Reagent combination, kit and method for protecting fragment integrity in DNA methylation detection

Abstract

The invention discloses a reagent combination for protecting fragment integrity in DNA methylation detection, the reagent combination is used for protecting fragment integrity in DNA methylation detection sample transformation, and the reagent combination comprises tetrahydrofurfuryl alcohol, L-ascorbyl palmitate and ethylenediamine tetraacetic acid; wherein the volume concentration of the tetrahydrofurfuryl alcohol is 10%-80%; the molar concentration of the L-ascorbyl palmitate is 10 mM to 100 mM; the molar concentration of the ethylenediamine tetraacetic acid is 1 mM to 10 mM. The invention also provides a kit and a method for DNA conversion by using the kit. According to the reagent combination, damage of sulfite to DNA in the transformation process can be reduced, DNA degradation is prevented, a transformed fragment is longer than that of a traditional transformation method, the DNA recovery concentration and the library concentration are higher, follow-up detection is facilitated, and the sensitivity of a follow-up detection method is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a reagent combination, a kit and a method for protecting fragment integrity in DNA methylation detection. Background Art

[0002] In the 1970s, scientists discovered the phenomenon of DNA methylation. DNA methyltransferases, through which methyl groups are added to specific DNA regions, primarily cytosine in CpG islands, to form 5-methylcytosine. Research has gradually revealed that DNA methylation plays a crucial role in numerous biological processes, including gene expression regulation, embryonic development, cell differentiation, and disease development.

[0003] Bisulfite has the property of reacting chemically with cytosine in DNA. Under specific conditions, bisulfite can deaminize unmethylated cytosine in DNA and convert it to uracil. However, methylated cytosine, protected by the methyl group, does not undergo this conversion. In 1992, Frommer et al. first reported a method for detecting methylation by treating DNA with bisulfite. By incubating DNA with a bisulfite solution under specific temperature and pH conditions, they successfully achieved the conversion of unmethylated cytosine. Then, combined with PCR amplification and sequencing techniques, they could determine the methylation status of the DNA sequence by detecting the presence of uracil.

[0004] However, there are some defects and deficiencies in bisulfite conversion, which are mainly reflected in the following aspects: 1. Impact on DNA Samples ① Sample damage and degradation: The bisulfite conversion process typically requires high temperatures and acidic conditions, which can easily lead to DNA chain breakage and degradation. This is especially true for long DNA fragments or poor-quality DNA samples, which can produce a large amount of DNA fragments, affecting the accuracy and integrity of subsequent PCR amplification and sequencing results.

[0005] ② Low recovery rate: During the bisulfite conversion process and subsequent purification steps, a large amount of DNA is often lost. Sometimes the DNA loss can be as high as 50% or even more. This is a serious problem for precious samples with small starting amounts. It may cause subsequent experiments to be unable to proceed due to insufficient DNA or the results to be inaccurate.

[0006] ③ Longer time: Traditional bisulfite conversion methods usually take a long time and may require overnight incubation. Zymo Research's commercial EZ DNA Methylation Gold Kit and EZ DNA Methylation-Lightning Kit also take 1-3 hours, which may also make DNA more susceptible to damage during long-term treatment.

[0007] 2. Limitations of Subsequent Analysis ① Increased difficulty in designing PCR detection primers: Too many short fragments may make the upstream and downstream sequences of the target region incomplete, making it difficult to find suitable primer binding sites, increasing the difficulty and complexity of primer design, which may lead to reduced primer specificity, nonspecific amplification, and affect amplification efficiency, reduced amplification efficiency, and the susceptibility of primer dimers.

[0008] ② Decreased accuracy of sequencing detection data: Too many short fragments may lead to inaccurate splicing during sequencing, resulting in a large number of sequencing errors and gaps, affecting the accurate interpretation of DNA sequences. Especially when detecting methylation sites, the methylation status may be misjudged, the genome coverage may be reduced, the sequencing depth may be uneven, and the bioinformatics analysis may be complicated.

[0009] Therefore, there is an urgent need to develop a reagent combination that can protect the integrity of fragments when converting DNA in DNA methylation detection. Summary of the Invention

[0010] In order to solve at least one of the above problems, the present invention provides a reagent combination, a kit and a method for protecting fragment integrity in DNA methylation detection.

