DNA extraction reagent, kit and extraction method

The technology of an extraction method and a kit, which is applied in the field of DNA extraction reagents, can solve the problems of complex steps, high chemical toxicity, and complicated operation process, and achieve the effects of low extraction cost, fast extraction method, and high nucleic acid quality

Active Publication Date: 2020-06-19
GENETRON HEALTH (BEIJING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the alkali cracking method usually uses corrosive sodium hydroxide to denature and then neutralizes the precipitation with acid; the phenol extraction method has complicated steps, and organic solvents such as phenol and chloroform are used for extraction, which has high chemical toxicity; the magnetic bead method uses magnetic beads Adsorption and solvent elution methods are used to extract DNA. The operation process requires the use of magnetic sleeves and magnets for adsorption, which limits the operating flux. It usually needs to be completed with the help of a fully automatic magnetic bead extractor; silica gel membrane spin column, using polymer resin membrane interception purification DNA, the whole process requires multiple high-speed centrifugation tube changes, which takes a long time
[0004] The main bottleneck in the widespread use of molecular diagnostics in medical laboratories is the requirement to purify nucleic acids from samples: commercial biological nucleic acid DNA release extraction kits generally use silica gel membrane spin columns and magnetic beads, and the operation process is complicated and time-consuming. At least It takes 1.5 hours, which is not conducive to rapid detection, especially in the detection of disease (cancer) treatment surgery. Therefore, it is urgent to develop a simple, fast and high-quality nucleic acid extraction method

Method used

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  • DNA extraction reagent, kit and extraction method
  • DNA extraction reagent, kit and extraction method
  • DNA extraction reagent, kit and extraction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 DNA Extraction and qPCR Detection of Fresh Brain Tumor Tissue

[0050] One, the rapid extraction method of DNA of the present application

[0051] 1. Add 25 mg of brain tumor tissue to 600 μL of lysate, then add glass beads, place in a crusher, grind and crush for 1 min, centrifuge at 14,000 rpm for 1 min after crushing, transfer 500 μl of supernatant to a spin column, and centrifuge at 14,000 rpm for 15 s ;This step takes 3 minutes;

[0052] Wherein, the composition and content of the lysate are:

[0053] 5mmol / L guanidine isothiocyanate;

[0054] 50mmol / L Tris-HCl;

[0055] 10mmol / L EDTA;

[0056] 150mmol / L NaCl;

[0057] 0.1% (mass percentage) SDS;

[0058] 5 μg / ml aprotinin (Aprotinin);

[0059] The volume percentage content is 1-1.3% TritonX-100.

[0060] 2. Transfer the silica gel adsorption membrane spin column to a new collection tube, add 500μl rinse solution 1, centrifuge at 14000rpm for 15s, and replace the collection tube; this step takes 30s...

Embodiment 2

[0095] Example 2 DNA Extraction of Frozen Brain Tumor Tissue and Detection of IDH1 R132H Mutation Point

[0096] Genomic DNA of 38 cases of frozen brain tumor tissues was extracted by using the rapid DNA extraction method and DNA kit (Qiagen Biotechnology Co., Ltd., product number: 69504) in Example 1 of the present invention, and the IDH1 R132H mutation point detection was performed.

[0097] The extracted sample was diluted to 10ng / μl, and 2μl was used for detection. Wherein, the detection system and reaction conditions are as shown in Example 1.

[0098] Result judgment:

[0099] BIO-Rad: Set the signal baseline to 200RFU. If the difference between the Ct values ​​of FAM signal and ROX signal is less than or equal to 10, it means that the test sample is positive, otherwise it is negative.

[0100] Test results such as Figure 4 As shown in Table 3, the minimum detection limit of IDH1 can reach 1%, and the rapid extraction method of the present invention is consistent wit...

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Abstract

The invention discloses a DNA extraction reagent, a kit and an extraction method. The invention firstly provides the DNA extraction reagent which comprises a lysis solution, a rinsing solution I, a rinsing solution II and an eluent, wherein the lysis solution comprises guanidine isothiocyanate, Tris-HCl, ethylene diamine tetraacetic acid (EDTA), NaCl, sodium dodecyl sulfate (SDS) and a protease inhibitor. The invention further provides the kit containing the DNA extraction reagent and the method for extracting DNA by using the DNA extraction reagent or the kit. The DNA extraction method is rapid, simple and convenient, is favorable for crushing fresh tissues or frozen tissues, is particularly suitable for rapid operation in surgery, has low relative extraction cost, has high quality of extracted nucleic acid, and can meet the detection requirements of qPCR, NGS, SNP typing and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a DNA extraction reagent, kit and extraction method. Background technique [0002] Nucleic acid amplification and detection technology is widely used, especially in the fields of disease diagnosis, microorganism and pathogenic substance detection. In the field of disease diagnosis, with the advent of the era of precision medicine, genetic testing technology is a necessary technical means for genetic disease diagnosis, prenatal testing, molecular typing and personalized medicine. In the diagnostic process, nucleic acid analysis has many advantages over traditional methods, such as enzyme or antibody analysis, with high sensitivity, high flexibility and short time. [0003] In the diagnostic process, nucleic acid extraction and purification is the basis of nucleic acid detection, and the quality of extracted and purified nucleic acid has an important impact on the accuracy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2527/125C12Q2527/127C12Q2523/32
Inventor 郑乔松王曼张凯华王思振
Owner GENETRON HEALTH (BEIJING) CO LTD
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