Complex enzyme digestion method for enriching methylated DNA (Deoxyribose Nucleic Acid) fragments

Abstract

The invention discloses a compound enzyme digestion method for enriching methylated DNA fragments. The compound enzyme digestion method for enriching methylated DNA fragments comprises the step of simultaneously carrying out methylated sensitive restriction enzyme digestion and single-stranded nucleic acid digestion in one reaction. According to the compound enzyme digestion method for enriching methylated DNA fragments, provided by the invention, single-stranded nuclease is introduced, so that methylated sensitive restriction enzyme digestion and single-stranded nucleic acid digestion are simultaneously carried out in one reaction, the interference of nucleic acid fragmentation on MSRE is effectively eliminated, and the accuracy of methylation detection based on MSRE is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemistry and molecular biology, and particularly relates to a complex enzyme cutting method for enriching methylated DNA fragments. BACKGROUND

[0002] As one of the core mechanisms of epigenetic modification, DNA methylation plays a key role in biological processes such as gene expression regulation, embryonic development, and tumor occurrence. Accurate detection of DNA methylation status has become a basic requirement for epigenetic research, and is also an important technical support for clinical applications such as tumor early screening and prenatal diagnosis. Existing methylation detection technologies mainly include three categories: chemical modification based on bisulfite conversion, enrichment based on methylation binding proteins, and enzyme cutting based on methylation sensitive restriction enzymes (MSRE). Among them, the MSRE enzyme (methylation sensitive restriction enzyme) cutting method is widely used in qPCR, capillary electrophoresis (CE) and next-generation sequencing (NGS) platforms due to its advantages of simple operation, strong specificity, controllable cost, etc.

[0003] The MSRE enzyme (methylation sensitive restriction enzyme) realizes methylation enrichment by specifically recognizing non-methylated sites and cutting double-stranded DNA. The core principle is that when a specific CpG site in the DNA chain is not methylated, the MSRE (such as HpaII, HhaI, etc.) can cut the site, while the methylated DNA resists enzyme cutting. After enzyme cutting treatment, the uncut methylated DNA fragments in the DNA sample can be selectively enriched through subsequent detection methods (such as qPCR amplification or NGS library construction). However, the traditional MSRE enzyme (methylation sensitive restriction enzyme) cutting method has significant defects in practical application - the insufficient enzyme cutting efficiency leads to an increased risk of false positives, severely restricting the detection sensitivity and reliability. This is also the main reason for restricting the large-scale application of MSRE method.

[0004] The incomplete cutting of the MSRE enzyme (methylation-sensitive restriction enzyme) can be attributed to two aspects: one is the restriction of enzyme cutting kinetics. The cutting efficiency of MSRE on double-stranded DNA is affected by enzyme activity, reaction time and substrate concentration and other factors. In the conventional operation, an excessive amount of enzyme preparation is used and the reaction time is prolonged to 4-6 hours. However, the increase of the amount of enzyme can significantly increase the experimental cost, and the prolongation of the time is easy to cause DNA degradation, especially irreversible damage to trace samples (such as cfDNA). In addition, single-stranded DNA interference can be caused by mechanical shearing, thermal denaturation or chemical treatment during DNA extraction, storage and transportation, which is easy to produce single-stranded regions or single-stranded hybrid structures. Since most MSRE (such as HpaII and HhaI) can only act on completely double-stranded DNA with high efficiency, the presence of single-stranded regions leads to inefficient recognition of target sites and only low-efficiency enzyme cutting, forming an "enzyme cutting blind area", thereby causing incomplete cutting of MSRE.

[0005] In order to break through the bottleneck of methylation detection technology, it is urgent to develop a methylation-sensitive restriction enzyme cutting optimization scheme which can ensure accuracy when detecting trace samples and has high efficiency, economy and platform compatibility. SUMMARY

[0006] In view of the above-mentioned deficiencies of the current methylation detection technology, the present application provides a composite enzyme cutting method for enriching methylated DNA fragments, so that the methylation-sensitive restriction enzyme cutting has high efficiency, economy and platform compatibility.

