Composition for gene methylation detection, kit, application and detection method

Abstract

The invention discloses a composition for gene methylation detection, a kit, application and a detection method, the composition comprises: 50-100 mM Tris HCl, the pH value of which is 9.0; 0.5 to 2.5 mM of dNTPs (deoxyribonucleoside 100 to 300 mM of KCl; 7.5 to 15 mM of MgCl2; 2.5%-5% of DMSO (dimethylsulfoxide); 100 to 300 mM of tetramethylammonium chloride; 2.0 to 5.0 mM of Ectocaine; and 2.0 to 4.0 M of proline. By using the composition and the gene methylation detection method provided by the invention, the enzyme digestion reaction and the qPCR reaction can be simultaneously carried out in one tube, so that not only is the efficiency of the enzyme digestion reaction and the specificity of gene methylation detection ensured, but also nucleic acid damage and loss of a large amount of nucleic acid caused by a sulfite conversion process are avoided; the sensitivity of gene methylation detection is obviously improved; meanwhile, the detection method has the characteristics of simplicity in operation and short time consumption.

Description

Technical Field

[0001] This invention belongs to the field of DNA methylation detection, specifically relating to compositions, kits, uses, and detection methods for gene methylation detection. Background Technology

[0002] DNA methylation refers to the selective addition of a methyl group to the cytosine of the C and G nucleotides in DNA, catalyzed by methyltransferases, to form 5-methylcytosine (5-mC). Numerous studies have shown that DNA methylation can alter chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression.

[0003] A growing body of research indicates a close relationship between abnormal DNA methylation and the development of cancer. Both abnormal DNA hypermethylation and hypomethylation can lead to abnormal gene expression, thereby promoting cancer development.

[0004] Currently, most DNA methylation products on the market use traditional sulfite conversion and quantitative real-time PCR (qPCR) techniques for detection. Traditional sulfite conversion uses sulfite to convert unmethylated cytosine (C) in nucleic acids to uracil (U), while methylated cytosine remains unchanged. After conversion, methylated CpG islands and unmethylated CpG islands show significant sequence differences. qPCR can detect the presence of target gene methylation in nucleic acid samples by designing methylation-specific primers, and this method has extremely high specificity. However, sulfite conversion causes significant damage to nucleic acid samples and results in substantial nucleic acid loss, greatly affecting detection sensitivity. Furthermore, sulfite conversion has limitations such as complex procedures, long conversion time, and the tendency for conversion reagent residues, which are unfavorable for downstream PCR detection.

[0005] To avoid the aforementioned limitations, a methylation-sensitive restriction endonuclease is introduced. This endonuclease can specifically recognize the cleavage site and perform the cleavage reaction. However, it cannot perform cleavage when the cytosine at the cleavage site is methylated. Therefore, this detection method can distinguish between methylated and unmethylated DNA. This detection method uses a process of first digesting and then qPCR. By replacing the harsh bisulfite conversion reaction with a mild digestion reaction, the nucleic acid damage caused by bisulfite conversion can be avoided. However, the reagents for the digestion reaction are generally incompatible with PCR reactions, and the nucleic acid still needs to be purified and washed after the digestion reaction, which carries the risk of nucleic acid loss. Furthermore, the process is cumbersome and time-consuming.

[0006] Therefore, it is essential to develop a detection method that allows for simultaneous enzyme digestion and qPCR reactions in the same tube. Summary of the Invention

[0007] To address at least one of the aforementioned problems, this invention provides a composition, kit, application, and detection method for gene methylation detection. Using the composition, kit, application, and detection method provided by this invention, enzyme digestion and qPCR reactions can be performed simultaneously in the same tube, ensuring the sensitivity and specificity of gene methylation detection. Furthermore, this detection method also boasts the advantages of simple operation and short processing time.

