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Nucleic acid purification method for DNA methylation analysis of human-derived stool

A technology for methylation and nucleic acid samples, applied in the field of obtaining nucleic acid samples for DNA methylation analysis, to achieve the effect of quick and easy operation

Active Publication Date: 2019-03-08
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-derived stool
  • Nucleic acid purification method for DNA methylation analysis of human-derived stool
  • Nucleic acid purification method for DNA methylation analysis of human-derived stool

Examples

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Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Comparison of the method of the present invention for total human fecal DNA and the method of Z brand commercial sulfite conversion kit

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

[0069] 1. The specific implementation steps of the nucleic acid purification method for DNA methylation analysis of the present invention:

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

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

[0072] (3) Seal the centrifuge tube in a constant temperature shaking incubator and incu...

Embodiment 2

[0109] Example 2 Comparison of the method of the present invention of paraffin DNA and the method of Z brand commercial sulfite conversion kit

[0110] Paraffin-embedded tissue DNA extraction kit ( DNA FFPE Tissue, 56404) to obtain FFPE-DNA.

[0111] 1. 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 processed into a 2.0mL centrifuge tube, the input volume is about 50μL;

[0113] (2) After adding 200μL of sulfite conversion solution and 50μL of protection solution to the centrifuge tube, turn it upside down to mix, 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, immediately remove the centrifuge tube and place it on ice for 5 minutes;

[0115] (4) Take out the centrifuge tube, mix it upside down, centrifuge immediately, place th...

Embodiment 3

[0152] Example 3 Comparison of the method of the present invention for plasma free DNA and the method of Z brand commercial sulfite conversion kit

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

[0154] 1. 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 processed into a 2.0mL centrifuge tube, the volume is 50μL;

[0156] (2) After adding 280 μL of sulfite conversion solution and 50 μL of protection solution to the centrifuge tube, turn it upside down to mix, 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, immediately remove the centrifuge tube and place it on ice for 5 minutes;

[0158] (4) Take out the centrifuge tube, mix it upside do...

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Abstract

The invention provides a nucleic acid purification method for DNA methylation analysis of human-derived stool. The method comprises steps as follows: an initial nucleic acid sample is subjected to denaturation treatment; a denaturation treatment product is subjected to conversion treatment for 40-50 min at the temperature of 75-80 DEG C, un-methylated cytosine in the denaturation treatment productis converted into uracil in the conversion treatment process, and methylated cytosine is still cytosine; a conversion treatment product is purified, and a nucleic acid sample for DNA methylation analysis is obtained; the conversion treatment is performed in the presence of sodium hydrogen sulfite, sodium sulfite and a protective agent. The method can be effectively applied to methylation analysisof the low- content human-derived DNA sample such as DNA methylation analysis of human-derived stool, converted DNA with high conversion rate and high quality is obtained, and fully-automatic and standard operation of methylation study is facilitated.

Description

Technical field [0001] The present invention relates to the field of nucleic acid purification. Specifically, the present invention relates to a method of nucleic acid purification for DNA methylation analysis of human fecal DNA. More specifically, the present invention relates to a method of obtaining nucleic acid samples for DNA methylation analysis. Background technique [0002] DNA methylation (DNAmethylation) refers to the transfer of active methyl groups to specific bases in the DNA chain using S-adenosylmethionine as a methyl donor under the catalysis of DNA methyltransferase (DNMT) On the 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 protein (Methyl-CpG Binding Proteins, MBDs) and DNA Under the action of DNAmethyltransferases (DNMTs), the cytosine at the 5'end of the CpG dinucleotide is converted into 5'methylcytosine. [00...

Claims

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Application Information

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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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