[0007] To achieve the above-mentioned purpose, the embodiments of the present application adopt the following technical solutions: A composite enzyme cutting method for enriching methylated DNA fragments, which comprises simultaneously performing methylation-sensitive restriction enzyme cutting and single-stranded nuclease digestion in one composite enzyme cutting reaction.

[0008] According to an aspect of the present application, the methylation-sensitive restriction enzyme uses a combination of one or both of HpaII and HhaI.

[0009] According to an aspect of the present application, the single-stranded nuclease uses a combination of one or more of exonuclease I, exonuclease VII, exonuclease T, S1 nuclease, P1 nuclease and mung bean nuclease.

[0010] According to an aspect of the present application, the single-stranded nuclease uses one or more of exonuclease I, exonuclease VII and mung bean nuclease which are compatible with the MSRE digestion system.

[0011] According to an aspect of the present application, the single-strand nuclease is used in combination with exonuclease I and mung bean nuclease based on the methylation-sensitive endonuclease; Alternatively, exonuclease I, exonuclease VII and mung bean nuclease are used in combination based on the methylation-sensitive endonuclease.

[0012] According to an aspect of the present application, the complex enzyme digestion method for enriching methylated DNA fragments uses 7.5 U / ul HpaII, 7.5 U / ul HhaI, 0.1-0.5 U / ul ExoI and 0.05-0.2 U / ul MBase.

[0013] According to an aspect of the present application, the digestion product obtained after simultaneous methylation-sensitive restriction endonuclease digestion and single-strand nuclease digestion is amplified by multiplex fluorescent PCR.

[0014] According to an aspect of the present application, the multiplex fluorescent PCR system comprises 2x amplification buffer 25ul, DNA polymerase 20U, primer mixture 250nM and MSRE digestion product 15ul.

[0015] According to an aspect of the present application, the multiplex fluorescent PCR system comprises a control site UBC without methylation digestion site and five detection sites with methylation digestion site, and the detection sites with methylation digestion site include SDHA, septin9, SDC2, NPY10 and IKZF.

[0016] According to an aspect of the present application, the primer sequence of the control site UBC without methylation digestion site is shown in SEQ NO. 4-SEQ NO. 6 and SEQ NO. 17; the primer sequence of the detection site SDHA with methylation digestion site is shown in SEQ NO. 1-SEQ NO. 3 and SEQ NO. 7-SEQ NO. 8; the primer sequence of the detection site septin9 with methylation digestion site is shown in SEQ NO. 9-SEQ NO. 10; the primer sequence of the detection site SDC2 with methylation digestion site is shown in SEQ NO. 11-SEQ NO. 12; the primer sequence of the detection site NPY10 with methylation digestion site is shown in SEQ NO. 13-SEQ NO. 14; and the primer sequence of the detection site IKZF with methylation digestion site is shown in SEQ NO. 15-SEQ NO. 16.

[0017] The advantages of the embodiment of the present application are as follows: by the above technical scheme, the single-strand nuclease is introduced into the enzyme digestion system, the methylation sensitive restriction enzyme digestion and single-strand nucleic acid digestion are simultaneously performed in one reaction, the single-strand interference region is effectively digested and degraded, the false positive signal caused by the single-strand nucleic acid is eliminated, the incomplete MSRE enzyme digestion problem is solved, and the accuracy, sensitivity and accuracy of the methylation detection on the cfDNA are improved; the enzymes with different activities are combined, the overall digestion effect is enhanced, the methylation enrichment efficiency is improved, the enzyme digestion reaction time is shortened, the non-specific degradation of the target DNA caused by the increase of the digestion intensity is avoided, and a high detection sensitivity of the detection system is ensured. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0019] Figure 1 A capillary electrophoresis detection result map of the complex enzyme digestion method for enriching methylated DNA fragments according to the present application, wherein A: normal human detection result, no amplification product at 5 detection sites; B: most colorectal cancer patient detection results have clear amplification products at multiple detection sites. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0021] Embodiment 1 DNA methylation detection by MSRE digestion is affected by DNA fragmentation High-methylation DNA, low-methylation DNA, fragmented high-methylation DNA and fragmented low-methylation DNA were taken as the samples to be detected, wherein the high-methylation DNA and the low-methylation DNA were purchased from Epigendx Company. The preparation method of the fragmented high-methylation DNA and the fragmented low-methylation DNA was as follows: 3000 rpm shaking for 15 seconds, 1 minute on ice, and repeating 10 times.

