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Method for extracting free DNA from human plasma

A technology of human plasma and blood plasma, which is applied in the field of extracting free DNA, can solve the problems of high cost, borrowing other instruments, and cumbersome enrichment steps, etc., and achieves the effect of simple method, high sensitivity, and large surface area

Pending Publication Date: 2019-09-20
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a method for extracting free DNA in human plasma to solve the problems of cumbersome enrichment steps, the need to borrow other instruments, and high cost in the existing methods for extracting free DNA in human plasma.

Method used

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  • Method for extracting free DNA from human plasma
  • Method for extracting free DNA from human plasma
  • Method for extracting free DNA from human plasma

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preparation example Construction

[0035] According to the present invention, the preparation method of the glassy carbon electrode 3D-PAAM-PA-PDA-H-GA-GCE modified by the three-dimensional polyacrylamide-phytic acid-polydopamine conductive hydrogel preferably includes:

[0036] 1) Take acrylamide, N,N-methylenebisacrylamide (MBA) and phytic acid (PA) solutions into the reactor, add water and shake well to obtain a mixed solution, pass argon gas and sonicate for 5 to 15 minutes Then add dopamine solution, add potassium persulfate (KPS) after 5-10 minutes, continue to sonicate for 10-20 minutes, add N,N,N,N-tetramethylethylenediamine (TEMED) and mix quickly to obtain a pregel gel. Condensate; the molar ratio of the acrylamide, phytic acid and dopamine is preferably 50:2:1, the N,N-methylenebisacrylamide is a crosslinking agent, and the mass fraction is preferably 0.038%; TEMED As an accelerator, the mass fraction is 0.012%;

[0037] 2), the glassy carbon electrode is pretreated, preferably: polish the glassy ca...

Embodiment 1

[0049] Example 1 Preparation of polyacrylamide-phytic acid-polydopamine conductive hydrogel modified glassy carbon electrode

[0050] Prepare 0.17mol / L dopamine, 6.0mol / L acrylamide, 0.02mol / L, N,N-methylenebisacrylamide (MBA) and pH 7.5, 0.54mol / L phytic acid monomer stock solution. Measure 667 μL of acrylamide (6.0 mol / L), 250 μL of MBA (0.02 mol / L), 250 μL of PA (pH 7.5, 0.54 mol / L) and 250 μL of ultrapure water, mix them evenly, pass argon gas, sonicate after 10 min and add 410 μL of dopamine solution, 85.2 mg of potassium persulfate (KPS) was added after 5 minutes, continued ultrasonication for 15 minutes, and 0.31 μL of N,N,N,N-tetramethylethylenediamine (TEMED) was added and quickly mixed to obtain a gel pre-coagulant;

[0051] Glassy carbon electrode pretreatment: Polish the electrode with 1.0, 0.3, 0.05 μm polishing powder on the polishing cloth, rinse with ultrapure water, and ultrasonicate in acetone, ethanol, and ultrapure water for 1 min, and use ultrapure Washed...

Embodiment 2

[0054] Example 2 Preparation of plasma extract and pretreatment of gel-modified glassy carbon electrode

[0055] Configure 10mmol / L Tris buffer and adjust the pH to 7.5.

[0056] Take 3 mL of human venous blood and place it in EDTA·K 2 In the anticoagulant tube, centrifuge at 3000rpm for 15min, absorb the upper layer of serum and place it in a 2mL centrifuge tube, after centrifuging at 3000rpm for 10min, absorb 1mL of the upper layer of plasma and place it in a 10mL reagent bottle.

[0057] Submerge the gel part of the gel-modified glassy carbon electrode tip prepared in Example 1 into the plasma, adjust the magnetic stirring speed to 120 rpm, take out the electrode after soaking for 45 minutes, transfer the plasma to a conductive extraction tube, and mix with the auxiliary electrode , reference electrode and working electrode are assembled together as figure 1 In the shown device, the electrolyte in the electrolytic cell is Tris solution, and the reference electrode and aux...

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Abstract

The invention provides a method for extracting free DNA from human plasma and belongs to the technical field of biological sample enrichment and extraction. The method includes: firstly, preparing a glassy carbon electrode modified by a working electrode 3D polyacrylamide-phytic acid-polydopamine conductive hydrogel; secondly, placing a human blood sample in an EDTA K2 anticoagulant tube, and absorbing the upper plasma as an extract after centrifugation; thirdly, adopting the electrode prepared in the first step to react with the plasma or a diluent of the plasma, soaking the electrode in the plasma or the diluent of the plasma, and adsorbing free DNA in the plasma by a chronoamperometric method; finally, taking out the electrode, and soaking the electrode in a little amount of Tris buffer to apply reverse current to the free DNA for desorption to obtain a free DNA solution extracted from blood. The method is simple and rapid in operation and high in enrichment efficiency, and can be successfully applied to subsequent detection and analysis.

Description

technical field [0001] The invention belongs to the technical field of biological sample extraction, and in particular relates to a method for extracting free DNA from human blood plasma. Background technique [0002] Cell-free DNA in plasma is a cell-free state, fragmented extracellular DNA, its content is small, and most of them are small fragments of DNA molecules. At present, cell-free DNA is widely used in tumor liquid biopsy. Accurate analysis of cell-free DNA in blood is of great significance for early treatment of tumors and screening and monitoring of disease recurrence. However, its application in clinical diagnosis is limited due to the difficulty in extraction due to its small content, large loss, and low detection sensitivity. Therefore, efficient extraction of cell-free DNA is of great significance for clinical medicine and scientific research. Although there are currently some methods for the extraction of free DNA in plasma, including spin column method, ma...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003Y02A50/30
Inventor 王振新张婳魏佳赵珍
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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