CN114410792B - Marker for kidney cancer screening, probe composition and application thereof - Google Patents

Marker for kidney cancer screening, probe composition and application thereof Download PDF

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CN114410792B
CN114410792B CN202210125008.5A CN202210125008A CN114410792B CN 114410792 B CN114410792 B CN 114410792B CN 202210125008 A CN202210125008 A CN 202210125008A CN 114410792 B CN114410792 B CN 114410792B
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连明
韩晓亮
王小奇
郭媛媛
刘栓平
魏闯
李永君
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Biochain Beijing Science and Technology Inc
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Abstract

The application discloses a marker for screening renal cancer, a probe composition and application thereof, wherein the marker is MCF2L. The marker can sensitively and specifically detect the methylation state of the gene, so that the marker can be used for detecting free DNA of peripheral blood, and the composition can be used for screening asymptomatic people in a non-invasive mode, reduces the harm caused by invasive detection, has higher sensitivity and accuracy and can realize real-time monitoring.

Description

Marker for kidney cancer screening, probe composition and application thereof
Technical Field
The application relates to the field of biotechnology, in particular to a marker for screening renal cancer, a probe composition and application thereof.
Background
Renal cancer is the malignancy of urinary system rank 3, whose mortality rates are among the leading major malignancies of urinary system 3, but precede prostate and bladder cancers.
Most patients do not have any pain or physical discomfort even from the early stage to the middle-late stage of the onset of the kidney cancer, and many patients have weak consciousness about cancer prevention, and the general investigation of the kidney cancer cannot cover all people. Although kidney cancer occupies a place in malignant tumors, if the kidney cancer can be detected as early as possible through scientific screening, the survival rate of early-stage kidney cancer patients in 5 years can reach 92 percent. If the disease is found later, the treatment process is more complex, and the patient is also physically uncomfortable and painful, and the disease is not necessarily controlled, so that the death rate is increased.
Early findings are critical to improving patient survival and cure rate, but currently lack accurate non-invasive diagnostic methods. Currently, DNA methylation has been demonstrated to be tissue specific, useful in early cancer detection, and can be traced to the primary tumor site based on the methylation profile of circulating tumor DNA (ctDNA).
Disclosure of Invention
The object of the present application is to provide a marker for detecting renal cancer, which can be used for screening renal cancer, is used for screening asymptomatic population in a non-invasive way, is used for prognosis detection of cancer patients, reduces the harm caused by invasive detection, and has higher sensitivity and accuracy, and a probe composition.
The specific technical scheme of the application is as follows:
1. a marker for detecting renal cancer, wherein the marker corresponding gene is MCF2L.
2. The marker according to item 1, wherein the nucleotide sequence of the marker is shown in SEQ ID NO.1, preferably the marker is a methylated marker.
3. A probe composition comprising a probe that targets methylation of a marker of item 1 or 2.
4. The probe composition of item 3, wherein the probe composition comprises a hypermethylated first probe composition for hybridization to a bisulfite converted hypermethylated region and a hypomethylated second probe composition for hybridization to a bisulfite converted hypomethylated region;
preferably, the first probe composition comprises n probes that hybridize to each nucleotide of the sense strand and/or the antisense strand of the bisulfite converted hypermethylated region;
preferably, the second probe composition comprises m probes that hybridize to each nucleotide of the sense strand and/or the antisense strand of the bisulfite converted hypomethylated region;
preferably, n and m are each any integer from 1 to 10;
preferably, the n-1 th probeWith x between needle and nth probe 1 Overlapping of nucleotides, preferably x 1 Is any integer from 0 to 100;
preferably, there is x between the m-1 th probe and the m-th probe 2 Overlapping of nucleotides, preferably x 2 Is any integer from 0 to 100;
further preferably, the first probe composition comprises one or two of SEQ ID NOS: 2-3 and the second probe composition comprises one or two of SEQ ID NOS: 4-5.
5. Use of a marker for the preparation of a kit for detecting renal cancer, wherein the marker-corresponding gene is MCF2L.
6. The use according to item 5, wherein the nucleotide sequence of the marker is shown in SEQ ID NO.1, preferably the marker is a methylated marker;
preferably, the probe composition is for targeting a post-methylation marker of renal cancer;
preferably, the probe composition is the probe composition of item 3 or 4.
7. A composition for use in the detection of renal cancer, comprising a nucleic acid for detecting methylation of the marker corresponding gene MCF2L.