[0022] The four samples were divided into experimental and control groups, with three repeated experiments. The experimental group was subjected to MSRE digestion, and the 20 μL MSRE digestion system included: HpaII 15U, HhaI 15U, 10x enzyme digestion buffer 2ul, 30 μg sample to be tested, and the 10x enzyme digestion buffer included: 500 mM NaAc, 100 mM MgAc2, 1 g / ml BSA, 200 nM Tris-Ac, pH 7.9. The control group used the same digestion system as above, but without HpaII and HhaI.

[0023] The digestion conditions were: incubation at 37°C for 2 hours, 60°C for 30 minutes, and 4°C storage.

[0024] The experimental groups 1-4 and control groups 1-4 after the above reaction were subjected to qPCR detection, and the target gene fragment SDHA and the internal reference gene UBC were detected. The composite amplification detection system included PCR reaction premix, internal standard, etc. The main components of the PCR reaction premix included hot-start Taq enzyme, amplification buffer, etc., and all the primers were mixed according to the experimental ratio to prepare a primer mixture. The PCR reaction system used a 20 μL basic system, wherein the primer Mix had a primer concentration of 100 μmol / L, and the primer probe sequences are shown in Table 1: Table 1

[0025] The SDHA fragment contains HpaII and HhaI enzyme cutting sites, which will be cut in the unmethylated state, so only the methylated state can produce an amplification signal in the qPCR reaction; UBC does not contain HpaII and HhaI enzyme cutting sites, and will not be cut whether methylated or not, and will produce an amplification signal in qPCR.

[0026] The PCR amplification system was prepared according to the components in Table 2, and after shaking and mixing, the samples were divided according to the sample quantity: Table 2

[0027] The reaction tubes were placed in the PCR amplification instrument reaction slot, and the reaction system was set to 20 µL. The PCR expansion was performed according to the following program: denaturation at 95°C for 5 min; 94°C denaturation for 10 s, 59°C annealing for 90 s for one cycle, a total of 25 cycles; then 60°C for 20 min, and after the end, the temperature was lowered to 4°C.

[0028] The obtained Ct values are shown in Table 3: Table 3

[0029] From the experimental results, it can be seen that the control group 1-4 without MSRE digestion has similar results, indicating that conventional PCR cannot distinguish whether the sample to be tested is methylated, and whether the sample to be tested is fragmented has no significant effect on PCR detection.

[0030] Compared with the control group 1-4 without digestion, the CT value of UBC in the experimental group 1-4 with MSRE digestion increases to a certain extent, indicating that the digestion step will cause a certain non-specific degradation to the DNA template, but the specific impact needs to be verified by experiment.

[0031] The CT value and ΔCT difference of SDHA between experimental group 1 and experimental group 2 are very significant, which shows that MSRE digestion can effectively distinguish whether the sample to be tested is methylated. It is noted that the ΔCT difference between experimental group 1 and control group 1 is not significant, while the ΔCT difference between experimental group 2 and control group 2 is significant, which shows that MSRE specifically digests the non-methylated double-stranded DNA, but not the methylated DNA.

[0032] Compared with experimental group 1, the ΔCT of experimental group 3 is basically the same, and the ΔCT of experimental group 4 is significantly lower, which is relatively close to the ΔCT value of experimental group 1. The reason for this result is that the template contains a large amount of single-stranded DNA, which cannot be digested by MSRE, whether it is methylated or not. These undigested single-stranded DNA as PCR template forms amplification and produces fluorescence signal, resulting in the decrease of CT value of SDHA and the decrease of ΔCT. From the results, it is difficult to effectively distinguish the results of experimental group 4 and experimental group 1, that is, fragmented non-methylated DNA will cause false positive signal, so that the methylation state cannot be effectively detected.