[0008] To achieve the above objectives, the present invention employs the following technical means: A first aspect of the present invention provides a composition for detecting gene methylation, the composition comprising: 50-100 mM Tris HCl, pH 9.0; 0.5-2.5 mM dNTPs; 100-300 mM KCl; 7.5-15 mM MgCl2; 2.5%-5% DMSO; 100-300 mM tetramethylammonium chloride; 2.0-5.0 mM ectoine; and 2.0-4.0 M proline.

[0009] In some embodiments of the present invention, the composition comprises: 80-100 mM Tris HCl, pH 9.0; 1-2 mM dNTPs; 200-300 mM KCl; 10-15 mM MgCl2; 3.0%-5.0% DMSO; 200-300 mM tetramethylammonium chloride; 2.0-3.0 mM ectoine; and 2.0-3.0 M proline.

[0010] In some embodiments of the present invention, the composition comprises: 100 mM Tris HCl, pH 9.0; 1.5 mM dNTPs; 300 mM KCl; 15 mM MgCl2; 3% DMSO; 250 mM tetramethylammonium chloride; 3.0 mM ectoine; and 2.5 M proline.

[0011] A second aspect of the invention provides the use of the composition described in the first aspect in the preparation of a gene methylation detection kit.

[0012] The present invention provides the use of the composition described in the first aspect in the preparation of a one-step gene methylation detection kit.

[0013] In this invention, the one-step gene methylation detection refers to mixing the nucleic acid sample to be tested with a reaction solution prepared using the composition of this invention, and simultaneously performing an enzyme digestion reaction and a qPCR reaction.

[0014] A third aspect of the present invention provides a kit for detecting gene methylation, the kit comprising the composition described in the first aspect.

[0015] In some embodiments of the present invention, the kit further includes a methylation-sensitive restriction endonuclease and a Taq enzyme. The methylation-sensitive restriction endonuclease includes, but is not limited to, one or more of HhaI, HpaII, and HinP1I.

[0016] In some embodiments of the present invention, the kit further includes nucleic acid primers and probes.

[0017] A fourth aspect of the present invention provides a method for detecting gene methylation based on the composition described in the first aspect, comprising the following steps: A one-step enzyme digestion-qPCR reaction mixture was prepared using the composition described in the first aspect; Set the reaction program, add the nucleic acid sample to be tested and the one-step enzyme digestion-qPCR reaction mixture to the reaction well, perform the test, and output the results.

[0018] In some embodiments of the present invention, the method includes preparing a one-step enzyme digestion-qPCR reaction mixture using a composition, a target gene forward primer, a reverse primer, a probe, a methylation-sensitive restriction endonuclease, a Taq enzyme, and water.

[0019] In some embodiments of the present invention, the composition accounts for 15%-20% of the final reaction system.

[0020] In some embodiments of the present invention, the concentration of the target gene forward primer and reverse primer in the final reaction system is 0.2–0.3 μM, and the volume ratio of the target gene forward primer, reverse primer and probe is 2:2:1.

[0021] In some embodiments of the present invention, the methylation-sensitive restriction endonuclease has a volume of 0.2-0.5 U / μL in the final reaction system.

[0022] In some embodiments of the present invention, the concentration of the Taq enzyme in the final reaction system is 0.075-0.125 U / μL.

[0023] In this invention, the "final reaction system" refers to a reaction system that can be directly used to simultaneously perform enzyme digestion and PCR on the test sample.

[0024] In some embodiments of the present invention, the forward and reverse primers for the target gene are not modified, the 5' end of the probe is modified with a reporter fluorescent group, and the 3' end is modified with a quencher group.

[0025] In some embodiments of the present invention, the reporting fluorescent group is selected from FAM, Cy5, VIC, ROX, and Cy3; the quenching group is selected from BHQ1, BHQ2, and BHQ3.

[0026] In some embodiments of the present invention, the reaction procedure includes an enzyme digestion reaction procedure and a qPCR reaction procedure.

