Ultrafast non-high-temperature DNA methylation conversion kit and use method thereof

Abstract

The invention discloses an ultra-fast non-high-temperature DNA methylation conversion kit and a use method thereof. The kit is composed of a conversion solution, a protection solution, a binding solution, a rinsing solution, a desulfonation solution, an eluent and a DNA adsorption column. The conversion liquid is prepared from ammonium hydrogen sulfite, sodium hydrogen sulfite and water; the protection liquid is prepared from hydroquinone and water; the binding liquid is prepared from guanidine hydrochloride, sodium chloride and water; the rinsing liquid is prepared from Tris, ethanol and water; the desulfonation liquid is prepared from sodium hydroxide, ethanol and water; the eluent is prepared from a Tris-HCl buffer solution, EDTA (Ethylene Diamine Tetraacetic Acid) and water; and the DNA adsorption column is made of a silicon matrix material. According to the method, the direct transformation time of DNA methylation can be remarkably shortened, meanwhile, the destructive effect of high temperature on a DNA sample is avoided in a non-high-temperature transformation mode, and high recovery rate and high transformation efficiency are guaranteed.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an ultra-fast non-high temperature DNA methylation conversion kit and a method for using the same. Background Art

[0002] DNA methylation is an epigenetic mechanism that adds a methyl group to the C-5 position of cytosine in dinucleotides through DNA methyltransferase, causing transcriptional activation or inhibition of different genes and regulating various cell functions. DNA methylation can change genetic expression without changing the DNA sequence. DNA methylation is a modification method of DNA epigenetics, which plays a regulatory role in gene expression. The DNA sequence before and after the gene promoter contains positive and negative regulatory units of gene expression. Generally speaking, the methylation of the functional unit sequence inhibits the function of the original functional unit. The genes that play an important role in tumor development include oncogenes (promoting tumor cell growth), tumor suppressor genes (inhibiting tumor cell growth), growth-promoting genes, and immune-regulating genes. Gene methylation detection has been increasingly used for early diagnosis of tumors. Generally speaking, its specificity and sensitivity are significantly higher than protein markers, and the presence of tumors can be detected earlier.

[0003] There are many methods for detecting DNA methylation levels, among which the most common methods are based on DNA bisulfite conversion technology. Unmethylated cytosine in DNA can be converted to uracil through bisulfite treatment, while methylated cytosine remains unchanged, thereby distinguishing methylated DNA from unmethylated DNA. Then, PCR, NGS and other methods are used to analyze which methylation sites of the DNA sequence to be tested have been methylated. Therefore, this technology can help researchers distinguish between methylated and unmethylated cytosine and provide single nucleotide resolution information for DNA methylation regions.

[0004] The current standard sulfite treatment takes a long time, requiring 3 to 4 hours to complete. In this way, together with nucleic acid extraction and PCR amplification, the entire methylation test generally requires a one-day test cycle, while clinicians and laboratories expect a PCR-based methylation test to be completed within a few hours. In addition, sulfite conversion at high temperatures will cause DNA to break and fragment, destroying the sample and causing a decrease in sample signal. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the technical defects of the background technology and provide an ultra-fast non-high temperature DNA methylation conversion kit and a method of using the same. The present invention provides a rapid, high-yield, high-conversion-efficiency DNA methylation conversion kit, which can significantly shorten the direct conversion time, and at the same time, through the non-high temperature conversion method, avoid the destructive effect of high temperature on DNA samples, and ensure high recovery rate and high conversion efficiency.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows: An ultra-fast non-high temperature DNA methylation conversion kit, the kit comprising a conversion solution, a protection solution, a binding solution, a rinsing solution, a desulfurization solution, an elution solution, and a DNA adsorption column; The conversion solution is prepared from ammonium bisulfite, sodium bisulfite, and water; The protective solution is prepared from hydroquinone and water; The binding solution is prepared from guanidine hydrochloride, sodium chloride, and water; The rinsing solution is prepared from Tris, ethanol, and water; The desulfurization liquid is prepared from sodium hydroxide, ethanol, and water; The eluent is prepared from Tris-HCl buffer, EDTA, and water; The DNA adsorption column is made of silicon matrix material; the silicon matrix material used in the DNA adsorption column can efficiently and specifically adsorb DNA and effectively remove impurity proteins.

[0007] Preferably, in the conversion solution, the concentration of the ammonium bisulfite is 1-6.5M, and the concentration of the sodium bisulfite is 1-6M.

[0008] Preferably, in the protective solution, the concentration of hydroquinone is 1-6M.

[0009] Preferably, in the combination solution, the concentration of the guanidine hydrochloride is 1-6M, and the concentration of the sodium chloride is 0.1-1M.