[0033] The above results show that DNA methylation can be detected by MSRE digestion; DNA fragmentation will affect the accuracy of methylation detection.

[0034] Example 2 Single-strand nuclease can eliminate the negative impact of DNA fragmentation Fragmented high-methylation DNA and fragmented low-methylation DNA were taken as samples to be tested, wherein the high-methylation DNA and low-methylation DNA were purchased from Epigendx company. The preparation method of the fragmented high-methylation DNA and the fragmented low-methylation DNA is: 3000 rpm shaking for 15 seconds, 1 minute on ice, repeating 10 times.

[0035] The above two samples were aliquoted into experimental group and control group, and three repeated experiments were set. The experimental group was subjected to MSRE digestion, and the MSRE digestion system is shown in Table 3, and the digestion conditions are: 37℃ incubation for 2 hours, 60℃ for 30 minutes, 4℃ storage.

[0036] MSRE digestion followed by digestion with single-strand nuclease, the single-strand nuclease digestion system is shown in Table 4: Table 4

[0037] The digestion conditions are: incubation at 37°C for 30 minutes.

[0038] The control group does not undergo single-strand nuclease digestion, and the other experimental conditions are the same.

[0039] The experimental groups 5-6 and the control group 5-6 after the above reaction are detected by qPCR, with three repeated experiments, 3 repeats per group of samples, and the obtained Ct values are shown in Table 5: Table 5

[0040] The control group 5-6 does not introduce single-strand nuclease for digestion. Due to the addition of one step of digestion, the template is diluted, and each CT value increases, but the ΔCT difference is small, and the results are similar to those of experimental groups 3-4 in Example 1, and the methylation state of the detection sample cannot be effectively judged from the ΔCT results.

[0041] The experimental groups 5-6 introduce single-strand nuclease for digestion, and the ΔCT difference is obvious, and the results are similar to those of experimental groups 1-2 in Example 1. This result is consistent with the expectation, that is, the addition of single-strand nuclease treatment effectively digests single-strand DNA, thereby eliminating the false positive results caused by single-strand DNA, so that the detection can effectively reflect the methylation state of the detection sample.

[0042] In this example, the CT values obtained by detection are generally larger than those in the corresponding reactions in Example 1. The larger CT values mean that the detection sensitivity is reduced, and the initial sample amount needs to be increased by several times. The reason why the CT values are larger is that the addition of one step of single-strand nuclease digestion dilutes the sample, that is, the template amount in qPCR is reduced, and on the other hand, it is speculated that the S1 nuclease also causes partial degradation of double-strand DNA while digesting single-strand DNA.

[0043] It is shown that the addition of single-strand nuclease digestion can eliminate the interference of single-strand nucleic acid on detection and ensure that methylation detection is effectively performed. At the same time, the addition of single-strand nuclease digestion also causes some negative effects, the most important of which is that the dilution of the sample and the degradation of double-strand DNA cause the CT values to be larger and the detection sensitivity to be reduced. And the addition of an independent digestion also increases the detection time, the detection cost, the operation complexity, and the risk of errors.

[0044] Example 3 Screening of single-strand nuclease available in MSRE digestion system It is required that the single-strand nuclease has the activity of digesting single-strand nucleic acid while not digesting double-strand DNA. Among the common tool enzymes, the following enzymes can meet the above conditions: exonuclease I (ExoI), exonuclease VII (ExoVII), exonuclease T (ExoT), S1 nuclease, P1 nuclease and mung bean nuclease (MBase).

[0045] The fragmented hypermethylated DNA and the fragmented hypomethylated DNA were taken as the samples to be detected, wherein the hypermethylated DNA and the hypomethylated DNA were purchased from Epigendx Company. The preparation method of the fragmented hypermethylated DNA and the fragmented hypomethylated DNA was as follows: oscillation at 3000 rpm for 15 seconds, 1 minute on ice, and repetition for 10 times.