[0027] In some embodiments of the present invention, the enzymatic digestion reaction program is 25-37℃ for 10-60 min; 80℃ for 15-25 min.

[0028] In some embodiments of the present invention, the qPCR reaction program is 95℃ for 1 min; 95℃ for 30 s-1 min, 60℃ for 45 s-1 min, for 38-45 cycles.

[0029] In some embodiments of the present invention, preferably, the enzyme digestion reaction program is 37°C for 60 min; 80°C for 20 min; the qPCR reaction program is 95°C for 1 min; 95°C for 30 s, 60°C for 1 min, for 40 cycles.

[0030] In some embodiments of the present invention, probe fluorescence signals are collected at 60°C during the qPCR reaction stage.

[0031] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: The composition and method for gene methylation detection provided by this invention avoid nucleic acid damage and loss of a large amount of nucleic acid during the sulfite conversion process, and significantly improve the sensitivity of gene methylation detection. At the same time, the composition provided by this invention has excellent enzyme digestion efficiency in the enzyme digestion reaction, ensuring the specificity of gene methylation detection.

[0032] Furthermore, the gene methylation detection method provided by this invention can perform enzyme digestion and qPCR reactions in the same tube, and is characterized by simple operation and short time consumption. It avoids the problems of cumbersome procedures and long time consumption caused by the sulfite conversion method and the detection method of enzyme digestion followed by qPCR. This method ensures the efficiency and specificity of one-step gene methylation detection. Compared with the bisulfite conversion method, the detection method of this invention avoids nucleic acid damage during the sulfite conversion process and nucleic acid loss caused by the purification and washing steps, and significantly improves the detection sensitivity. Attached Figure Description

[0033] Figure 1 The results of Ct detection of the target TWIST1 at different concentrations and different compositions of positive reference samples in Example 1 are shown. Figure 2 The Ct detection results of different compositions of the target TWIST1 negative reference in Example 1 are shown; Figure 3 The Ct results for the positive reference, negative reference, and blank control in different compositions of the target TWIST1 in Example 2 are shown. Detailed Implementation

[0034] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials disclosed herein and cited therein are incorporated herein by reference. Many equivalent techniques of specific embodiments of the invention described herein will be recognized or can be understood by ordinary experimentation by those skilled in the art. These equivalents will be included in the claims.

[0036] The technical solution of this application will be further described in detail below with reference to specific embodiments.

[0037] Example 1 1. Target TWIST was selected as the target gene for testing, and ACTB was selected as the internal control gene. The amplification fragments and primer / probe designs are shown in the table below:

[0038] 2. Reference sample: (1) Positive references: References of different concentrations were prepared using human hyper-methylation reference standard average meththy≈100% methylated DNA from Kebai Biotechnology. The specific concentrations were 0.2 ng / μL, 0.1 ng / μL, 0.02 ng / μL and 0.01 ng / μL.

[0039] (2) Negative reference: Human genomic DNA with no target gene methylation verified by sequencing, at a concentration of 2.0 ng / μL.

[0040] 3. Preparation of different compositions: According to the table below, compositions-1 and-2, which can simultaneously perform enzyme digestion and qPCR reactions, are prepared as follows: Composition-1: 100 mM Tris HCl (pH 9.0); 1.5 mM dNTPs; 300 mM KCl; 15 mM MgCl2; 3% DMSO.

[0041] Composition-2: 100mM Tris HCl (pH 9.0); 1.5mM dNTPs; 300mM KCl; 15mM MgCl2; 3% DMSO; 250mM tetramethylammonium chloride; 3mM ectoine; 2.5M proline.

[0042] 4. Detection Method According to the table below, prepare the reaction mixture that can simultaneously perform enzyme digestion and qPCR, as follows:

[0043] Methylation-sensitive restriction endonucleases (MIXs) include, but are not limited to, one or more of HhaI, HpaII, and HinP1I.