8. The composition of item 7, wherein the nucleotide sequence of the marker is set forth in SEQ ID NO. 1.
9. The composition of item 7 or 8, wherein the nucleic acid comprises the probe composition of item 3 or 4;
preferably, the nucleic acid comprises:
a primer that is a fragment of at least 9 nucleotides in a target sequence of the marker, the fragment comprising at least one CpG dinucleotide sequence;
preferably, the nucleic acid further comprises:
a probe that hybridizes under moderately stringent or stringent conditions to at least 15 nucleotide fragments in a target sequence of the marker, the fragments comprising at least one CpG dinucleotide sequence;
preferably, the composition further comprises an agent that converts the unmethylated cytosine base at position 5 of the target sequence of the marker to uracil;
preferably, the nucleic acid for detecting methylation of a target sequence of a marker further comprises:
blocking agents that preferentially bind to target sequences in the unmethylated state.
10. A kit comprising a reagent for detecting a marker according to item 1 or 2 or a probe composition according to item 3 or 4 or a composition according to any one of items 7 to 9.
11. A chip comprising the marker of item 1 or 2 or the probe composition of item 3 or 4 or the composition of item 7 or 8.
ADVANTAGEOUS EFFECTS OF INVENTION
The inventor of the application utilizes the epigenomic and bioinformatics technology to find a methylation gene related to kidney cancer by analyzing genome methylation data of the kidney cancer, determines a target sequence of methylation abnormality of the methylation gene of the kidney cancer, and can sensitively and specifically detect the methylation state of the gene through the target sequence of the methylation gene, so that the methylation gene can be used for detecting free DNA of peripheral blood.
The composition is used for screening asymptomatic people in a non-invasive mode, harm caused by invasive detection is reduced, and the composition has higher sensitivity and accuracy and can realize real-time monitoring.
Detailed Description
The present application is described in detail below. While specific embodiments of the present application are shown, it should be understood that the present application may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
It should be noted that, throughout the specification and claims, the terms "include" and "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth the preferred embodiment for carrying out the present application, but is not intended to limit the scope of the present application in general, as the description proceeds. The scope of the present application is defined by the appended claims.
The application provides a marker for detecting kidney cancer, wherein the marker corresponding gene is MCF2L.
The corresponding gene refers to a gene corresponding to a marker, and in the application, the gene corresponding to the marker is MCF2L.
In one embodiment, the nucleotide sequence of the marker is shown in SEQ ID NO.1, preferably the marker is a methylated marker.
Wherein the sequence of the marker is a sequence which is not converted by bisulfite.
The present application provides a probe composition comprising a probe that targets methylation of the marker.
Methylation refers to methylation of the 5 th carbon atom on cytosine in CpG dinucleotides, and is taken as a stable modification state, and can inherit new generation progeny DNA along with the replication process of DNA under the action of DNA methyltransferase, so that the methylation of the gene promoter region can lead to silence transcription of cancer suppressor genes during DNA methylation, and the methylation is closely related to tumor occurrence. Aberrant methylation includes hypermethylation of cancer suppressor genes and DNA repair genes, hypomethylation of repeated sequence DNA, imprinting loss of certain genes, which are associated with the occurrence of a variety of tumors.
Methylation as described herein can be methylation level, degree of methylation, or methylation state, and when analyzing methylation of such target sequences, one skilled in the art can use quantitative determination methods to determine methylation.
The probe is single-stranded or double-stranded DNA with a length of tens to hundreds or even thousands of base pairs, which can utilize the denaturation, renaturation and high precision of base complementary pairing of molecules, and can be combined with (hybridized with) complementary unlabeled single-stranded DNA or RNA in a sample to be tested in a hydrogen bond manner to form a double-stranded complex (hybrid). After washing off the unpaired and bound probe, the hybridization reaction results can be detected by a detection system such as an autoradiography or an enzyme-linked reaction. In this application, the region that complementarily binds or hybridizes to a probe is a specific target region, and a plurality of probes are combined into a probe composition.
In one embodiment, the probe composition comprises a hypermethylated first probe composition for hybridization to a bisulfite converted hypermethylated region and a hypomethylated second probe composition for hybridization to a bisulfite converted hypomethylated region.
The hypermethylation means that after the marker is converted by bisulfite, a base C is changed into a base U, but if the marker is a base CG, the base C is kept unchanged;
the hypomethylation means that after the marker is converted by bisulfite, all bases CG are not methylated, and the bases C are changed into the bases U.
