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Nucleic acid extraction method and kit

A nucleic acid extraction reagent and extraction method technology, which are applied in the field of nucleic acid extraction methods and kits to achieve the effect of ensuring broad-spectrum applicability

Active Publication Date: 2021-01-08
上海慕柏生物医学科技有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the application of high-throughput sequencing technology has continuously found that DNA extraction should not only be based on the integrity, concentration, purity and acquisition efficiency of nucleic acid fragments, but should also consider the comprehensiveness of nucleic acid acquisition in complex environments and complex biological sample types. sex, unbiased sex questions

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  • Nucleic acid extraction method and kit
  • Nucleic acid extraction method and kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] In order to observe whether different DNA extraction methods affect the results in the field of high-throughput sequencing, two DNA extraction methods were selected, one is a DNA extraction method based on environmental samples published by Gordon et al., as a representative of traditional extraction methods The other method is the fecal DNA extraction kit (product number: D4015) produced by Omega Bio-tek, which is a representative method of DNA extraction from silicon matrix spin columns (abbreviation: Omega kit). Reasons for selection: 1) The traditional extraction method has been used for many years, and it is still recognized, which means that the results are closer to real-time data and are widely accepted. 2) The Omega kit has the main process of the traditional spin column DNA extraction method, which can provide a good reference for subsequent verification of the efficacy of the ingredients of the present invention. Therefore, two extraction methods were used to...

Embodiment 2

[0126] In order to determine whether each chemical component or its mixture of this method plays the role it should play in the DNA extraction process. The Omega kit with a similar extraction process is replaced with the reagents or components of each extraction process of this method to compare the extraction effect, so as to determine whether the reagents in this method have volatilized their due effect in the link.

[0127] Preparation of reagents for this method:

[0128] 1) Nucleic acid chemical lysis solution: 4M guanidine isothiocyanate solution; 10% w / v N-lauroyl sarcosine sodium salt solution; 5% (w / v) N - Lauroyl acid sodium salt solution.

[0129] 2) Solutions related to impurity removal: 150mM ammonium acetate solution; 120mM ammonium aluminum sulfate dodecahydrate solution (containing 30% v / v PVPP); 20mg / mL proteinase K room temperature storage solution: 25mM Tris-base, 10mM calcium chloride; 200mM Sodium chloride, 50% v / v glycerin, HCl to adjust the pH to 8.0. ...

Embodiment 3

[0177] In order to observe whether this method can more comprehensively obtain the DNA information of various species of samples, the traditional method and this method were used to compare the two sets of DNA extraction processes, and to observe whether there were differences in the final sequencing and bioinformatics analysis results.

[0178] The stool sample of volunteer 11 was selected as the target sample of this test, and the traditional method and the method of the present invention were used to extract the stool DNA respectively, and each method extracted 3 replicates. According to the 16s rRNA gene library construction method officially provided by Illumina (https: / / support.illumina.com.cn / content / dam / illumina-marketing / documents / products / other / 16s-metagenomics-faq-1270-2014-003 .pdf) Complete the library construction of the Miseq sequencing platform and complete the sequencing.

[0179] Bioinformatics analysis was performed as follows.

[0180] The raw data (Rawdat...

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Abstract

The present invention relates to the field of nucleic acid extraction, particularly to a nucleic acid extraction method and a kit. The kit comprises a nucleic acid chemical cracking solution and an inhibition component removing solution, wherein the nucleic acid chemical cracking solution comprises guanidinium isothiocyanate, N-lauroyl sarcosine sodium salt and PBS; and the inhibiting component removing solution comprises a mixed solution of an aluminum ammonium sulfate dodecahydrate solution and cross-linked polyvinylpyrrolidone (PVPP) and ammonium acetate. The nucleic acid extraction methodcomprises the following steps of chemically cracking a sample by adopting the nucleic acid chemical cracking solution, removing impurities, adsorbing the nucleic acid, and performing washing, eluting,and collecting to obtain the nucleic acid. According to the method, comprehensiveness and unbiased property in nucleic acid extraction are fully considered, purity and integrity of nucleic acid are also considered, loss in the process is reduced to ensure concentration, nesting can be formed for different types of biological or environmental samples, and broad-spectrum applicability of the extraction method is guaranteed.

Description

technical field [0001] The invention relates to the field of nucleic acid extraction, in particular to a nucleic acid extraction method and a kit. Background technique [0002] Deoxyribonucleic acid (DNA) is the main genetic material of living things. In the fields of molecular biology and biochemistry, DNA is widely used as the research object or target. However, obtaining DNA from environmental samples (such as soil, water, sand, etc.) and biological samples (such as excreta, tissues, body fluids, etc.) is the first step in carrying out relevant research experiments. At present, the means of obtaining DNA can be roughly divided into the following categories: the use of alcohols to precipitate DNA based on traditional molecular biology methods; the column adsorption of DNA based on silica gel matrix materials; -OH and carboxyl groups -COOH) to adsorb DNA, etc. According to different DNA acquisition methods, convenient and fast kits have also emerged, such as spin column ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2527/125C12Q2521/537
Inventor 孙佳瑞王玲任红燕
Owner 上海慕柏生物医学科技有限公司
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