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Nucleic acid extraction method by virtue of paramagnetic particle method

An extraction method, the technology of the magnetic bead method, applied in the biological field, can solve the problems that it is difficult to effectively improve the nucleic acid extraction efficiency and nucleic acid purity, and restrict the development of the magnetic bead method, so as to improve the extraction efficiency and nucleic acid purity, improve the nucleic acid extraction efficiency, The effect of reducing the impact

Inactive Publication Date: 2016-12-21
广东国盛医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In fact, when the biological sample to be tested is saliva, it is still difficult to effectively improve the nucleic acid extraction efficiency and nucleic acid purity in the existing magnetic bead method for nucleic acid extraction, even through lysing, binding, washing and separation. Restricted the development of magnetic bead method for saliva nucleic acid extraction

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  • Nucleic acid extraction method by virtue of paramagnetic particle method
  • Nucleic acid extraction method by virtue of paramagnetic particle method
  • Nucleic acid extraction method by virtue of paramagnetic particle method

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1: Nucleic acid extraction and detection using saliva as a biological sample

[0025] 1. Saliva sample processing: The saliva collection tube is the saliva preservation tube of Guangdong Guosheng Medical Technology Co., Ltd. Take 12 saliva samples without pretreatment, mix them upside down several times, and then add them directly into the lysate system for lysate digestion.

[0026] 2. Reagents:

[0027] a. Lysis solution: 10~150mM Tris-HCl, 1~10M chaotropic salt, 0.1~10% anionic surfactant, 1~20% nonionic surfactant, 0.1~4% CTAB And 1 ~ 100mM chelating agent composition. It is further optimized to be 50mM Tris-HCl, 3M potassium thiocyanate, 5% dodecylbenzenesulfonic acid, 11% Tween 20, 0.5% CTAB and 30mM sodium citrate;

[0028] b. Magnetic beads-binding solution: a mixed solution obtained by uniformly dispersing 20ul magnetic beads in 200ul isopropanol;

[0029] c. Washing solution A: composed of 50mM Tris-HCl, 3M potassium thiocyanate, 0.5% CTAB, 2% PVP ...

Embodiment 2

[0036] Example 2: Nucleic acid extraction and detection using blood as a biological sample

[0037] 1. Blood sample processing: Blood was collected in blood preservation tubes of Guangdong Guosheng Medical Technology Co., Ltd. Collect 10 human blood samples, mix them evenly upside down, and then directly add to the lysate system for lysate digestion.

[0038] 2. Reagents:

[0039] a. Lysis solution: 10~150mM Tris-HCl, 1~10M chaotropic salt, 0.1~10% anionic surfactant, 1~20% nonionic surfactant, 0.1~4% CTAB And 1 ~ 100mM chelating agent composition. It is further optimized as a mixture of 100mM Tris-HCl, 8M guanidine hydrochloride, 0.5% sodium laurate, 3% Triton-X-100, 3% CTAB and 100mM aminotriacetic acid;

[0040] b. Magnetic beads-binding solution: a mixture obtained by uniformly dispersing 20ul of magnetic beads in 200ul of ethanol;

[0041] c. Washing solution A: composed of 100mM Tris-HCl, 10M lithium chloride, 3% CTAB, 0.2% PVP and 40% ethanol;

[0042] d. Washing s...

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Abstract

The invention discloses a nucleic acid extraction method by virtue of a paramagnetic particle method. The nucleic acid extraction method comprises the following steps: sub-packaging 'N' [mu]M (M refers to mol / L) of sample ('N' is less than or equal to 300 and greater than or equal to 50), 'N' [mu]M of lysate and '0.1N' [mu]M of protease K in a No.1 nucleic acid extraction plate, conducting a cracking reaction at 70 DEG C for 10-60min, and then adding '1.1N' [mu]M of magnetic particle-binding buffer; sub-packaging '2.5N' [mu]M of washing solution A, '2.5N' [mu]M of washing solution B and '0.25-0.5N' [mu]M of TE solution in No.2, No.3 and No.4 nucleic acid extraction plates; and under the action of magnetic force, adsorbing magnetic particles in the No.1 nucleic acid extraction plate by virtue of a magnetic bar, conducting transferring, releasing, rapid mixing and magnetic absorption and sequentially processing the magnetic particles by virtue of the No.2, No.3 and No.4 nucleic acid extraction plates, so as to obtain extracted nucleic acid. With the application of the method disclosed by the invention, the influence of impurities on nucleic acid extraction from saliva and other biological samples is effectively reduced, so that nucleic acid extraction efficiency and nucleic acid purity are improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a magnetic bead nucleic acid extraction method. Background technique [0002] With the development of molecular biology such as gene detection and disease diagnosis, the extraction effects of traditional nucleic acid extraction methods such as phenol-chloroform method, salting-out method, and filter membrane spin column method can no longer meet the needs of development. The magnetic bead nucleic acid extraction method has been more and more widely used because of its advantages of batch operation, automatic extraction, and short time. [0003] The operation steps of magnetic bead nucleic acid extraction mainly include: (1) Lysis; (2) Binding; (3) Washing; (4) Separation of four major parts. Specifically, cells or tissues release DNA / RNA under the action of lysate, and the surface-modified superparamagnetic silica nano-magnetic beads specifically bind to the released DNA / RNA to form...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 邓杏飞曾汉兵霍云龙
Owner 广东国盛医学科技有限公司
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