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Gene methylation analysis method and product and application

An analysis method, a methylation technology, applied in the field of gene analysis, can solve the problems of increasing nucleic acid loss, high DNA loss, and high probability of contamination, and achieve the effects of reducing degradation and loss, simplifying the operation process, and facilitating automatic operation

Pending Publication Date: 2019-08-23
JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The whole process requires multiple washings and replacement of reaction tubes, which is cumbersome to operate, and the loss of DNA is high, and the probability of contamination is high
[0004] The above two processes of the commercial kit have been greatly improved compared with the traditional method, but it still takes nearly 8 hours to obtain the detection template on the machine; in addition, for some samples with relatively scarce tumor-specific circulating DNA Complicated manipulations can greatly increase nucleic acid loss
This poses a challenge to existing DNA extraction and transformation techniques

Method used

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  • Gene methylation analysis method and product and application
  • Gene methylation analysis method and product and application
  • Gene methylation analysis method and product and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Detection of methylation of septin9 gene

[0063] Sample pretreatment: Put equal amounts of interrupted Hela and Jurkat cell DNA into 2 ml of healthy human plasma samples;

[0064] Concentration: Add 4 ml of lysate (the lysate consists of 5.22M guanidine isothiocyanate, 0.42M TE buffer and 17% triton) and 40 microliters of magnetic beads (beaver, hydroxyl magnetic beads), Shake at room temperature for 10 minutes, discard the supernatant after absorbing the magnetic beads on the magnetic stand.

[0065] Conversion: add 320 microliters of conversion reagent (the conversion reagent is 5.34M ammonium bisulfite, 0.53M sodium sulfite, 0.05M protective agent (protective agent component 6-hydroxyl-2,5,7,8-tetramethyl The magnetic beads were resuspended in a mixture of tetrahydrofuran solution of chroman-2-carboxylic acid)), and the above mixture was incubated at 85° C. for 40 minutes.

[0066] Re-concentration: 1 ml of binding solution (7M guanidine hydrochloride) was added t...

Embodiment 2

[0070] In this example, the effects of synchronous addition of lysate and magnetic beads and sequential addition of lysate and magnetic beads were compared. The remaining steps of the method of sequentially adding magnetic beads are the same as those in Example 1, and nucleic acid concentration is performed on the same batch of samples. The obtained DNA was detected by real-time fluorescent PCR for methylation, and the detection results are shown in figure 2 . The results show that magnetic beads can be added simultaneously with the lysate in the sample, which will not affect the effect of lysis and binding, so that the binding step and lysis step can be completed at the same time, thereby greatly simplifying the nucleic acid extraction step. figure 2 It shows that the cleavage and binding are carried out synchronously on the effect of nucleic acid concentration.

Embodiment 3

[0072] DNA is directly transformed after enrichment by magnetic beads

[0073] Into 2mL of healthy human plasma samples into the interrupted Hela cell DNA. 1 part is processed completely according to embodiment 1, and 1 part of step (1) is processed according to the following steps:

[0074] Concentration: add 4 ml of lysate and 40 μl of magnetic beads, shake at room temperature for 10 minutes, discard the supernatant after the magnetic frame absorbs the magnetic beads, and then wash with 100 μl of buffer (TE buffer, pH=8.0) take off.

[0075] Transformation: Add 220 microliters of transformation reagent (the transformation reagent is ammonium bisulfite of 7.8M, sodium sulfite of 0.78M, DNA protectant of 0.07M (protectant component 6-hydroxyl-2,5,7,8 - a mixture of tetramethylchroman-2-carboxylic acid in diethylene glycol dimethyl ether solution), the above mixture was incubated at 85° C. for 40 minutes.

[0076] Re-concentration: add 1 ml of binding solution (7M guanidine ...

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Abstract

The invention relates to a gene methylation analysis method and product and application and provides a DNA methylation analysis method. The DNA methylation analysis method comprises the steps that 1)a biologic sample with DNA is in contact with a carrier with a nucleic acid adsorption capacity and a lysis solution with a nucleic acid adsorption capacity at the same time, and the DNA is gathered on the carrier; 2) the carrier, adsorbing the DNA, obtained in the first step is in direct contact with a conversion reagent, and accordingly one or more non-methylated cytosine basic groups of the DNAgathered on the carrier are converted into cytosine or other basic groups, different from cytosine, detectable in hybridization; 3) a combination solution is used for treating a mixed solution obtained in the second step, so that the converted DNA is gathered on the carrier again. The invention further provides a corresponding gene methylation detection method, a kit and application.

Description

technical field [0001] The invention belongs to the field of gene analysis. Specifically, the present invention relates to gene methylation detection methods, products and applications. Background technique [0002] DNA methylation is one of the most important epigenetic modifications in the regulation of gene transcription. This modification plays an important role in biological processes related to development and disease. Gene methylation is used as a molecular marker for early cancer diagnosis and monitoring of disease progression. In recent years, a variety of methods have been designed to detect and distinguish methylated sequences in normal and cancer samples. These methods are basically based on nucleic acid extraction, bisulfite conversion, and bisDNA (bisulfite-converted DNA) obtained after recovery for detection. [0003] A variety of commercial products have appeared on the market, including the extraction kit from Qiagen and the transformation and purificatio...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2523/308C12Q2523/125
Inventor 王弢张田田王冬华巴兆粉
Owner JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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