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Nucleic acid purification method for DNA methylation analysis of human stool

A technology of methylation and feces, applied in the field of obtaining nucleic acid samples for DNA methylation analysis, achieving a high conversion rate

Active Publication Date: 2021-11-02
SHANGHAI REALBIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the bisulfite conversion methods currently on the market need further research

Method used

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  • Nucleic acid purification method for DNA methylation analysis of human stool
  • Nucleic acid purification method for DNA methylation analysis of human stool
  • Nucleic acid purification method for DNA methylation analysis of human stool

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] The method of the present invention of embodiment 1 human source feces total DNA is compared with Z brand commercialization sulfite conversion kit method

[0068] Acquisition of total DNA from human stool: select the stool samples of several patients with colorectal cancer, and obtain highly methylated DNA of the target gene through a nucleic acid extraction kit (produced by Shanghai Ruiyi Biotechnology Co., Ltd., No. 20180535). Human fecal total DNA.

[0069] One, the specific implementation steps of the nucleic acid purification method for DNA methylation analysis of the present invention:

[0070] (1) Add 2 μg of DNA to be processed (sample1, 2, 3) into a 2.0 mL centrifuge tube, and control the input volume range to 20-100 μL;

[0071] (2) After adding 190 μL of sulfite conversion solution and 30 μL of protective solution, cover the centrifuge tube tightly, mix it upside down, and centrifuge immediately;

[0072] (3) Seal the centrifuge tube and place it in a const...

Embodiment 2

[0109] The method of the present invention of embodiment 2 paraffin DNA is contrasted with Z brand commercialization sulfite conversion kit method

[0110] Paraffin DNA was extracted using a paraffin-embedded tissue DNA extraction kit ( DNA FFPE Tissue, 56404), to obtain FFPE-DNA.

[0111] One, the specific implementation steps of the nucleic acid purification method for DNA methylation analysis of the present invention:

[0112] (1) Add 500ng of FFPE-DNA to be treated in a 2.0mL centrifuge tube, the input volume is about 50μL;

[0113] (2) After adding 200 μL of sulfite conversion solution and 50 μL of protective solution, cover the centrifuge tube tightly, mix it upside down, and centrifuge immediately;

[0114] (3) Seal the centrifuge tube and place it in a constant temperature shaking incubator, and incubate at 95°C for 5 minutes; to prevent DNA renaturation, take out the centrifuge tube immediately and place it on ice for 5 minutes;

[0115] (4) Take out the centrifug...

Embodiment 3

[0152] The method of the present invention of embodiment 3 plasma free DNA is contrasted with Z brand commercialization sulfite conversion kit method

[0153] The plasma DNA was extracted using a free peripheral blood DNA extraction kit (VAHTS Serum / Plasma CircμLating DNA Kit, N902-01) to obtain cfDNA.

[0154] One, the specific implementation steps of the nucleic acid purification method for DNA methylation analysis of the present invention:

[0155] (1) Add 20ng of DNA to be treated into a 2.0mL centrifuge tube, with a volume of 50μL;

[0156] (2) After adding 280 μL of sulfite conversion solution and 50 μL of protective solution, cover the centrifuge tube tightly, mix it upside down, and centrifuge immediately;

[0157] (3) Seal the centrifuge tube and place it in a constant temperature shaking incubator, and incubate at 95°C for 5 minutes; to prevent DNA renaturation, take out the centrifuge tube immediately and place it on ice for 5 minutes;

[0158] (4) Take out the ce...

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Abstract

The present invention proposes a nucleic acid purification method for DNA methylation analysis of human feces, the method comprising: denaturing the initial nucleic acid sample; subjecting the denatured product to a temperature of 75-80°C for 40-50 minutes conversion treatment, during the conversion treatment process, the unmethylated cytosine in the denatured treatment product is converted into uracil, and the methylated cytosine is still cytosine; the conversion treatment product is purified so as to obtain A nucleic acid sample for DNA methylation analysis; wherein, the conversion treatment is carried out in the presence of sodium bisulfite, sodium sulfite and a protective agent. This method can be effectively used for methylation analysis of low-content human DNA samples, such as human stool DNA methylation analysis, to obtain high conversion rate and high-quality converted DNA, which is conducive to the full automation of methylation research and standardized operations.

Description

technical field [0001] The present invention relates to the field of nucleic acid purification, specifically, the present invention relates to a method for purifying nucleic acid for analysis of DNA methylation in human feces, and more specifically, the present invention relates to a method for obtaining a nucleic acid sample for DNA methylation analysis. Background technique [0002] DNA methylation refers to the transfer of active methyl groups to specific bases in the DNA chain under the catalysis of DNA methyltransferase (DNMT) using S-adenosylmethionine as the methyl donor. chemical modification process. In the study of human epigenetics, the most common is the methylation modification of cytosine in CpG dinucleotides. The main process is: in CpG methylation binding proteins (Methyl-CpG Binding Proteins, MBDs) and DNA Under the action of methyltransferases (DNAmethyltransferases, DNMTs), the cytosine at the 5' end of the CpG dinucleotide is converted into 5' methylcyto...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2523/125C12Q2563/143C12Q2563/149
Inventor 王美丛刘晶秦楠
Owner SHANGHAI REALBIO TECH CO LTD
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