[0011] In order to achieve the above object, the present invention adopts the following technical means: A first aspect of the present invention provides a reagent combination for protecting fragment integrity in DNA methylation detection, wherein the reagent combination protects fragment integrity during DNA methylation detection sample conversion, and the reagent combination includes tetrahydrofurfuryl alcohol, L-ascorbyl palmitate and ethylenediaminetetraacetic acid.

[0012] In some embodiments of the present invention, in the reagent combination, the volume concentration of tetrahydrofurfuryl alcohol is 10%-80%; the molar concentration of the L-ascorbyl palmitate is 10mM-100mM; and the molar concentration of the ethylenediaminetetraacetic acid is 1mM-10mM.

[0013] In some embodiments of the present invention, in the reagent combination, the molar ratio of L-ascorbyl palmitate to EDTA is 10:1.

[0014] In some preferred embodiments of the present invention, in the reagent combination, the volume concentration of tetrahydrofurfuryl alcohol is 80%; the molar concentration of L-ascorbyl palmitate is 100 mM; and the molar concentration of ethylenediaminetetraacetic acid is 10 mM.

[0015] The present invention also provides use of the reagent combination of the first aspect in preparing a kit for detecting DNA methylation.

[0016] A second aspect of the present invention provides a kit comprising the reagent combination described in the first aspect.

[0017] In some preferred embodiments of the present invention, the reagent combination includes tetrahydrofurfuryl alcohol, L-ascorbyl palmitate and ethylenediaminetetraacetic acid, including a volume concentration of 10%-80% tetrahydrofurfuryl alcohol, a molar concentration of 10mM-100mM L-ascorbyl palmitate and a molar concentration of 1mM-10mM ethylenediaminetetraacetic acid, and the molar ratio of L-ascorbyl palmitate to ethylenediaminetetraacetic acid is 10:1.

[0018] In some embodiments of the present invention, the conversion kit further comprises a conversion solution, a binding solution, a desulfurization solution, a washing solution, and an elution solution; The conversion solution contains bisulfite, and the bisulfite is selected from one or more of sodium bisulfite, potassium bisulfite, ammonium bisulfite or ammonium sulfite; The binding solution contains 5-8M guanidine hydrochloride, 0.5-1M sodium chloride, and 40-50% isopropanol, and is prepared using nuclease-free water; The desulfurization solution contains 0.5-1M sodium hydroxide, 0.5-1% Triton X-100, and 70-75% ethanol, and is prepared using nuclease-free water; The washing solution contains 15-20 mM Tris-HCl, 0.5-1 mM ethylenediaminetetraacetic acid, and 75-80% ethanol, and is prepared using nuclease-free water; The eluent contains 5-10 mM Tris-HCl and 0.1-0.15 mM ethylenediaminetetraacetic acid and is prepared with nuclease-free water.

[0019] In some embodiments of the present invention, the kit further comprises magnetic beads, which are silanol magnetic beads with a particle size of 400-600 nm.

[0020] The third aspect of the present invention provides a method for DNA transformation using the kit described in the second aspect, comprising the following steps: S1. Add the reagent combination described in the second aspect, the conversion solution described in the second aspect, and nuclease-free water to the sample DNA to be processed, and mix thoroughly and centrifuge; S2. The treated sample DNA is subjected to thermal cycling reaction on a PCR thermal cycler; S3. The magnetic beads described in the second aspect are added to the reaction product of step S2 and mixed with the binding solution described in the second aspect, followed by incubation reaction. The magnetic beads after the reaction are washed with a washing solution, and then mixed with a desulfonation solution and incubated for reaction. The magnetic beads after the reaction are washed with a washing solution, and then eluted with an elution solution to obtain a converted DNA sample.

[0021] In some embodiments of the present invention, in step S2, the thermal cycling reaction conditions are 95-98° C. and the reaction time is 4-6 min.

[0022] In some embodiments of the present invention, the step of washing with a washing solution is repeated 1-2 times.

[0023] In some embodiments of the present invention, the sample DNA to be processed is a sheared DNA fragment with a length of 180-220 bp.

