Method and kit for transforming and purifying DNA in DNA methylation detection process

A detection process and methylation technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as DNA fragmentation, false positive DNA methylation detection results, and difficulty in mechanization, and improve detection efficiency. , The effect of saving detection energy consumption and saving detection cost

Pending Publication Date: 2021-10-26
奥明(杭州)基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Obviously, the existing transformation methods have the following defects: 1) DNA transformation takes a long time, and some studies report more than 3 hours, which is not conducive to rapid detection; 2) DNA transformation protection agents are required, and the protection agent components generally contain Environmentally unfriendly substances such as p-diphenol; 3) The temperature of DNA conversion is relatively high. When the conversion temperature is close to 90°C or above, it is very likely to cause false positive results in DNA methylation detection. At the same time, DNA fragmentation is serious and the recovery rate Less than 40%; 4) Adsorption column method is used for DNA purification after conversion, and the operation is not easy to realize mechanization

Method used

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  • Method and kit for transforming and purifying DNA in DNA methylation detection process

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Effect test

Embodiment 1

[0026] In order to achieve the above object, the method steps of DNA conversion and purification in the DNA methylation detection process ( figure 1 )for:

[0027] 1) In the supersaturated sodium bisulfite / sodium hydroxide solution, add the DNA sample without adding any DNA protecting agent;

[0028] 2) Carry out DNA transformation at 70°C, and the transformation time is 20 minutes;

[0029] 3) After step 2) is completed, add an appropriate amount of 2mol / L citric acid + 3mol / L sodium citrate + 6mol / L guanidine hydrochloride, and use magnetic beads to absorb the converted DNA;

[0030] 4) For the DNA adsorbed by the magnetic beads in the step 3), the purified DNA is obtained by washing with 8mol / L sodium perchlorate+dehydrated ethanol, isopropanol and tris(hydroxymethyl)aminomethane-hydrochloric acid solution respectively;

[0031] 5) The purity and concentration of the purified DNA were tested, which met the requirements for further DNA methylation detection.

Embodiment 2

[0033] In order to achieve the above object, the method steps of DNA conversion and purification in the DNA methylation detection process ( figure 1 )for:

[0034] 1) In supersaturated sodium sulfite / ammonium sulfite monohydrate / sodium bisulfite solution, add DNA sample without adding any DNA protectant;

[0035] 2) Carry out DNA transformation at 70°C, and the transformation time is 20 minutes;

[0036] 3) After step 2) is completed, add an appropriate amount of absolute ethanol + 3mol / L sodium chloride + 1mol / L tris(hydroxymethyl)aminomethane-hydrochloric acid solution, and use magnetic beads to absorb the transformed DNA;

[0037] 4) For the DNA adsorbed by the magnetic beads in step 3), wash with 1 mol / L sodium chloride + 0.5% sodium lauryl sulfate + isopropanol, absolute ethanol and nucleic acid-free water respectively to obtain Purified DNA;

[0038] 5) The purity and concentration of the purified DNA were tested, which met the requirements for further DNA methylation...

Embodiment 3

[0040] In order to achieve the above object, the method steps of DNA conversion and purification in the DNA methylation detection process ( figure 1 )for:

[0041] 1) In supersaturated sodium bisulfite / ammonium bisulfite monohydrate / ammonium bisulfite solution, add DNA sample without adding any DNA protectant;

[0042] 2) Carry out DNA transformation at 70°C, and the transformation time is 20 minutes;

[0043] 3) After step 2) is completed, add an appropriate amount of 2mol / L citric acid + 4mol / L guanidine hydrochloride solution, and use magnetic beads to absorb the transformed DNA;

[0044] 4) For the DNA adsorbed by magnetic beads in step 3), pass through 1mol / L sodium chloride+sodium lauryl sulfate+absolute ethanol with a mass percentage of 0.5%, isopropanol and tris(hydroxymethyl)aminomethane respectively - washing with hydrochloric acid solution to obtain purified DNA;

[0045] 5) The purity and concentration of the purified DNA were tested, which met the requirements ...

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Abstract

The invention discloses a method and a kit for transforming and purifying DNA in a DNA methylation detection process. According to the method, a DNA sample in the methylation detection process is directly converted by using supersaturated sulfite solution, so that the use of a DNA protective agent is avoided, the DNA conversion temperature is reduced to 30-70 DEG C, meanwhile, the conversion time is greatly shortened to 5-20 minutes. In addition, with the help of a binding agent, the transformed DNA is adsorbed and separated through magnetic beads, so that the purification process of the transformed DNA is simplified. The kit can greatly improve the DNA methylation detection efficiency and reduce the detection cost.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method and kit for DNA conversion and purification in the process of DNA methylation detection. Background technique [0002] Tumor can be said to be a genetic disease. Studies have shown that changes in DNA promoter methylation patterns most likely play a key role in tumorigenesis and cancer progression. At present, many research institutions have begun to use DNA methylation detection in the diagnosis of various cancers. At present, the general method of DNA conversion and purification in the process of DNA methylation detection is to treat the extracted cfDNA with a sulfite solution at a temperature of about 90°C under the protection of a DNA protecting agent, and then use the adsorption column method to convert the DNA. of purification. [0003] Obviously, the existing transformation methods have the following defects: 1) DNA transformation takes a long time, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2523/125C12Q2523/308
Inventor 童云广唐正清张书祖唐姗姗任永丽
Owner 奥明(杭州)基因科技有限公司
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