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Silica gel absorption column extraction kit and method for free DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid) of blood plasma or blood serum

A technology of silica gel adsorption and kits, which is applied in the biological field and can solve problems such as high prices

Inactive Publication Date: 2018-05-25
江苏然科生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most important product of the silica gel membrane adsorption column method is QIAamp Circulating Nucleic Acid Kit from Qiagen, which is very expensive because it has a very large monopoly in the field of free DNA or RNA extraction.

Method used

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  • Silica gel absorption column extraction kit and method for free DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid) of blood plasma or blood serum
  • Silica gel absorption column extraction kit and method for free DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid) of blood plasma or blood serum
  • Silica gel absorption column extraction kit and method for free DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid) of blood plasma or blood serum

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Experimental program
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Effect test

Embodiment 1

[0101] The preparation of embodiment 1 reagent

[0102] 1. Preparation of lysate

[0103] Prepare 50mM Tris-HCl, 4M guanidine isothiocyanate, 1M sodium chloride, 5mM EDTA, 2% triethanolamine dodecyl sulfate and 1% dimethyl sulfoxide solution respectively by conventional preparation methods, mix them uniformly, adjust The lysate was obtained after the pH reached 6.5.

[0104] Wherein, the substance concentration of Tris-HCl can realize the technical purpose of the present invention at 10-100mM, for example, it can be: 10mM, 20mM, 30mM, 40mM, 60mM, 70mM, 80mM, 90mM, etc.;

[0105] The amount concentration of guanidine isothiocyanate can realize the technical purpose of the present invention at 3-5M, for example can be: 3M, 3.5M, 4M, 4.5M, 5M etc.;

[0106] The substance concentration of sodium chloride can realize the technical purpose of the present invention at 0.5-1.5M, for example can be: 0.5M, 0.7M, 0.9M, 1.1M, 1.3M, 1.5M etc.;

[0107] The substance concentration of EDT...

Embodiment 2

[0128] The extraction of nucleic acid in embodiment 2 blood plasma or serum

[0129] 1. Preparation of plasma samples:

[0130] Take fresh EDTA anticoagulated blood, centrifuge at 900G for 10 minutes, and absorb the supernatant; centrifuge the supernatant at 15,800G for 10 minutes, absorb the supernatant to obtain plasma, divide the prepared plasma into 4 parts on average, and set aside.

[0131] Among them, 2 parts of the mixed plasma were extracted with the method and kit of the present invention, and the remaining 2 parts of the mixed plasma were extracted with Qiagen's QIAamp Circulating Nucleic Acid Kit to extract plasma free DNA and RNA.

[0132] 2. Lysis of plasma samples

[0133] Add an equal volume of lysate to 4ml plasma, add 320μl proteinase K, mix for 30s, and incubate at 60°C for 30 minutes.

[0134] Wherein, the volume ratio of the amount of plasma, lysate and proteinase K can realize the technical purpose of the present invention in the scope of 0.5-1.5:0.5-1....

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Abstract

The invention discloses a silica gel absorption column extraction kit and method for free DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid) of blood plasma or blood serum and relates to the fieldof molecular biology. The kit comprises proteinase K, isopropyl alcohol, absolute ethyl alcohol and threefold-distilled water, and further comprises a lysis solution containing a nucleic acid separation accelerant, an adsorption solution containing a nucleic acid adsorption accelerant and a washing solution; the method is mainly carried out by utilizing the kit. By adopting the kit provided by the invention, the free DNA and RNA in the blood plasma or the blood serum can be rapidly and completely released, and denatured protein keeps a dissolved state; under the action of the adsorption solution, the free DNA and RNA are efficiently adsorbed on a silica gel absorption column; after the silica gel absorption column is washed by the washing solution, the free DNA and RNA with relatively high purity and concentration are obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit and method for extracting plasma or serum free DNA and RNA through a silica gel adsorption column. technical background [0002] Free DNA, referred to as cf DNA (Free circulating / Cell-free DNA), was first discovered in 1948. It is a cell-free state, fragmented extracellular DNA, and mainly exists in body fluids such as blood, synovial fluid, and cerebrospinal fluid. At present, the application of cell-free DNA in non-invasive prenatal detection and liquid biopsy of tumors are two hot fields in clinical practice. In addition, the application value of cell-free DNA in diseases such as organ transplantation, stroke, autoimmune disease and myocardial infarction has become increasingly clear. [0003] Free RNA (cell-freeRNA, cfRNA) is the transcription product existing outside the cell, including mRNA and microRNA. MicroRNA is found in a variety of human body fluids (plasma, serum...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/101C12Q2521/537C12Q2523/308C12Q2527/125
Inventor 黄钧昱
Owner 江苏然科生物技术有限公司
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