[0024] In some embodiments of the present invention, the method comprises the following steps: S1. Add the sample DNA to be processed into centrifuge tube A, add the conversion solution, protective agent and nuclease-free water, shake the liquid thoroughly to mix, and centrifuge briefly to the bottom of the tube; S2. Transfer half of the liquid to centrifuge tube B, cover the tube, and centrifuge briefly to the bottom of the tube; S3, perform thermal cycling reaction on centrifuge tube B on a PCR thermal cycler; S4. Transfer the reaction product to centrifuge tube C, add magnetic beads and binding solution, mix well, and incubate at room temperature for 1-10 minutes; S5. After instant centrifugation, place centrifuge tube C on a magnetic rack and magnetically absorb for 1-10 minutes. After the solution in the centrifuge tube has clarified, carefully remove the supernatant. S6. Add washing solution to centrifuge tube C, mix well, centrifuge briefly, and place centrifuge tube C on a magnetic rack. Allow the magnetic rack to absorb for 1-10 minutes. After the solution in centrifuge tube C becomes clear, carefully remove the supernatant. S7. Add desulfurization solution to centrifuge tube C, mix well, and heat and incubate at 37°C for 5-20 minutes; S8. After instant centrifugation, place centrifuge tube C on a magnetic rack and magnetically absorb for 1-10 minutes. After the solution in centrifuge tube C becomes clear, carefully remove the supernatant. S9. Add washing solution to centrifuge tube C, mix well, centrifuge briefly, and place centrifuge tube C on a magnetic rack. Magnetize for 1-10 minutes. After the solution in the centrifuge tube is clear, carefully remove the supernatant. S10. Place centrifuge tube C on the magnetic rack and dry at room temperature for 1-10 min. S11. Add elution buffer to centrifuge tube C, mix well, centrifuge briefly, and then heat and elute at 37°C for 1-5 minutes. S12. Place centrifuge tube C on a magnetic stand for 1-5 minutes. After the solution in the centrifuge tube has clarified, carefully pipette the supernatant into centrifuge tube D to obtain the converted DNA sample.

[0025] The present application also provides a method for DNA methylation detection, which comprises first performing sample conversion processing using the method described in the third aspect, and then performing methylation analysis on the converted sample DNA: detecting the DNA methylation status by detecting uracil in the processed DNA sample.

[0026] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a reagent combination for protecting fragment integrity during DNA bisulfite conversion, including tetrahydrofurfuryl alcohol, L-ascorbyl palmitate and ethylenediaminetetraacetic acid. The protective agent composed of these components can reduce sulfite damage to DNA and prevent DNA degradation. In addition, the converted fragments are longer than those of traditional conversion methods, and the DNA recovery concentration and library concentration are higher, which is more conducive to subsequent detection and improves the sensitivity of subsequent detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Shown are the Qsep400 library fragment distribution diagrams of Example 1 and Comparative Examples 1-4 of the present invention; Figure 2 Shown are the Qsep400 library fragment distribution diagrams of Examples 2-5 of the present invention; Figure 3 The figure shows the distribution of Qsep400 library fragments under four different storage conditions in Example 6 of the present invention; Figure 4 Shown are the Qsep400 library fragment distribution diagrams of Example 7 and Comparative Examples 11-12 of the present invention; Figure 5 qPCR test results of Example 8 and Comparative Examples 13-14 of the present invention are shown; Figure 6 qPCR test results of two groups of experiments in Example 9 of the present invention are shown; Figure 7 The figure shows the qPCR detection results of two groups of experiments in Example 10 of the present invention. DETAILED DESCRIPTION

[0028] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs, and the disclosures herein and the materials they cite are hereby incorporated by reference. Those skilled in the art will recognize or be able to ascertain, through routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0030] The technical solution of the present application will be further described in detail below in conjunction with specific implementation methods.

[0031] Example 1 1. Reagent Preparation Conversion solution: 1.983 M sodium bisulfite, 0.917 M ammonium sulfite, 9.87 M ammonium bisulfite; Protective agent: 10 mM L-ascorbyl palmitate, 10% tetrahydrofurfuryl alcohol, 1 mM ethylenediaminetetraacetic acid; Magnetic beads: Si-hydroxy magnetic beads, mass fraction 15%, particle size 500 nm; Binding solution: 8 M guanidine hydrochloride, 1 M sodium chloride, 50% isopropanol, nuclease-free water; Desulfurization solution: 1 M sodium hydroxide, 1% Triton X-100, 75% ethanol, nuclease-free water; Washing solution: 20 mM Tris-HCl, 1 mM EDTA, 80% ethanol, nuclease-free water; Elution buffer: 10 mM Tris-HCl, 0.1 mM EDTA, nuclease-free water; 2. Sample DNA Pretreatment Take 100 ng of unmethylated lambda DNA and shear the main peak to about 200 bp using a Covaris M220 ultrasonic shearer for later use.