[0044] After preparation, use a pipette to thoroughly mix the prepared MIX.

[0045] 5. Sample addition and loading: (1) Add the positive / negative reference samples to the PCR reaction tubes, 5 μL per well; then add the reaction MIX prepared in step 4 above, 15 μL per well, cover the PCR reaction tubes, centrifuge briefly, and then run the PCR reaction tubes.

[0046] (2) Place the above PCR reaction tubes into a real-time PCR instrument and select the appropriate detection channel: select the FAM channel to detect TWIST1; select the Cy5 channel to detect the internal reference gene ACTB (without restriction enzyme site).

[0047] (3) Set up the qPCR reaction program as follows:

[0048] FAM and Cy5 signals were collected at 60°C during the PCR phase.

[0049] 6. Data export and test results: (1) After the PCR reaction is completed, the instrument automatically saves the results. The instrument can be automatically analyzed using its built-in software (or the baseline and threshold can be manually adjusted for analysis) and the Ct value can be exported.

[0050] (2) The following are the mean Ct values ​​for different sample types, concentrations, and target detections, as detailed in the table below:

[0051] 7. Data Conclusions: Experimental results are as follows Figure 1 and Figure 2 As shown.

[0052] The results show that when using the composition-2 provided by the present invention to detect the positive reference material, the TWIST1 gene can be detected under the condition of 0.01 ng / μL positive reference material. Moreover, the Ct values ​​of the positive reference material at different concentrations are all better for composition-2 than those of composition-1, that is, the detection performance of composition-2 provided by the present invention is significantly better.

[0053] Under negative reference conditions (2.0 ng / μL), the mean Ct value of the TWIST1 gene detected using the composition-1 provided by this invention was 37.4, indicating that the enzyme digestion efficiency was insufficient under the composition-1 condition, resulting in some targets not being cleaved by the restriction endonuclease; while under the composition-2 condition, the TWIST1 gene was not detected (not detected is replaced by Ct value = 40.0), indicating that the enzyme digestion reaction efficiency was better under this condition.

[0054] The combined results of positive and negative reference samples indicate that the composition-2 provided by this invention exhibits excellent detection sensitivity and specificity.

[0055] Example 2 Based on the optimal composition-2 in Example 1, we further investigated the key substances in composition-2 that affect the sensitivity and specificity of the one-step detection method.

[0056] 1. Detection method: In this scheme, a control was performed compared to composition-2: Composition-2: 100mM Tris HCl (pH 9.0); 1.5mM dNTPs; 300mM KCl; 15mM MgCl2; 3% DMSO; 250mM tetramethylammonium chloride; 3mM ectoine; 2.5M proline.

[0057] The detection method and steps are the same as in Example 1, except that the positive reference concentration is 2.0 ng / μL; and the composition formulation is different, as detailed below: Composition-3: 100mM Tris HCl (pH 9.0); 1.5mM dNTPs; 300mM KCl; 15mM MgCl2; 3% DMSO; 3mM ectoine; 2.5M proline.

[0058] Composition-4: 100mM Tris HCl (pH 9.0); 1.5mM dNTPs; 300mM KCl; 15mM MgCl2; 3% DMSO; 250mM tetramethylammonium chloride; 2.5M proline.

[0059] Composition-5: 100 mM Tris HCl (pH 9.0); 1.5 mM dNTPs; 300 mM KCl; 15 mM MgCl2; 3% DMSO; 250 mM tetramethylammonium chloride; 3 mM ectoine.

[0060] 2. Data export and test results: (1) After the PCR reaction is completed, the instrument automatically saves the results. The instrument can be automatically analyzed using its built-in software (or the baseline and threshold can be manually adjusted for analysis) and the Ct value can be exported.