[0010] Preferably, in the rinse solution, the concentration of Tris is 1-2 M, and the concentration of ethanol is 70%-95% (v / v).

[0011] Preferably, in the desulfurization solution, the concentration of sodium hydroxide is 0.1% to 0.5%, and the concentration of ethanol is 70% to 95% (v / v).

[0012] Preferably, in the eluent, the concentration of the Tris-HCl buffer is 10-100 mM and the pH is 7.0-9.0, and the concentration of the EDTA is 0.5-2 mM.

[0013] The method for using the ultra-fast non-high temperature DNA methylation conversion kit as described above comprises the following steps: (1) Transformation: Add 50-200 μl of transformation solution, 20-40 μl of protection solution, and 10-50 μl of DNA sample to be processed into a PCR tube, mix well, and perform non-high temperature transformation treatment; (2) Binding: Add 300-800 μl of nucleic acid binding solution to the transformed product, mix well and transfer all the liquid to the DNA adsorption column; (3) First rinse: Add 300-800 μL of rinse solution to the adsorption column, centrifuge at 12000 rpm (~13400 × g) for 45 seconds, discard the filtrate, and place the adsorption column back in the collection tube; (4) Desulfonation: Add 300-800 μl of desulfonation solution to the adsorption column, place at room temperature for 5 min, centrifuge at 12000 rpm (~13400 × g) for 45 sec, discard the filtrate, and put the adsorption column back into the collection tube; (5) Second and third rinses: Add 300-800 μL of rinse solution to the adsorption column, centrifuge at 12000 rpm (~13400 × g) for 45 seconds, discard the filtrate, add 300-800 μL of rinse solution, and repeat this step once; (6) Drying: Place the adsorption column in a collection tube and centrifuge at 12,000 rpm (~13,400 × g) for 2 min to remove as much rinse solution as possible; transfer the adsorption column to the prepared 1.5 ml centrifuge tube and open the lid to dry; (7) Elution: Add 10~100µl of elution buffer for elution. After elution, place the sample in a new centrifuge tube and store it at -20℃.

[0014] Preferably, in step (1), the conversion temperature is 70-85°C, more preferably 80°C, because too low a conversion temperature will lead to insufficient conversion rate, and high temperature will destroy the DNA structure, resulting in fragmentation. 80°C can prevent DNA damage and achieve ultra-short time conversion; the conversion time is 30 minutes.

[0015] Preferably, in step (1), the DNA sample to be processed is one of human genomic DNA, animal genomic DNA, plant genomic DNA or free DNA in body fluids of the above species.

[0016] The reagent and method of the present invention can make the methylation pre-treatment sample complete the conversion at ultra-fast speed, while ensuring nucleic acid integrity and conversion efficiency, and is applicable to various types of samples, and the kit components can be transported and stored at room temperature, greatly reducing transportation and storage costs, effectively saving conversion time, and methylation conversion can be completed in only about 30min, greatly shortening the sample processing time. The kit of the present invention also includes a unique conversion protection liquid, which not only ensures the nucleic acid yield, but also optimizes the nucleic acid fragment length, can be applicable to downstream applications, and can fully meet market demand.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The reagent of the present invention has stable performance, simple preparation, convenient use and easy operation; (2) Due to the use of relevant protective agents, the kit of the present invention can complete the sulfite conversion effect at a constant temperature of 70-85°C, which can prevent DNA from being broken and fragmented due to high temperature, and shorten the DNA methylation conversion time to 30-40 minutes, greatly shortening the conversion time while ensuring the conversion efficiency; (3) The present invention has low requirements for instruments and equipment, and is suitable for research institutions at all levels to perform bisulfite conversion on DNA samples; the conversion rate of unmethylated cytosine to uracil is as high as over 99%, and the obtained nucleic acid after bisulfite treatment has relatively complete fragments and high conversion rate, and the product can be used for downstream PCR and NGS detection; (4) The kit components of the present invention can be stored at room temperature and can be transported at room temperature, greatly reducing labor costs; (5) The kit of the present invention can be used for various sample types: including genomic DNA from feces, genomic DNA from animals, genomic DNA from plants, or free DNA in the body fluids of the above species. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The CT values ​​of qPCR after the samples were transformed by the reagent of the present invention (Example 1) and the comparative kit (Comparative Example 1). DETAILED DESCRIPTION

[0019] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments and accompanying drawings. It should be understood that these embodiments are only used to further illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art may make some non-essential changes or adjustments to the present invention, which still belong to the protection scope of the present invention.

[0020] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art.