[0032] 3. Conversion Process (1) Add 170 μL of the above-prepared conversion solution and 10 μL of the protective agent to a 200 μL centrifuge tube, then add the pretreated sample DNA. If the volume is less than 200 μL, add nuclease-free water to make up the volume to 200 μL. Vortex the liquid thoroughly and centrifuge it to the bottom of the tube. (2) Divide the liquid into two equal parts into another 200 μL centrifuge tube, cover the tube, and centrifuge it to the bottom of the tube; (3) Perform thermal cycling on a PCR thermal cycler at 98°C for 5 min. (4) Transfer the reaction product to a 1.5 mL centrifuge tube, add 10 μL of magnetic beads and 600 μL of binding solution, shake or pipette to mix, and incubate at room temperature for 1-10 min; (5) After instant centrifugation, place the centrifuge tube on a magnetic rack and magnetically absorb it for 1-10 minutes. After the solution in the centrifuge tube is clear, carefully remove the supernatant. (6) Add 100-400 μL of freshly prepared washing solution to the centrifuge tube, shake or pipette to mix, centrifuge briefly, place the centrifuge tube on a magnetic rack, and magnetically absorb for 1-10 minutes. After the solution in the centrifuge tube is clear, carefully remove the supernatant; (7) Add 50-200 μL of desulfurization solution to the centrifuge tube, shake or pipette to mix, and heat and incubate at 37°C for 5-20 minutes; (8) After instant centrifugation, place the centrifuge tube on a magnetic rack and magnetically absorb it for 1-10 minutes. After the solution in the centrifuge tube is clear, carefully remove the supernatant; (9) Add 100-400 μL of freshly prepared washing solution to the centrifuge tube, shake or pipette to mix, centrifuge briefly, place the centrifuge tube on a magnetic rack, and magnetically absorb for 1-10 minutes. After the solution in the centrifuge tube is clear, carefully remove the supernatant; (10) Repeat step (7) twice; (11) Place the centrifuge tube on a magnetic rack and dry at room temperature for 1-10 min. (12) Add 20 μL of elution buffer to the centrifuge tube, shake or pipette to mix, centrifuge briefly, and heat at 37°C for 1-5 min. (13) Place the centrifuge tube on a magnetic stand for 1-5 min. After the solution in the centrifuge tube has clarified, carefully pipette 20 μL of the supernatant into a new 1.5 mL centrifuge tube.

[0033] 10 ng of converted DNA was used to construct a single-stranded methylation library using the Novozymes EpiArt® DNA Methylation Library Kit for Illumina V3. The library construction steps were carried out according to the instructions. The concentration of the converted DNA and the constructed library were tested, and the fragment distribution results are shown in Table 1. The fragment distribution of the Qsep400 library is shown in Figure 1 shown.

[0034] Comparative Example 1 The test samples, other reagents used in the transformation process, the transformation process, and the library construction process were the same as those in Example 1, except that no protective agent was added.

[0035] The concentration of the converted DNA and the library after construction were tested and the fragment distribution results are shown in Table 1. The fragment distribution of the Qsep400 library is shown in Figure 1 shown.

[0036] Comparative Example 2 The test samples, other reagents used in the transformation process, the transformation process, and the library construction process were the same as those in Example 1, except that the protective agent was 10 mM ascorbic acid.

[0037] The concentration of the converted DNA and the library after construction were tested and the fragment distribution results are shown in Table 1. The fragment distribution of the Qsep400 library is shown in Figure 1 shown.

[0038] Comparative Example 3 The test samples, other reagents used in the transformation process, the transformation process, and the library construction process were the same as those in Example 1, except that the protective agent was 10 mM hydroquinone.

[0039] The concentration of the converted DNA and the library after construction were tested and the fragment distribution results are shown in Table 1. The fragment distribution of the Qsep400 library is shown in Figure 1 shown.

[0040] Comparative Example 4 The test samples, other reagents used in the conversion process, the conversion process, and the library construction process were the same as in Example 1, except that the protective agent was 10 mM quinolone dimethacrylate, The concentration of the converted DNA and the library after construction were tested and the fragment distribution results are shown in Table 1. The fragment distribution of the Qsep400 library is shown in Figure 1 shown.