[0061] (2) The mean Ct values ​​for different target detections of different sample types in this embodiment are shown in the table below:

[0062] 3. Data Conclusions: Under positive reference (2.0 ng / μL) and negative reference (2.0 ng / μL) conditions, the differences between different compositions were detected using the TWIST1 gene assay. Specific test results are as follows: Figure 3 As shown.

[0063] The results showed that (1) Composition-3 reduced tetramethylammonium chloride compared to composition-2. In the positive reference, the detection Ct value of composition-3 was significantly earlier than that of composition-2, but the detection Ct value was also earlier in the blank test; indicating that the reduction of tetramethylammonium chloride in composition-2 led to a significant decrease in the specificity of PCR detection.

[0064] (2) Compared with composition-2, composition-4 has reduced ectoine. In the positive reference, the detection Ct value of composition-4 is significantly later than that of composition-2, that is, the reduction of ectoine in the composition leads to poor PCR amplification performance.

[0065] (3) Composition-5 reduced proline compared to composition-2. The detection results of composition-5 and composition-2 in the positive reference were not significantly different, but the Ct value of composition-5 was detected much earlier in the negative reference, indicating that the enzyme digestion efficiency of composition-5 was worse during the enzyme digestion reaction.

[0066] In summary, reducing tetramethylammonium chloride in Composition-2 leads to decreased PCR detection specificity, reducing ectoine leads to decreased PCR amplification performance, and reducing proline leads to decreased enzyme digestion reaction. Therefore, tetramethylammonium chloride, ectoine, and proline are the key substances in the composition, and the three substances have a synergistic effect. The effect is best when all three are present.

[0067] Example 3 This embodiment explores the differences in effectiveness between the reagent kit and detection method of the present invention and the bisulfite conversion method.

[0068] 1. Preparation of samples with different methylation rates Positive and negative reference samples were mixed in different proportions to prepare samples with different methylation rates: Sample-1, Sample-2, Sample-3, Sample-4, Sample-5, Sample-6, and Sample-7. The total nucleic acid concentration was consistent across all samples. See the table below for details:

[0069] 2. Detection method of the present invention: The method and steps are the same as in Example 1, except that in this example, only composition 2 is used for testing.

[0070] 3. Sulfite conversion method: (1) Sulfite conversion: The ZYMO EZ DNA Methylation-Gold™ Kit was used to convert samples with different methylation rates prepared in step 1 to sulfite.

[0071] Take 20 μL of the sample prepared in step 1 for sulfurous conversion, strictly follow the instructions of the kit, and finally elute with 20 μL.

[0072] (2) Target primer design: The detection primers used after sample processing via sulfite conversion are different from those used in the detection method involved in this invention. The amplification fragments of TWIST1 and ACTB used in the sulfite conversion method, and their primer / probe design, are as follows:

[0073] (3) qPCR detection: ① Preparation of the reaction mixture:

[0074] ② Sample addition and instrument loading: Add 5 μL of the sulfite-converted nucleic acid sample to each PCR reaction tube; then add 15 μL of the above PCR reaction MIX to each well, cap the PCR reaction tube, centrifuge briefly, and then run the PCR reaction tube.

[0075] ③ Set up the qPCR reaction program as follows:

[0076] FAM and Cy5 signals were collected at 60°C during the PCR phase.

[0077] (4) Data export: After the qPCR reaction is completed, the instrument automatically saves the results, which can be automatically analyzed using the instrument's built-in software (or manually analyzed by adjusting the baseline and threshold). The detection Ct values ​​corresponding to the samples from the sulfite conversion method and the detection method involved in this invention are then exported.

[0078] (5) Test results and conclusions: Samples with different methylation rates were tested using the kit and detection method of this invention and the traditional sulfite conversion method, respectively. The results are shown in the table below.

[0079]

[0080] According to the CLSI EP17 standard, the limit of detection (LoD) threshold is 95%. Based on the above test results, the respective TWIST1 limits of detection for the two methods at a DNA concentration of 2.0 ng / μL can be determined: 0.25% for the method of this invention, and 1% for the sulfite conversion method.