Since the methylation status varies from person to person, the sequence of the tag converted by bisulfite varies, one extreme case of each tag is shown here, i.e. all CG of the segment is in hypermethylation status, and the hypermethylation status sequence of its complementary strand:
the sequence of one extreme case of SEQ ID NO.1 is shown as SEQ ID NO. 6;
the sequence of the extreme case of the complementary strand of SEQ ID NO.1 is shown as SEQ ID NO. 7.
Similarly, since each person has a different methylation state, an extreme case is shown here, in which all CG is in hypomethylated state, and the sequence of hypomethylated states of their complementary strands is also shown:
the sequence of one extreme case of SEQ ID NO.1 is shown as SEQ ID NO. 8;
the sequence of the extreme case of the complementary strand of SEQ ID NO.1 is shown as SEQ ID NO. 9;
in one embodiment, the first probe composition comprises n probes that hybridize to each nucleotide of the sense strand and/or the antisense strand of the bisulfite converted hypermethylated region.
The second probe composition includes m probes that hybridize to each nucleotide of the sense strand and/or the antisense strand of the bisulfite converted hypomethylated region.
The number of probes in the first probe composition and the second probe composition is not limited in any way, and those skilled in the art can select the number as desired, for example, m and n may be any integer of 1 to 10, and m and n may be the same or different.
For example, m and n may be any integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, preferably m=n=2.
In one embodiment, there is x between the n-1 th probe and the n-th probe 1 Overlapping of nucleotides, preferably x 1 Is any integer from 0 to 100;
preferably, there is x between the m-1 th probe and the m-th probe 2 Overlapping of nucleotides, preferably x 2 Is any integer from 0 to 100.
Wherein x is 1 And x 2 May be the same or different, when x 1 When 0, it is indicated that the tail of the n-1 th probe is connected with the head of the n-th probe, and similarly, when x 2 When 0, it is indicated that the tail of the m-1 th probe is connected to the head of the m-th probe.
The probe composition is hybridized with the marker converted by the bisulfite, wherein the high-methylation first probe composition is hybridized with a high-methylation region, and the low-methylation second probe composition is hybridized with a low-methylation region, so that the methylation level of a target sequence can be detected efficiently and accurately, and the probe composition can be used for screening renal cancer.
In one embodiment, the hypermethylated first probe composition comprises one or both of SEQ ID NOS: 2-3.
The hypomethylated second probe composition comprises one or two of SEQ ID NOS: 4-5.
The application provides application of a marker in preparation of a kit for detecting kidney cancer, wherein the marker corresponds to MCF2L.
In one embodiment, the nucleotide sequence of the marker is selected from one of the markers shown in SEQ ID NOS.1-28, preferably the marker is a methylated marker.
The application provides the use of a probe composition for targeting a marker after methylation of kidney cancer in the preparation of a kit for detecting kidney cancer.
In one embodiment, the probe composition is the probe composition described above.
The application provides a composition for detecting kidney cancer, which comprises nucleic acid for detecting methylation of a marker corresponding gene MCF2L, and preferably the nucleotide sequence of the marker is shown as SEQ ID NO. 1.
In one embodiment, the nucleic acid comprises a probe composition as described above.
In one embodiment, the nucleic acid comprises:
a primer that is a fragment of at least 9 nucleotides in a target sequence of the marker, the fragment comprising at least one CpG dinucleotide sequence.
Wherein, if bisulfite is used to convert NDA in a sample to be tested, the nucleic acid for detecting methylation of the target sequence of the marker comprises a fragment of at least 9 nucleotides in the sequence after bisulfite conversion of the target sequence of the marker, said fragment comprising at least one CpG dinucleotide sequence.
In one embodiment, the nucleic acid further comprises:
a probe that hybridizes under moderately stringent or stringent conditions to at least 15 nucleotide fragments in a target sequence of the marker, the fragments comprising at least one CpG dinucleotide sequence.
In one embodiment, the composition further comprises an agent that converts the unmethylated cytosine base at position 5 of the target sequence of the marker to uracil, e.g., the agent can be bisulfite or the like; preferably, the nucleic acid for detecting methylation of a target sequence of a marker further comprises:
blocking agents that preferentially bind to target sequences in the unmethylated state.