[0041] Table 1 DNA, library concentration and fragment distribution results after conversion in Example 1 and Comparative Examples 1-4

[0042] The results showed that the main peak of the fragment obtained by conversion with the protective agent composed of L-ascorbyl palmitate, tetrahydrofurfuryl alcohol and ethylenediaminetetraacetic acid in Example 1 was 360 bp, which was significantly longer than the 291 bp in Comparative Example 1, proving the protective effect of the protective agent combination on DNA fragments.

[0043] Example 2 The test samples, reagents used in the transformation process, and the transformation process and library construction process were the same as in Example 1, and a parallel experiment was performed once.

[0044] The concentration of the converted DNA and the library after construction were tested and the fragment distribution results are shown in Table 2. The fragment distribution of the Qsep400 library is shown in Figure 2 shown.

[0045] Example 3 The test samples, other reagents used in the transformation process, and the transformation and library construction processes were the same as in Example 1, except that the protective agents were 100 mM L-ascorbyl palmitate and 10 mM ethylenediaminetetraacetic acid; The concentration of the converted DNA and the library after construction were tested and the fragment distribution results are shown in Table 2. The fragment distribution of the Qsep400 library is shown in Figure 2 shown.

[0046] Example 4 The test samples, other reagents used in the transformation process, and the transformation and library construction processes were the same as in Example 1, except that the protective agent was 100 mM L-ascorbyl palmitate, 80% tetrahydrofurfuryl alcohol, and 10 mM EDTA. The concentration of the converted DNA and the library after construction were tested and the fragment distribution results are shown in Table 2. The fragment distribution of the Qsep400 library is shown in Figure 2 shown.

[0047] Example 5 The test samples, other reagents used in the conversion process, and the conversion and library construction processes were the same as those in Example 1, except that the protective agent was 80% tetrahydrofurfuryl alcohol and 10 mM ethylenediaminetetraacetic acid. The concentration of the converted DNA and the library after construction were tested and the fragment distribution results are shown in Table 2. The fragment distribution of the Qsep400 library is shown in Figure 2 shown.

[0048] Table 2 DNA, library concentration and fragment distribution results after conversion in Example 2-5

[0049] The results showed that the protective agent composed of tetrahydrofurfuryl alcohol, ethylenediaminetetraacetic acid and L-ascorbyl palmitate had an excellent protective effect on DNA fragments at different concentrations, and the main peak of the fragment reached above 360bp; when the reagent combination lacked tetrahydrofurfuryl alcohol, the main peak of the fragment was small, and when the reagent combination lacked L-ascorbyl palmitate, the DNA recovery concentration and library concentration were low; among them, the protective agent composed of 100mM L-ascorbyl palmitate, 80% volume concentration of tetrahydrofurfuryl alcohol, and 10mM ethylenediaminetetraacetic acid, under the joint action of all components, had the strongest protective effect on DNA fragments, with the main peak of the fragment reaching 370bp, and the DNA recovery concentration and library concentration were the highest, with the average fragment reaching 387.2bp.

[0050] Example 6 50 μg of unmethylated lambda DNA was sheared to about 200 bp using a Covaris M220 ultrasonic shearer as the test sample for this example and related comparative examples. 100 ng was used in each experiment. The storage conditions of the protective agent were investigated: The protective agent is 100 mM L-ascorbyl palmitate, 80% tetrahydrofurfuryl alcohol, and 10 mM ethylenediaminetetraacetic acid; Storage conditions are divided into four groups: ①37℃+light; ②37℃+dark; ③room temperature+light; ④room temperature+dark; DNA conversion test: Other reagents used in the conversion process, as well as the conversion process and library construction process were the same as in Example 1.

[0051] The concentration of the converted DNA and the library after construction were tested and the fragment distribution results are shown in Table 3. The fragment distribution of the Qsep400 library is shown in Figure 3 shown.

[0052] Comparative Example 5 The test samples, storage conditions of the protective agent, other reagents used in the transformation process, and the transformation and library construction processes were the same as those in Example 6, except that the protective agent was 100 mM L-ascorbyl palmitate.

[0053] The concentration of the converted DNA and the library after construction and the fragment distribution results are shown in Table 3.

[0054] Comparative Example 6 The test samples, storage conditions of the protective agent, other reagents used in the transformation process, and the transformation and library construction processes were the same as those in Example 6, except that the protective agent was 100 mM quinolone dimethacrylate.

[0055] The concentration of the converted DNA and the library after construction and the fragment distribution results are shown in Table 3.