[0046] The above two samples were aliquoted, and an alternative single-strand nuclease was additionally added to divide into complex experimental groups 1-12 and complex enzyme digestion control groups 1-2, and three repeated experiments were set to perform complex enzyme digestion.

[0047] The control group 1-2 and the experimental groups 1-12 after the above reaction were detected by qPCR, and the target gene fragment SDHA and the internal reference gene UBC were detected, and the added nucleases, the digestion time and the obtained Ct values of the reaction were as shown in Table 6: Table 6

[0048] It can be known from the experimental data of the complex control group 1 and the complex experimental groups 2-6 that the ΔCT of the complex experimental groups 1, 2, 5 and 6 has different degrees of increase compared with the ΔCT of the complex control group 1, which indicates that the single-strand nucleic acid is effectively digested. The ΔCT of the complex experimental groups 3 and 4 is basically the same as that of the experimental 1, which indicates that the single-strand nucleic acid has only limited digestion or no digestion.

[0049] It can be known from the experimental data of the complex control group 2 and the complex experimental groups 7-12 that the CT values of the complex experimental groups 9 and 11 have a large increase compared with the CT value of the complex control group 2, which indicates that the complex enzyme digestion system can significantly cause the degradation of double-strand DNA. The CT values of the complex experimental groups 7, 8, 10 and 12 are basically consistent with or slightly increased compared with the CT value of the complex control group 2, which indicates that the added single-strand nuclease does not additionally increase the degradation of double-strand DNA.

[0050] It can be known from the above that Exo1, ExoVII and MBase are single-strand nucleases that can work with MSRE.

[0051] The CT value of the complex control group 2 is increased greatly compared with the experimental group 3 of example 2, which shows that increasing the MSRE digestion time will cause non-specific degradation of double-stranded methylated DNA, that is, simply increasing the MSRE digestion intensity will not only not eliminate the single-stranded false positive signal but also reduce the detection sensitivity.

[0052] Example 4 Preference of complex digestion system Take the fragmented hypomethylated DNA as the sample to be detected, divide the above sample into equal parts, into a complex digestion control group and complex digestion experimental groups 1-7, and set three repeated experiments, and perform complex enzyme digestion.

[0053] The experimental group is subjected to MSRE digestion, and 20 μL of the MSRE digestion system comprises: HpaII 15U, HhaI 15U, 10x enzyme digestion buffer 2u1, 30 ng of sample to be detected, and one or more enzymes are additionally added for digestion, wherein the 10x enzyme digestion buffer comprises 500 mM sodium acetate, 100 mM magnesium acetate, 1 g / ml bovine serum albumin, 200 nM Tris-Ac, 10 nM zinc sulfate, and the pH of the 10x enzyme digestion buffer is 7.9.

[0054] The complex digestion control group and the complex digestion experimental groups 1-7 after the above reaction are subjected to qPCR detection, and the target gene fragment SDHA and the internal reference gene UBC are detected at the same time, and the setting, digestion conditions and CT values of the complex digestion control group and the complex digestion experimental groups 1-7 are shown in Table 7: Table 7

[0055] The experimental results of the complex digestion control group and the experimental group 4 in example 1 are basically the same, which shows that only MSRE digestion treatment cannot eliminate the false positive signal caused by DNA fragmentation.

[0056] The ΔCT values of the complex digestion experimental groups 1-3 are increased to different degrees compared with the ΔCT of the complex digestion control group, which shows that the single-stranded nuclease digests the single-stranded nucleic acid while the MSRE works. The ΔCT of the complex digestion experimental group 3 using MBase is relatively larger, which indicates that the digestion of single-stranded nucleic acid is better. The CT values of SDHA of the complex digestion experimental groups 1-3 are about 34-37, which shows that there are still a considerable number of SDHA fragments that can be amplified after digestion, that is, the false positive signal caused by fragmentation has not been completely eliminated.

[0057] In the complex digestion experiment groups 4-7, multiple single-strand nucleases were combined to completely eliminate false positive signals caused by fragmentation. Compared with the results of complex digestion experiment groups 1-3, the CT value of UBC changed less, and the CT value and ΔCT of SDHA increased significantly. The results of complex digestion experiment groups 5 and 7 were the best, the ΔCT value was larger, and the CT value of SDHA was more than 40, close to undetectable, that is, the false positive signals caused by fragmentation were basically completely eliminated.