The blocker is used for improving the amplification specificity of the PCR amplification primer, the 5 '-end of the blocker nucleotide sequence and the 3' -end nucleotide sequence of the forward or reverse primer have an overlapping region of more than or equal to 5 nucleotides, the blocker is complementary with the forward or reverse primer and the same strand of target gene target sequence DNA, the melting temperature of the blocker is higher than that of the forward or reverse primer by more than (including) 5 ℃, and the nucleotide sequence of the blocker comprises at least one CpG dinucleotide sequence and is complementary with the sequence of the target gene target sequence DNA which is not subjected to methylation after the conversion of the bisulfite. Thus, when the genomic DNA of the biological sample to be detected is a mixture of methylated and unmethylated state, especially in the case where the DNA in the methylated state is far less than the DNA in the unmethylated state, the DNA in the unmethylated state is converted by bisulfite and preferentially binds to the blocker, and thus the DNA template binds to the PCR obligation, and thus PCR amplification does not occur, whereas the DNA in the methylated state does not bind to the blocker and thus the primer set, PCR amplification occurs, and then the fragment obtained by the amplification is detected directly or indirectly.
The present application provides a kit comprising the above-described marker or the above-described probe composition or the above-described composition.
In one embodiment, the kit further comprises a container for holding a biological sample of a subject.
In one embodiment, the kit further comprises instructions for use and interpretation of the test results.
The biological sample may be, for example, peripheral blood whole blood, plasma or serum.
The method for detecting the methylation level of a target sequence using the above-described kit is not limited in any way, and one skilled in the art can select as desired, for example, the present application provides a method for detecting the methylation level of a marker target sequence using the above-described kit, which comprises the steps of:
collecting a sample of a subject;
extracting and purifying DNA in the sample;
constructing a DNA library for sequencing against the purified DNA sample;
transforming said constructed DNA library with bisulfite;
pre-PCR amplifying the bisulfite converted DNA library;
performing hybridization capture on the sample amplified by the pre-PCR by using the probe composition;
amplifying the hybridized and captured product by utilizing PCR;
performing high-throughput second-generation sequencing on the PCR amplified product after hybridization capture;
analyzing the sequencing data to determine the methylation level of the sample;
calculating a threshold value for each marker based on methylation of an existing sample, interpreting the patient's disease based on the methylation level of a certain marker of the sample, if the methylation level of a certain marker of the sample exceeds the threshold value, it is a cancer sample, if it is below the threshold value, it is a healthy human sample.
Also for example, the present application provides a method for detecting the methylation level of a target sequence of a marker using the kit described above, comprising the steps of:
(1) Extracting peripheral blood of a subject, and separating plasma or serum;
(2) Extracting free DNA from plasma or serum;
(3) Treating the free DNA obtained in step (2) with a reagent to convert the unmethylated cytosine base at position 5 to uracil or other bases, i.e., to convert the unmethylated cytosine base at position 5 of the target sequence of the marker to uracil or other bases, the converted bases differing from the unmethylated cytosine base at position 5 in hybridization performance and being detectable;
(4) Contacting the free DNA treated in step (3) with a DNA polymerase and primers for the target sequence of the marker such that the target sequence of the treated marker is amplified to produce amplified products or not amplified; the target sequence of the treated marker, if subjected to DNA polymerization, produces amplification products; the target sequence of the treated marker is not amplified if DNA polymerization does not occur;
(5) Detecting the amplified product with a probe;
(6) Determining the methylation status of at least one CpG dinucleotide of the target sequence of the marker based on the presence or absence of the amplification product, thereby determining the methylation level of the target sequence of the marker.
The present application provides a chip comprising the above-described marker or the above-described probe composition or the above-described composition.
The sequencing principle of the chip, also called a gene chip, is a hybridization sequencing method, namely a method for determining the sequence of nucleic acid by hybridizing with a group of nucleic acid probes with known sequences, wherein probes with target nucleotides with known sequences are immobilized on the surface of a substrate. When the nucleic acid sequence with fluorescent mark in the solution is complementarily matched with the nucleic acid probe at the corresponding position on the gene chip, a group of probe sequences with complete complementation of the sequences are obtained by determining the probe position with the strongest fluorescence intensity.
The chip is prepared by mainly taking a glass sheet or a silicon wafer as a carrier, and sequentially arranging oligonucleotide fragments or cDNA (complementary deoxyribonucleic acid) serving as probes on the carrier by adopting an in-situ synthesis and microarray method.
The chip is based on signal detection of DNA sequence hybridization after bisulfite treatment, wherein unmethylated cytosine is changed into uracil, methylated cytosine is kept unchanged, uracil is converted into thymine, and finally chip hybridization is carried out; finally, judging the type of the added base according to the fluorescence color, and further determining whether the locus is methylated.
The present application provides a method of screening for renal cancer comprising:
detecting methylation level of a marker
Determining the risk of the subject to suffer from kidney cancer based on the methylation level, wherein the marker corresponding gene is MCF2L.
