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Lysate, nucleic acid extraction kit, and nucleic acid extraction method for nucleic acid extraction

The technology of a lysate and a kit is applied in the field of lysate for nucleic acid extraction, which can solve the problems of complex nucleic acid extraction process, easy residue of proteinase K, unfavorable detection accuracy, etc., so as to reduce the requirements of experimental equipment, simple steps, realize automation and The effect of high-throughput quantization

Active Publication Date: 2020-01-14
HANGZHOU BIGGER FISH BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First of all, the commonly used lysate for nucleic acid extraction by the magnetic bead method usually contains proteinase K, which needs to be used to digest the protein in biological samples, especially for whole blood samples, proteinase K must be added to ensure high-quality extraction Nucleic acid, proteinase K is easy to remain, the extraction time is longer, and the extraction process needs to be heated
Secondly, in the nucleic acid extraction operation of the prior art, the lysis of the sample and the binding of magnetic beads and DNA are often carried out step by step, and the corresponding kits often include separately packaged lysate and binding liquid, which not only makes the nucleic acid extraction process Complicated, and greatly increases the probability of nucleic acid contamination, which is not conducive to subsequent detection and detection accuracy

Method used

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  • Lysate, nucleic acid extraction kit, and nucleic acid extraction method for nucleic acid extraction
  • Lysate, nucleic acid extraction kit, and nucleic acid extraction method for nucleic acid extraction
  • Lysate, nucleic acid extraction kit, and nucleic acid extraction method for nucleic acid extraction

Examples

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Embodiment 1

[0060] In this example, the saliva collected by the applicant was taken as a sample from a normal person, and the nucleic acid in the sample was extracted. The nucleic acid extraction process of the present embodiment is as follows:

[0061] 1. Take 100 μL of the sample and add it to a centrifuge tube, add 400 μL of lysate to the centrifuge tube, and supercis-hydroxyl nanomagnetic particles with a particle size of 200 nm, mix at room temperature for 4 minutes, place the centrifuge tube on a magnetic stand, and magnetically absorb it for 20 seconds. Absorb the supernatant; wherein, the composition of the lysate is: sodium acetate 0.5M, EDTA 1mM, guanidine hydrochloride 2M, TritonX-100 0.25%, PEG8000 1%, and the pH of the lysate is 7.4;

[0062] 2. Add 500 μL of the first washing buffer solution to the centrifuge tube, wash and mix for 1 min, place the centrifuge tube on the magnetic stand, magnetically absorb it for 20 seconds, and absorb the supernatant; wherein, the compositi...

Embodiment 2

[0066] In this embodiment, the sample used, the nucleic acid extraction process, the first washing buffer, the second washing buffer, and the composition of the eluent are the same as in Example 1. The difference is that the composition of the lysate is: acetic acid Sodium 1M, EDTA 3mM, guanidine hydrochloride 4M, TritonX-100 0.5%, PEG8000 2%, the pH of the lysate is 7.0.

Embodiment 3

[0068] In this embodiment, the sample used, the nucleic acid extraction process, the first washing buffer, the second washing buffer, and the composition of the eluent are the same as in Example 1. The difference is that the composition of the lysate is: acetic acid Sodium 2M, EDTA 5mM, guanidine hydrochloride 5M, TritonX-100 1%, PEG8000 3%, the pH of the lysate is 7.5.

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Abstract

The invention discloses a lysate, nucleic acid extraction kit, and nucleic acid extraction method for nucleic acid extraction. The lysate for the nucleic acid extraction is prepared from 0.5 to 2M ofsodium salt, 2 to 5M of guanidine salt, 1 to 5mM of a metal ion complexing agent, 0.25% to 1% of a nonionic surfactant, and 1% to 3% of PEG, and the molecular weight of the PEG is 6000 to 10000 Da, preferably 8000 Da. The lysate has a reasonable ratio, cells can be fully and effectively lysed, the lysis efficiency is high, so that the nucleic acids in the cells can be fully released, and the nucleic acids with higher concentration and purity can be obtained, and the quality and extraction efficiency of the nucleic acids are improved advantageously; and according to the lysate and the kit, digestion and removal of protein can be conducted without the aid of proteinase K during the entire nucleic acid extraction process, alcohols are not usedfor precipitate the nucleic acids,the obtained nucleic acids have high purity, and fragments are complete.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid extraction, and in particular relates to a lysate for nucleic acid extraction, a nucleic acid extraction kit and a nucleic acid extraction method. Background technique [0002] Nucleic acid is a kind of biopolymer, which is an essential component of all known life. It exists widely in animal and plant cells and microorganisms. It is divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). At present, the detection of nucleic acid is inseparable from various research and detection fields of molecular biology. The extraction and purification of nucleic acid is the basis of molecular biology research and clinical detection. [0003] Existing nucleic acid extraction methods mainly include alkaline lysis, phenol-chloroform extraction, chelating resin method, spin column membrane adsorption method, and magnetic bead method. Among them, the alkaline lysis method has the disadvantages of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1013C12Q1/6806
Inventor 黄宵谢廉毅李明
Owner HANGZHOU BIGGER FISH BIOTECHNOLOGY CO LTD
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