[0056] Comparative Example 7 The test samples, storage conditions of the protective agent, other reagents used in the transformation process, and the transformation and library construction processes were the same as those in Example 6, except that the protective agent was 100 mM ascorbic acid.

[0057] The concentration of the converted DNA and the library after construction and the fragment distribution results are shown in Table 3.

[0058] Comparative Example 8 The test samples, storage conditions of the protective agent, other reagents used in the transformation process, and the transformation and library construction processes were the same as those in Example 6, except that the protective agent was 100 mM hydroquinone.

[0059] The concentration of the converted DNA and the library after construction and the fragment distribution results are shown in Table 3.

[0060] Comparative Example 9 The test samples, storage conditions of the protective agent, other reagents used in the transformation process, and the transformation and library construction processes were the same as those in Example 6, except that the protective agent was tetrahydrofurfuryl alcohol at a volume concentration of 80%.

[0061] The concentration of the converted DNA and the library after construction and the fragment distribution results are shown in Table 3.

[0062] Comparative Example 10 The test samples, storage conditions of the protective agent, other reagents used in the transformation process, and the transformation and library construction processes were the same as those in Example 6, except that the protective agent was 10 mM EDTA.

[0063] The concentration of the converted DNA and the library after construction and the fragment distribution results are shown in Table 3.

[0064] Table 3 DNA, library concentration and fragment distribution results after conversion in Example 6 and Comparative Examples 5-10

[0065] The results showed that the protectant composed of L-ascorbyl palmitate, tetrahydrofurfuryl alcohol, and EDTA was effective and stable under both light and 37°C conditions, with temperature and light having minimal impact on the protectant's effectiveness. Furthermore, when L-ascorbyl palmitate, tetrahydrofurfuryl alcohol, and EDTA were used alone, the post-conversion DNA concentration, library concentration, and fragment distribution were all inferior to those of the protectant composed of all three reagents. The three components of the reagent combination, L-ascorbyl palmitate, tetrahydrofurfuryl alcohol, and EDTA, exhibited significant synergistic effects.

[0066] Example 7 The protective agent, other reagents, transformation process, and library construction used in the transformation process were the same as those in Example 6, with the following differences: 1 μg of human genomic gDNA was used as the test sample, and the main peak was sheared to approximately 200 bp using a Covaris M220 ultrasonic shearer. 100 ng was used in each experimental group.

[0067] The concentration of the converted DNA and the library after construction were tested, and the fragments were detected as shown in Table 4. The fragments of the Qsep400 library were as follows: Figure 4 shown.

[0068] Comparative Example 11 The same test sample as in Example 7 was used for transformation using Zymo Research's EZ DNA MethylationGold Kit. The concentration of the transformed DNA and the constructed library were tested, and the fragments were analyzed. The results are shown in Table 4. The fragments of the Qsep400 library are shown in Table 4. Figure 4 shown.

[0069] Comparative Example 12 The same test sample as in Example 7 was used for transformation using Zymo Research's EZ DNA Methylation-Lightning Kit. The concentration of the transformed DNA and the constructed library were tested, and the fragments were analyzed. The results are shown in Table 4. The fragments of the Qsep400 library are shown in Table 4. Figure 4 shown.

[0070] Table 4 DNA, library concentration and fragment distribution results after conversion in Example 7, Comparative Examples 11 and 12

[0071] The results showed that compared with Zymo's GOLD and Lightning kits, the recovery rate, library yield, main peak size of fragments and average fragment size of Example 7 were all greater than those of the finished kits.

[0072] Example 8 Based on the sequence of the ACTB gene in the human genome, probe primers were designed according to the rule that unmethylated C is converted to U (amplified to T) for specific methylation qPCR detection. The reaction system consisted of 10 μL of 2× qPCR Mix, 1.8 μL of primers (10 μM), 3.2 μL of HO, and 5 μL of transformed DNA. The reaction was performed on an ABI7500 with the following protocol: 98°C for 3 minutes, followed by 45 cycles of (95°C for 10 seconds, 58°C for 30 seconds). The input for each transformation was 100 ng.

[0073] The protective agent, other reagents used in the transformation process and the transformation process are the same as those in Example 6. The transformed DNA is subjected to qPCR detection. The qPCR detection results are as follows: Figure 5 shown.

[0074] Comparative Example 13 The same test sample as in Example 8 was used for transformation using Zymo Research's EZ DNA MethylationGold Kit. The transformed DNA was subjected to qPCR detection. The qPCR detection results were as follows: Figure 5 shown.