Examples
The materials used in the test and the test methods are generally and/or specifically described herein, and in the examples which follow,% represents wt%, i.e., weight percent, unless otherwise specified. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Example 1 screening markers
1) Sample collection: the 450k methylated chip cancer tissue data in TCGA was downloaded, and included in 7769 cancer tissue samples from 26 tumors, including adrenocortical carcinoma (80), bladder urothelial carcinoma (409), acute myeloid leukemia (140), brain low grade glioma (654), breast carcinoma (740), cervical carcinoma (286), colorectal carcinoma (348), esophageal carcinoma (183), uveal melanoma (80), head and neck squamous cell carcinoma (527), renal carcinoma (660), liver carcinoma (377), lung adenocarcinoma (425), lung squamous carcinoma (372), diffuse large B-cell lymphoma (29), ovarian serous cyst adenocarcinoma (10), pancreatic carcinoma (184), mesothelioma (116), prostate carcinoma (488), skin melanoma (104), sarcoma (117), gastric carcinoma (397), testicular carcinoma (134), thymus carcinoma (94), thyroid carcinoma (506), endometrial carcinoma (309). For healthy people, the blood plasma of 38 healthy people was collected in the Bohr's way, and genome-wide methylation sequencing was performed (Whole Genome Bisulfite Sequencing, WGBS).
2) Candidate marker screening: for healthy plasma samples, the third quartile (Q3), also known as the "greater quartile", of the beta value of each probe corresponding to the 450K corresponding region was calculated, and the sites with Q3<0.02 were screened, resulting in List1. For 450K chip organization data, calculating a first quartile (Q1) of beta value of each probe corresponding to the 450K corresponding region, wherein Q1 is also called as smaller quartile, screening sites of Q1>0.1, and obtaining a result as List2. Taking the intersection of List1 and List2 yields 65739 differentially methylated regions
3) And (3) marker selection: among the above markers, the markers specific for kidney cancer were selected to obtain 123 markers. Meanwhile, the difference between the methylation level of the 450k chip kidney cancer tissue (660) and the methylation level of the paracancerous tissue (210) in TCGA is more than 0.2, and 23 differential methylation areas are finally obtained.
4) And (3) marker verification: probe capture was designed for the 23 differential methylation regions, and verification was performed using bohr honest plasma sample data (number of renal cancer samples=17, number of healthy human samples=32), to finally obtain 1 marker capable of distinguishing renal cancer from healthy human. The sequence of the polypeptide is shown as SEQ ID NO. 1.
Customizing a probe composition (panel) comprising a hypermethylated first probe composition and a hypomethylated second probe composition according to the obtained target sequence region, wherein the first probe composition comprises two probes for a marker and the first probe composition comprises a nucleotide sequence as shown in SEQ ID NO:2-3 for SEQ ID NO: 1; the second probe composition comprises two probes, for SEQ ID NO.1, the second probe composition comprises the nucleotide sequences as shown in SEQ ID NO. 4-5.
Then, the sample is verified in a plasma sample, and the experimental detection method is as follows:
cfdna extraction purification
1.1.1. Plasma sample preparation:
the blood samples were centrifuged at 2000g for 10min at 4℃and the plasma was transferred to a new centrifuge tube. The plasma samples were centrifuged at 16000g for 10min at 4℃and the next step was performed depending on the type of collection tube used, which was the other one used in the experiment.
TABLE 1
1.1.2. Cleavage and binding
1.1.2.1. The binding solution/bead mixture was prepared according to the following table and then thoroughly mixed.
TABLE 2
An appropriate volume of plasma sample was added.
1.1.2.2. The plasma sample and binding solution/bead mixture are thoroughly mixed.
1.1.2.3. The cfDNA was bound to the magnetic beads by sufficient binding on a spin mixer for 10 min.
1.1.2.4. The binding tube was placed on a magnetic rack for 5min until the solution became clear and the beads were fully adsorbed on the magnetic rack.
1.1.2.5. The supernatant was carefully discarded with a pipette, the tube was kept on the magnetic rack for several minutes, and the residual supernatant was removed with a pipette.
1.1.3. Washing
1.1.3.1. The beads were resuspended in 1ml of wash solution.
1.1.3.2. The resuspension was transferred to a new non-adsorbed 1.5ml centrifuge tube. The binding tube remains.
1.1.3.3. The centrifuge tube containing the bead resuspension was placed on a magnetic rack for 20s.
1.1.3.4. The separated supernatant was aspirated and the binding tube was washed, and the washed residual beads were collected again into a heavy suspension, discarding the lysis/binding tube.