[0021] Example 1 An ultra-fast non-high temperature DNA methylation conversion kit, the kit comprising a conversion solution, a protection solution, a binding solution, a rinsing solution, a desulfurization solution, an elution solution, and a DNA adsorption column; The conversion solution is prepared from ammonium bisulfite, sodium bisulfite, and water; wherein the concentration of ammonium bisulfite is 3M, the concentration of sodium bisulfite is 6M, and finally the volume is fixed to 1000mL; The protective solution is prepared from hydroquinone and water; the concentration of hydroquinone is 3M, and the volume is finally fixed to 1000mL; The combination solution is prepared from guanidine hydrochloride, sodium chloride, and water; wherein the concentration of guanidine hydrochloride is 1M, the concentration of sodium chloride is 1M, and finally the volume is fixed to 1000mL; The rinsing solution is prepared from Tris, ethanol, and water; wherein the concentration of Tris is 1.5M, and the concentration of ethanol is 80% (v / v); The desulfurization solution is prepared from sodium hydroxide, ethanol, and water; wherein the concentration of sodium hydroxide is 0.5% and the concentration of ethanol is 70% (v / v); The eluent is prepared from Tris-HCl buffer, EDTA, and water; wherein the concentration of the Tris-HCl buffer is 10 mM, and the pH is 7.0-9.0, and the concentration of EDTA is 1 mM.

[0022] The DNA adsorption column is made of silicon matrix material and is commercially available (Biowax BW-BD1009T).

[0023] The method for using the ultra-fast non-high temperature DNA methylation conversion kit as described above comprises the following steps: taking a DNA sample, extracting, converting and purifying the DNA sample, and the specific implementation steps are as follows: (1) Transformation: Add 100 μl of transformation solution, 20 μl of protection solution and 20 μl of DNA sample to be processed into a PCR tube, mix well and perform constant temperature transformation treatment (80°C, 30 min); (2) Binding: Add 500 μl of nucleic acid binding solution to the transformed product, mix well and transfer all the liquid to the DNA adsorption column; (3) First rinse: Add 600 μL of rinse solution to the adsorption column, centrifuge at 12000 rpm (~13400 × g) for 45 seconds, discard the filtrate, and place the adsorption column back in the collection tube; (4) Desulfonation: Add 600 μl of desulfonation solution to the adsorption column, place at room temperature for 5 min, centrifuge at 12000 rpm (~13400 × g) for 45 sec, discard the filtrate, and put the adsorption column back into the collection tube; (5) Second and third rinses: Add 600 μl of rinse solution to the adsorption column, centrifuge at 12000 rpm (~13400 × g) for 45 seconds, discard the filtrate, add 600 μl of rinse solution, and repeat this step once; (6) Drying: Place the adsorption column in a collection tube and centrifuge at 12,000 rpm (~13,400 × g) for 2 min to remove as much rinse solution as possible; transfer the adsorption column to the prepared 1.5 ml centrifuge tube and open the lid to dry; (7) Elution: Add 30 µl of elution buffer and store the sample in a new centrifuge tube at -20°C.

[0024] In step (1), the DNA sample to be processed, i.e., the DNA template source is gDNA extracted from fresh tissue; the gDNA sample is extracted according to the instructions of the corresponding DNA extraction kit.

[0025] Two fresh tissue samples (with known clinical information) collected from clinical hospitals were selected for testing, as shown in Table 1.

[0026] Table 1 DNA templates used in the present invention

[0027] The present invention selects human internal reference gene ACTB as the detection object, and designs forward primer AF, reverse primer AR and probe AP for the internal reference gene (bisACTB) sequence after bisulfite conversion. The primer and probe sequences are shown in Table 2.

[0028] Table 2 Primer and probe sequences used in the present invention

[0029] The reaction system used in the present invention is shown in Table 3.

[0030] Table 3 Reaction system used in the present invention

[0031] The PCR reaction program used in the present invention is shown in Table 4.

[0032] Table 4 PCR reaction program used in the present invention

[0033] Comparative Example 1 Two fresh tissue DNA samples in Table 1 were selected and processed using the commercially available DNA bisulfite conversion kit (Cat. No. DP215) from Tiangen Biochemical Company (as a comparison reagent). The specific steps were strictly followed in accordance with the kit instructions.

[0034] Results Analysis By comparing Example 1 with Comparative Example 1, the present invention comprehensively compares the performance of the kit from the CT value of (bisACTB). The comparison of the test results of the kits of Example 1 and Comparative Example 1 is shown in Table 5.

[0035] Table 5 Comparison of test results of the kit of Example 1 and Comparative Example 1

[0036] Figure 1 It is a BisACTB detection curve diagram of DNA converted by treating a nucleic acid sample using the method described in the kit of Example 1 of the present invention and DNA converted using a commercially available nucleic acid methylation conversion kit (Tiangen Biotechnology) of Comparative Example 1.