[0075] Comparative Example 14 The same test sample as in Example 8 was used for transformation using Zymo Research's EZ DNA Methylation-Lightning Kit. The transformed DNA was subjected to qPCR detection. The qPCR detection results were as follows: Figure 5 shown.

[0076] The results showed that the CT value obtained by qPCR detection of DNA converted by the method of the present application was smaller, indicating that the subsequent detection sensitivity was higher and the conversion effect was better than that of commercially available finished test kits.

[0077] Example 9 Based on the promoter sequence of the Septin 9 gene in human genomic DNA, a CpG-rich fragment was identified. The corresponding primers and probes were designed and synthesized according to the primer and probe design principles for methylation-specific qPCR. They were named Septin 9-F, Septin 9-R, and Septin 9-P. Two aliquots of 10 ng of plasma-free DNA extracted from the blood of colorectal cancer patients, designated as samples 1 and 2, and two aliquots of plasma-free DNA extracted from the blood of healthy subjects, designated as samples 3 and 4, were used for the following two experiments.

[0078] Experimental group 1: Samples 1 and 3 were transformed using the EZ DNA Methylation-Lightning Kit, and the transformed DNA was detected by qPCR.

[0079] Experimental Group 2: The protective agent used in Samples 2 and 4, the other reagents used in the transformation process, and the transformation process were the same as in Example 6. The transformed DNA was subjected to qPCR detection. The qPCR detection results were as follows: Figure 6 shown.

[0080] Prepare the reaction system according to the formula in the following table:

[0081] The qPCR reaction was performed according to the following procedure using the ABI7500 qPCR instrument:

[0082] Experimental Group 2: The protective agent used in Samples 2 and 4, the other reagents used in the transformation process, and the transformation process were the same as in Example 6. The transformed DNA was subjected to qPCR detection. The qPCR detection results were as follows: Figure 6 shown.

[0083] The results showed that when plasma cell-free DNA was used as a sample, the DNA converted by the method of this application was subjected to specific methylation qPCR detection. Compared with the EZ DNA Methylation-Lightning Kit, the CT value was smaller and the subsequent detection sensitivity was higher.

[0084] Example 10 Based on the promoter sequence of the PAX 1 gene in human genomic DNA, a CpG-rich fragment was identified. Following the principles of methylation-specific qPCR primer and probe design, corresponding primers and probes were designed and synthesized, designated PAX 1-F, PAX 1-R, and PAX 1-P. Two aliquots (100 ng) of DNA extracted from cervical exfoliated cells of cervical cancer patients, designated Samples 5 and 6, and two aliquots of plasma-free DNA extracted from healthy individuals, designated Samples 7 and 8, were used for the following two sets of experiments.

[0085] Experimental Group 1: Samples 5 and 7 were transformed using the EZ DNA Methylation-Lightning Kit, and the transformed DNA was tested by qPCR.

[0086] Experimental Group 2: The protective agent used in samples 6 and 8, the other reagents used in the transformation process, and the transformation process were the same as in Example 6. The transformed DNA was subjected to qPCR detection. The qPCR detection results were as follows: Figure 7 shown.

[0087] Prepare the reaction system according to the formula in the following table:

[0088] The qPCR reaction was performed according to the following procedure using the Yarui MA6000 qPCR instrument:

[0089] Experimental Group 2: The protective agent used in samples 6 and 8, the other reagents used in the transformation process, and the transformation process were the same as in Example 6. The transformed DNA was subjected to qPCR detection. The qPCR detection results were as follows: Figure 7 shown.

[0090] The results showed that the subsequent detection effect of qPCR detection of DNA converted by the method of the present application was better than that of the EZ DNA Methylation-Lightning Kit on the market.

[0091] In Example 9 and Example 10, different qPCR instruments were used for detection, and the conclusions obtained were consistent.

[0092] In Examples 9 and 10, probe primers were designed based on the methylation status of the cancer patient's DNA. During the conversion process, methylated cytosine in the cancer patient's sample DNA would not be converted by the conversion reagent, whereas methylation did not occur in the DNA of a healthy person, and unmethylated cytosine would be converted by the reagent. When amplification was performed using the corresponding methylation-specific qPCR primer probes, the cancer patient's sample had an amplification curve, while the healthy person's blood sample did not.

[0093] Example 11 Two 100 ng DNA samples extracted from pancreatic tissue of pancreatic cancer patients were collected, designated as Sample 9 and Sample 10. The following two sets of experiments were performed.