[0058] It is shown that the preferred digestion combination is the combination of ExoI and MBase based on HpaII and Hhal, or the combination of ExoI, ExoVII and MBase based on HpaII and Hhal. Considering that the effects of the two schemes are similar, the cost of ExoVII is slightly higher, and the control CT value is slightly larger, it is shown that it causes certain double-stranded DNA degradation, and the optimal digestion combination is determined as: the combination of ExoI and MBase based on HpaII and Hhal.

[0059] Example 5 Take 64 cases of colorectal cancer patients as the experimental group, including 5 cases of clinical stage I, 24 cases of stage II, 27 cases of stage III, and 8 cases of stage IV; take 60 healthy people as the control group.

[0060] Collect 10 ml of peripheral blood from all patients and healthy people, separate the plasma, and extract cfDNA.

[0061] The extracted cfDNA is subjected to MSRE combined with single-strand nuclease complex digestion. The complex digestion system is shown in Table 8 below: Table 8

[0062] Among them, the 10x enzyme digestion buffer contains 500mM sodium acetate, 100mM magnesium acetate, 1g / ml bovine serum albumin, 200nM Tris-Ac, 10nM zinc sulfate, and the pH of the 10x enzyme digestion buffer is 7.9.

[0063] Digestion reaction conditions: 30 degrees for 30 minutes, 37 degrees for 30 minutes, 60 degrees for 30 minutes, and 4 degrees for storage.

[0064] After digestion, the digestion products are amplified by multiplex fluorescent PCR.

[0065] The multiplex fluorescent PCR reaction system is shown in Table 9 below: Table 9

[0066] The primer sequences used are shown in Table 10 below: Table 10

[0067] PCR reaction conditions: pre-denaturation 95 degrees for 15 minutes; denaturation 95 degrees for 30 seconds, annealing 60 degrees for 90 seconds, extension 72 degrees for 60 seconds for 35 cycles; final extension 72 degrees for 10 minutes, 4 degrees for storage.

[0068] The amplification product was detected by capillary electrophoresis on a genetic analyzer, QD550 internal standard and formamide were mixed at a ratio of 2.5:100, 12.5 μL of the mixture was added to a 96-well plate, 1 μL of the amplification product sample or allele standard was added, and the mixture was allowed to stand for several minutes, denatured at 95°C for 3 min, immediately ice-bathed for 3 min, and then placed in an ABI5000xL sequencer for detection.

[0069] The capillary electrophoresis result is shown in FIG. 5. Figure 1 As shown in FIG. 5, the normal person detection result has no amplification product at the 5 detection sites, as shown in A; most colorectal cancer patients have clear amplification products at multiple detection sites, as shown in B. Figure 1 Figure 1 Figure 1

[0070] According to the peak height of the corresponding product in the electrophoresis result, the methylation state of the detection sample was determined. When the peak height of the control site was greater than 1000 RFU and the peak height of the detection site was greater than 100 RFU, the site was determined to be positive. According to the above standard, the number of positive sites of the detection sample was counted, and the results are shown in Table 11. Table 11

[0071] The methylation state of the sample was determined according to the number of positive sites in the 5 detection sites. If 2 or more sites were positive, the sample was determined to be positive, otherwise it was determined to be negative. According to this standard, the sensitivity of the detection system for colorectal cancer patients was 81.3% (42 / 64), and the specificity was 96.7% (58 / 60).

[0072] This enzyme digestion method uses a small number of sites (5) to achieve high sensitivity and specificity, which indicates that the overall detection process is effective, and the MSRE digestion combined with single-strand nuclease is effective.

