention and the technical effects produced in conjunction with the examples to fully understand the purpose, characteristics and effects of the present invention.

[0005] Technical solution: The rapid free DNA enrichment method based on the magnetic bead method of the present invention comprises the following steps: (1) Take plasma, add proteinase K, mix thoroughly, and incubate; (2) Add magnetic beads and binding solution, shake, centrifuge briefly, and let stand until the magnetic beads are completely attached to the wall; (3) Discard all supernatant; (4) Add elution buffer, shake, centrifuge briefly, continue to place on the magnetic stand, and let it stand for five minutes until the magnetic beads are completely attached to the wall; (5) Take the supernatant and transfer it to a sample storage tube for enrichment.

[0006] Furthermore, the plasma is human plasma, and the dosage is 1-10 mL, preferably 4 mL.

[0007] Furthermore, the concentration of the proteinase K solution is 10-40 mg / mL, and the dosage is 150-200 μl, preferably: the concentration is 20 mg / mL, and the dosage is 200 μl.

[0008] Furthermore, the magnetic beads are ferroferric oxide magnetic microbeads with positively charged surfaces coated with amino groups.

[0009] Furthermore, the binding solution is 15-150mM Tris-HCl and 20%-30% PEG400, preferably 20mM Tris-HCl and 25% PEG400.

[0010] Furthermore, the eluent is 18-22 mM Tris-HCl, 0.8-1 M NaCl, 4-6 mM EDTA-2Na, preferably 20 mM Tris-HCl, 1 M NaCl, 5 mM EDTA-2Na.

[0011] The reagents involved in the method are proteinase K, amino magnetic beads, a binding solution, and an eluent. The proteinase K solution contains proteinase K, Tris-HCl, and CaCl2; the magnetic bead solution contains ferroferric oxide magnetic microbeads coated with amino groups and deionized water.

[0012] This method can rapidly and efficiently adsorb large amounts of free DNA in body fluids by coating magnetic beads with specific positively charged groups. Through electrostatic adsorption, the magnetic beads carrying positively charged amino groups specifically bind to the negatively charged phosphate groups of free DNA. This method improves the disadvantage of common magnetic beads on the market that preferentially bind to long fragments. It has the advantages of high extraction efficiency, high nucleic acid purity, strong reproducibility, and low loss of short DNA fragments. In addition, the extraction method is simple and easy to operate. The free DNA obtained is of stable and reliable quality and can be directly used for PCR, qPCR, ddPCR, and second-generation sequencing library construction.

[0013] The following are the preferred extraction steps: Take three clean 15 ml centrifuge tubes, add 4 ml of plasma, add 200 μl of proteinase K, vortex mix thoroughly, and incubate at 60°C for 30 min; add 20 μl of the above-mentioned magnetic beads and 5 ml of binding buffer, vortex for ten minutes, centrifuge briefly, place on a magnetic rack and let it stand for five minutes until the magnetic beads are completely attached to the wall; use a pipette to discard all the supernatant; add 60 μl of the above-mentioned eluate, vortex for ten minutes, centrifuge briefly, continue to place on a magnetic rack, and let it stand for five minutes until the magnetic beads are completely attached to the wall; take the above supernatant and transfer them to clean sample storage tubes respectively; take the supernatant DNA liquid for Qsep detection to test its fragment length and purity.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The improved method established in this study achieves multiple optimization breakthroughs in cell-free DNA (cfDNA) enrichment technology: First, through innovative technical improvements, experimental costs can be significantly reduced; second, the optimized operational process significantly improves operational convenience; most notably, the DNA purity is high, and the QSep results show no interference from impurities, demonstrating the feasibility of this method. Furthermore, the method can be used with any positively charged magnetic beads, demonstrating its strong practicality. These technical improvements provide high-quality templates for downstream molecular analysis (including NGS sequencing and digital PCR), demonstrating their significant value in clinical applications such as liquid biopsy and prenatal diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the QSep test result diagram for sample 1; Figure 2 This is the QSep test result diagram for sample 2; Figure 3 This is the QSep test result diagram of sample 3. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be further described below.

[0017] The reagents used include: The body fluid used for extraction is human plasma, and the amount used is 4 mL.

[0018] The proteinase K solution used for protein digestion has a concentration of 20 mg / mL and is used in an amount of 200 μl.

[0019] The magnetic bead solution comprises ferroferric oxide magnetic microbeads with positively charged surfaces coated with amino groups and deionized water; the solution is vortexed thoroughly and the usage volume is 20 μl.

[0020] The binding solution is 20mM Tris-HCl and 25% PEG400; The eluent is 20 mM Tris-HCl, 1 M NaCl, 5 mM EDTA-2Na; The following are the extraction steps: Take three clean 15 ml centrifuge tubes, add 4 ml of plasma, add 200 μl of proteinase K, vortex thoroughly to mix, and incubate at 60°C for 30 min; Add 20 μl of the above magnetic beads and 5 ml of binding solution, vortex for ten minutes, centrifuge briefly, and let stand on a magnetic rack for five minutes until the beads are completely attached to the wall; Use a pipette to discard all the supernatant; Add 60 μl of the above elution solution, vortex for ten minutes, centrifuge briefly, continue to place on the magnetic stand, and let it stand for five minutes until the magnetic beads are completely attached to the wall; Take the above supernatant and transfer it into clean sample storage tubes respectively; The supernatant DNA liquid (samples 1, 2, and 3) was taken for Qsep testing to check the fragment length and purity.

[0021] The above description is merely a preferred embodiment of the present invention and does not limit the present invention in any way. Any person skilled in the art who, without departing from the scope of the present invention, makes any equivalent substitution, modification, or other changes to the technical solution and technical content disclosed in the present invention shall be deemed to be within the scope of the present invention and still fall within the scope of protection of the present invention.

Claims

1. A rapid free DNA enrichment method based on magnetic beads, characterized in that: The steps include: (1) Take plasma, add proteinase K solution, mix thoroughly, and incubate; (2) Add magnetic beads and binding solution, shake, centrifuge, and let stand until the magnetic beads are completely attached to the wall; (3) Aspirate and discard all supernatant; (4) Add elution buffer, shake, centrifuge, and let stand until the magnetic beads are completely attached to the wall; (5) Take the supernatant and transfer it to the sample for enrichment.

2. The method for rapid free DNA enrichment based on magnetic beads according to claim 1, characterized in that: The plasma is human plasma, and the dosage is 1-10 mL, preferably 4 mL.

3. The rapid free DNA enrichment method based on magnetic beads according to claim 1, characterized in that: The proteinase K solution has a concentration of 10-40 mg / mL and a dosage of 150-200 μl, preferably a concentration of 20 mg / mL and a dosage of 200 μl.

4. The method for rapid free DNA enrichment based on magnetic beads according to claim 1, characterized in that: The magnetic beads are ferroferric oxide magnetic microbeads with positively charged surfaces and coated with amino groups.

5. The method for rapid free DNA enrichment based on magnetic beads according to claim 1, characterized in that: The binding solution is 15-150 mM Tris-HCl and 20%-30% PEG400, preferably 20 mM Tris-HCl and 25% PEG400.

6. The method for rapid free DNA enrichment based on magnetic beads according to claim 3, characterized in that: The eluent is 18-22 mM Tris-HCl, 0.8-1 M NaCl or 4-6 mM EDTA-2Na, preferably 20 mM Tris-HCl, 1 M NaCl or 5 mM EDTA-2Na.

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