[0094] Experimental Group 1: Sample 9 was transformed using the EZ DNA Methylation-Lightning Kit, and the concentration of the transformed DNA was quantified using the Novozymes ssDNA Qubit reagent. 10 ng of the transformed DNA was quantified using the Novozymes EpiArt ® The library was constructed using the DNA Methylation Library Kit for Illumina V3. The library concentration was quantified and the yield was calculated using the Novozymes dsDNA Qubit reagent, and then the library was sent to the Illumina platform for sequencing.

[0095] Experimental Group 2: The protective agent used for sample 10, the remaining reagents used in the transformation process, and the transformation process were the same as in Example 6. The library construction and quantification reagents were the same as in Experimental Group 1. The transformed DNA was quantified, library constructed, and sequenced. The sequencing results were analyzed by bioinformatics analysis (Insert Size Peak), as shown in Table 5: Table 5 Transformed DNA, library concentrations, and bioinformatics analysis results in experimental groups 1 and 2

[0096] The results showed that the recovery concentration, library concentration and fragment length obtained by the method of the present application were better than those of the EZDNA Methylation-Lightning Kit.

[0097] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the present application.

Claims

1. A reagent combination for protecting fragment integrity in DNA methylation detection, characterized in that: The reagent combination protects fragment integrity during the conversion process of DNA methylation detection samples, and the reagent combination includes tetrahydrofurfuryl alcohol, L-ascorbyl palmitate and ethylenediaminetetraacetic acid.

2. The reagent combination according to claim 1, characterized in that In the reagent combination, the volume concentration of tetrahydrofurfuryl alcohol is 10%-80%; the molar concentration of the L-ascorbyl palmitate is 10mM-100mM; and the molar concentration of the ethylenediaminetetraacetic acid is 1mM-10mM.

3. The reagent combination according to claim 1, characterized in that In the reagent combination, the volume concentration of tetrahydrofurfuryl alcohol is 80%; the molar concentration of L-ascorbyl palmitate is 100 mM; and the molar concentration of ethylenediaminetetraacetic acid is 10 mM.

4. Use of the reagent combination according to any one of claims 1 to 3 in preparing a kit for DNA methylation detection.

5. A kit, characterized in that: The kit comprises the reagent combination according to any one of claims 1 to 3.

6. The kit according to claim 5, wherein: The conversion kit also includes a conversion solution, a binding solution, a desulfurization solution, a washing solution and an elution solution; The conversion solution contains bisulfite, and the bisulfite is selected from one or more of sodium bisulfite, potassium bisulfite, ammonium bisulfite or ammonium sulfite; The binding solution contains 5-8M guanidine hydrochloride, 0.5-1M sodium chloride, and 40-50% isopropanol, and is prepared using nuclease-free water; The desulfurization solution contains 0.5-1M sodium hydroxide, 0.5%-1% Triton X-100, and 70%-75% ethanol, and is prepared using nuclease-free water; The washing solution contains 15-20 mM Tris-HCl, 0.5-1 mM ethylenediaminetetraacetic acid, and 75%-80% ethanol, and is prepared using nuclease-free water; The eluent contains 5-10 mM Tris-HCl and 0.1-0.15 mM ethylenediaminetetraacetic acid and is prepared with nuclease-free water.

7. The kit according to claim 6, wherein: The kit further comprises magnetic beads, which are silanol magnetic beads with a particle size of 400-600 nm.

8. A method for DNA transformation using the kit according to claim 7, characterized in that: The steps include: S1. Add the sample DNA to be processed into the reagent combination, conversion solution and nuclease-free water in the kit and mix thoroughly and centrifuge; S2. The treated sample DNA is subjected to thermal cycling reaction on a PCR thermal cycler; S3, the magnetic beads and the binding solution are added to the reaction product in step S2, mixed and incubated for reaction, the magnetic beads after the reaction are washed with a washing solution, and then added with a desulfonation solution and mixed and incubated for reaction, the magnetic beads after the reaction are washed with a washing solution, and then eluted with an elution solution to obtain a converted DNA sample.

9. The method according to claim 8, characterized in that: In step S2, the thermal cycle reaction conditions are 95-98° C. and the reaction time is 4-6 min.

10. The method according to claim 8, characterized in that: The sample DNA to be processed is a sheared DNA fragment with a length of 180-220 bp.

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