[0073] ​​​The advantages of the embodiment of the present application are as follows: by the above technical scheme, the single-strand nuclease is introduced into the enzyme cutting system, the methylation sensitive restriction enzyme cutting and single-strand nucleic acid digestion are simultaneously carried out in one reaction, the single-strand interference region is effectively digested and degraded, the false positive signal caused by the single-strand nucleic acid is eliminated, the incomplete MSRE enzyme cutting problem is solved, and the accuracy, sensitivity and accuracy of the methylation detection on the cfDNA are improved; the enzymes with different activities are combined, the overall digestion effect is enhanced, the methylation enrichment efficiency is improved, the enzyme cutting reaction time is shortened, the non-specific degradation of the target DNA caused by the increase of the digestion intensity is avoided, and a high detection sensitivity of the detection system is ensured.

[0074] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A complex enzyme digestion method for enriching methylated DNA fragments, characterized in that, The combined enzyme digestion method for enriching methylated DNA fragments includes simultaneous methylation-sensitive restriction endonuclease digestion and single-stranded nuclease digestion in a single combined enzyme digestion reaction.

2. The complex enzyme digestion method for enriching methylated DNA fragments according to claim 1, characterized in that, The methylation-sensitive restriction endonuclease used is one or a combination of two of HpaII and HhaI.

3. The complex enzyme digestion method for enriching methylated DNA fragments according to claim 1, characterized in that, The single-stranded nuclease used is one or a combination of two or more of the following: exonuclease I, exonuclease VII, exonuclease T, S1 nuclease, P1 nuclease, and mung bean nuclease.

4. The complex enzyme digestion method for enriching methylated DNA fragments according to claim 3, characterized in that, The single-stranded nuclease used is one or more of the following: exonuclease I, exonuclease VII, and mung bean nuclease, all of which are compatible with the MSRE digestion system.

5. The complex enzyme digestion method for enriching methylated DNA fragments according to claim 4, characterized in that, The single-stranded nuclease is based on a methylation-sensitive endonuclease and uses exonuclease I and mung bean nuclease in combination. Alternatively, exonuclease I, exonuclease VII, and mung bean nuclease can be used in combination with methylation-sensitive endonuclease.

6. The complex enzyme digestion method for enriching methylated DNA fragments according to any one of claims 1-5, characterized in that, The complex enzyme digestion method for enriching methylated DNA fragments uses 7.5 U / ul HpaII, 7.5 U / ul HhaI, 0.1-0.5 U / ul ExoI, and 0.05-0.2 U / ul MBase.

7. The complex enzyme digestion method for enriching methylated DNA fragments according to claim 1, characterized in that, The digestion products obtained after simultaneous methylation-sensitive restriction endonuclease digestion and single-stranded nuclease digestion were amplified by multiplex fluorescent PCR.

8. The complex enzyme digestion method for enriching methylated DNA fragments according to claim 7, characterized in that, The multiplex fluorescent PCR system includes: 25 μL of 2× amplification buffer, 20 U of DNA polymerase, 250 nM of primer mixture, and 15 μL of MSRE digestion product.

9. The complex enzyme digestion method for enriching methylated DNA fragments according to claim 8, characterized in that, The multiplex fluorescent PCR system includes a control site UBC without methylation restriction sites and five detection sites containing methylation restriction sites, namely: SDHA, septin9, SDC2, NPY10, and IKZF.

10. The complex enzyme digestion method for enriching methylated DNA fragments according to claim 9, characterized in that, The primer sequences for the control site UBC, which does not contain methylated restriction enzyme sites, are shown in SEQ NO.4-SEQ NO.6 and SEQ NO.17; the primer sequences for the detection site SDHA, which contains methylated restriction enzyme sites, are shown in SEQ NO.1-SEQ NO.3 and SEQ NO.7-SEQ NO.8; the primer sequences for the detection site septin9, which contains methylated restriction enzyme sites, are shown in SEQ NO.9-SEQ NO.10; the primer sequences for the detection site SDC2, which contains methylated restriction enzyme sites, are shown in SEQ NO.11-SEQ NO.12; the primer sequences for the detection site NPY10, which contains methylated restriction enzyme sites, are shown in SEQ NO.13-SEQ NO.14; and the primer sequences for the detection site IKZF, which contains methylated restriction enzyme sites, are shown in SEQ NO.15-SEQ NO.16.

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