[0037] The results show that the method of Example 1 of the present invention shortens the DNA conversion time. The comparison kit requires 60 minutes to complete the conversion when the DNA is >500 ng, while the present kit can complete the conversion in 40 minutes, while also ensuring the DNA conversion recovery rate.

[0038] The present invention provides a kit for pretreatment of gene methylation detection samples, which greatly shortens the direct conversion time and completes the entire process of DNA methylation conversion experiment within 30-40 minutes. The non-high temperature conversion method avoids the destructive effect of high temperature on DNA samples and ensures high recovery rate and high conversion efficiency.

[0039] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. An ultra-fast non-high temperature DNA methylation conversion kit, characterized in that: The kit is composed of a conversion solution, a protection solution, a binding solution, a rinsing solution, a desulfurization solution, an elution solution, and a DNA adsorption column; The conversion solution is prepared from ammonium bisulfite, sodium bisulfite, and water; The protective solution is prepared from hydroquinone and water; The binding solution is prepared from guanidine hydrochloride, sodium chloride, and water; The rinsing solution is prepared from Tris, ethanol, and water; The desulfurization liquid is prepared from sodium hydroxide, ethanol, and water; The eluent is prepared from Tris-HCl buffer, EDTA, and water; The DNA adsorption column is made of silicon matrix material.

2. An ultra-fast non-high temperature DNA methylation conversion kit as claimed in claim 1, characterized in that: In the conversion solution, the concentration of the ammonium bisulfite is 1-6.5M, and the concentration of the sodium bisulfite is 1-6M.

3. An ultra-fast non-high temperature DNA methylation conversion kit as claimed in claim 1, characterized in that: In the protective solution, the concentration of hydroquinone is 1-6M.

4. The ultra-fast non-high temperature DNA methylation conversion kit according to claim 1, characterized in that: In the combination solution, the concentration of guanidine hydrochloride is 1-6M, and the concentration of sodium chloride is 0.1-1M.

5. The ultra-fast non-high temperature DNA methylation conversion kit according to claim 1, characterized in that: In the rinse solution, the concentration of Tris is 1-2M, and the concentration of ethanol is 70%-95%.

6. The ultra-fast non-high temperature DNA methylation conversion kit according to claim 1, characterized in that: In the desulfurization solution, the concentration of the sodium hydroxide is 0.1% to 0.5%, and the concentration of the ethanol is 70% to 95%.

7. The ultra-fast non-high temperature DNA methylation conversion kit according to claim 1, characterized in that: In the eluent, the concentration of the Tris-HCl buffer is 10-100 mM and the pH is 7.0-9.0, and the concentration of the EDTA is 0.5-2 mM.

8. A method for using an ultra-fast non-high temperature DNA methylation conversion kit according to any one of claims 1 to 7, characterized in that: The steps include: (1) Transformation: Add 50-200 μl of transformation solution, 20-40 μl of protection solution, and 10-50 μl of DNA sample to be processed into a PCR tube, mix well, and perform non-high temperature transformation treatment; (2) Binding: Add 300-800 μl of nucleic acid binding solution to the transformed product, mix well and transfer all the liquid to the DNA adsorption column; (3) First rinse: Add 300-800 μL of rinse solution to the adsorption column, centrifuge at 12,000 rpm for 45 seconds, discard the filtrate, and place the adsorption column back in the collection tube; (4) Desulfonation: Add 300-800 μl of desulfonation solution to the adsorption column, place at room temperature for 5 min, centrifuge at 12,000 rpm for 45 sec, discard the filtrate, and place the adsorption column back into the collection tube; (5) Second and third rinses: Add 300-800 μL of rinse solution to the adsorption column, centrifuge at 12,000 rpm for 45 seconds, discard the filtrate, add 300-800 μL of rinse solution, and repeat this step once; (6) Drying: Place the adsorption column in a collection tube, centrifuge at 12,000 rpm for 2 min, and remove the rinse solution; transfer the adsorption column to the prepared 1.5 ml centrifuge tube and open the lid to dry; (7) Elution: Add 10~100µl of elution buffer for elution. After elution, place the sample in a new centrifuge tube and store it at -20℃.

9. The method for using the ultra-fast non-high temperature DNA methylation conversion kit according to claim 8, characterized in that: In step (1), the conversion temperature is 70-85°C, and the conversion time is 30 minutes.

10. The method for using the ultra-fast non-high temperature DNA methylation conversion kit according to claim 8, characterized in that: In step (1), the DNA sample to be processed is one of human genomic DNA, animal genomic DNA, plant genomic DNA or free DNA in body fluids of the above species.

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