[0081] The results showed that the detection capability of the method of the present invention is superior to that of the bisulfite conversion method. This may be because the chemical reaction and purification steps of the bisulfite conversion method cause significant loss of methylated DNA, which is not conducive to the detection of low copy number targets. In contrast, the method of the present invention reduces DNA damage through gentler enzymatic treatment and avoids nucleic acid loss caused by purification steps through a one-step reaction, ultimately improving the sensitivity of DNA methylation detection.

[0082] Furthermore, in this embodiment, the detection method of the present invention and the sulfite conversion method were used, and no methylation rate was detected in the sample (sample-7) and the blank sample, indicating that the detection method of the present invention and the traditional sulfite conversion method both have good specificity.

[0083] Example 4 A kit for detecting gene methylation, comprising a composition for detecting gene methylation, a methylation-sensitive restriction endonuclease, and a Taq enzyme.

[0084] The composition for gene methylation detection includes: 50-100mM Tris HCl, pH 9.0; 0.5-2.5mM dNTPs; 100-300mM KCl; 7.5-15mM MgCl2; 2.5%-5% DMSO; 100-300mM tetramethylammonium chloride; 2.0-5.0mM ectoine; and 2.0-4.0M proline.

[0085] Methylation-sensitive restriction endonucleases include, but are not limited to, one or more of HhaI, HpaII, and HinP1I.

[0086] The method for gene methylation detection using the above kit is the same as in Example 1.

[0087] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by this application.

Claims

1. A composition for the detection of gene methylation, characterized in that: The composition comprises: 50-100 mM Tris HCl, pH 9.0; 0.5-2.5 mM dNTPs; 100-300 mM KCl; 7.5-15 mM MgCl2; 2.5%-5% DMSO; 100-300 mM tetramethylammonium chloride; 2.0-5.0 mM ekdotin; 2.0-4.0 M proline.

2. The composition for gene methylation detection according to claim 1, characterized in that: The composition comprises: 80-100 mM Tris HCl, pH 9.0; 1-2 mM dNTPs; 200-300 mM KCl; 10-15 mM MgCl2; 3.0%-5.0% DMSO; 200-300 mM tetramethylammonium chloride; 2.0-3.0 mM ekdotin; 2.0-3.0 M proline.

3. Use of the composition of any one of claims 1-2 in the preparation of a kit for detecting gene methylation.

4. A kit for detection of genetic methylation, characterized by: The kit comprises the composition of any one of claims 1-2.

5. The kit for detecting gene methylation according to claim 4, characterized in that: The kit further comprises a methylation-sensitive restriction enzyme and Taq enzyme.

6. A method for detecting methylation of a gene based on the composition according to any one of claims 1 to 2, characterized in that, The method comprises the following steps: The one-step enzyme digestion-qPCR reaction mixture is prepared by using the composition of any one of claims 1-2. The reaction program is set, the nucleic acid sample to be detected and the one-step enzyme digestion-qPCR reaction mixture are added into the reaction well, and the machine is detected to output the result.

7. The method of claim 6, wherein: The method comprises using the composition, the forward primer and the reverse primer of the target gene, the probe, the methylation-sensitive restriction enzyme, the Taq enzyme, and water to prepare the one-step enzyme digestion-qPCR reaction mixture.

8. The method of claim 6, wherein: The reaction program comprises an enzyme digestion reaction program and a qPCR reaction program.

9. The method of claim 8, wherein: The enzyme digestion reaction program is 25-37℃ for 10-60 min; 80℃ for 15-25 min.

10. The method of claim 8, wherein: The qPCR reaction program is 95℃ for 1 min; 95℃ for 30 s-1 min, 60℃ for 45 s-1 min, 38-45 cycles.

11. The method according to any one of claims 6-10, characterized by: The probe signal is collected at 60℃ in the qPCR reaction stage.

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