1.1.3.5. The tube was placed on a magnet rack for 2min until the solution became clear, the beads were collected on the magnet rack and the supernatant was removed with a 1ml pipette.
1.1.3.6. The tube was left on the magnet rack and the remaining liquid was removed as much as possible with a 200. Mu.L pipette.
1.1.3.7. The tube was removed from the magnet holder, 1ml of wash solution was added and vortexed for 30s.
1.1.3.8. The solution was allowed to settle for 2min on a magnetic rack, the beads were collected on the magnetic rack, and the supernatant was removed with a 1ml pipette.
1.1.3.9. The tube was left on the magnet rack and the residual liquid was removed thoroughly with a 200 μl pipette.
1.1.3.10. The tube was removed from the magnet holder, 1ml 80% ethanol was added, and vortexed for 30s.
1.1.3.11. The solution was allowed to settle for 2min on a magnetic rack and the supernatant was removed with a 1ml pipette.
1.1.3.12. The tube was left on the magnet holder and the residual liquid was removed with a 200. Mu.L pipette.
1.1.3.13. The above 1.1.3.10.— 1.1.3.12 steps were repeated with 80% ethanol once, and the supernatant was removed as much as possible.
1.1.3.14. The tube was left on the magnetic rack and the beads were dried in air for 3-5 minutes.
1.1.4. Elution of cfDNA
1.1.4.1. The eluent was added according to the following table.
TABLE 3 Table 3
1.1.4.2. Vortex for 5min, place on a magnetic rack for 2min, the solution becomes clear, and suck cfDNA in the supernatant.
1.1.4.3. The purified cfDNA was used immediately or the supernatant was transferred to a new centrifuge tube and stored at-20 ℃.
Disruption and purification of gDNA:
1.2.1. according to the Qubit concentration, 2. Mu.g of gDNA was taken, added with water to 125. Mu.l, added to a covaries 130. Mu.l disruption tube, and the procedure was set: 50W,20%,200 cycles, 250s.
1.2.2. After the interruption, 1 μl of the sample is taken and subjected to fragment detection by using Agilent2100, and after normal interruption, the main peak of the sample detection is about 150bp-200bp.
For cfDNA samples, agilent2100 performed fragment detection, and direct Qubit was used for subsequent experiments.
1.3. Terminal repair, 3' end plus "a":
1.3.1. 20ng of the cut gDNA or cfDNA was added to a PCR tube, and the mixture was supplemented to 50. Mu.l with nuclease-free water, and the following reagents were added and vortexed to mix well:
TABLE 4 Table 4
Component (A) Volume of
gDNA/cfDNA 50μl
Stop repair and A tailing buffer 7μl
Termination repair and A tailing enzyme mixture 3μl
Total volume of 60μl
1.3.2. The following procedure was set up for the reaction on the PCR instrument: the temperature of the hot cover is 85 ℃.
TABLE 5
Temperature (temperature) Time
20℃ 30min
65℃ 30min
4℃ ∞
1.4. Joint connection and purification:
1.4.1. the linker was diluted in advance to the appropriate concentration with reference to the following table:
TABLE 6
1.4.2. The following reagents were prepared according to the following table, gently blotted and mixed, and centrifuged briefly:
TABLE 7
Component (A) Volume of
End repair, addition of "A" reaction product 60μl
Joint 5μl
Nuclease-free water 5μl
Connection buffer solution 30μl
DNA ligase 10μl
Total volume of 110μl
1.4.3. The following procedure was set up for the reaction on the PCR instrument: there is no thermal cover.
TABLE 8
Temperature (temperature) Time
20℃ 30min
4℃ ∞
1.4.4. Adding purified magnetic beads for experiment (AgencourtAMPure XP magnetic beads are taken to room temperature in advance, and are vibrated and mixed uniformly for standby) according to the following system:
TABLE 9
Component (A) Volume of
Joint connection product 110μl
AgencourtAMPure XP bead 110μl
Total volume of 220μl
1.4.4.1. Gently sucking and beating, and mixing for 6 times.
1.4.4.2. Standing at room temperature for 5-15min, and placing the PCR tube on a magnetic rack for 3min to clarify the solution.
1.4.4.3. The supernatant was removed, the PCR tube was placed on a magnetic rack, 200. Mu.l of 80% ethanol solution was added to the PCR tube, and the mixture was allowed to stand for 30 seconds.
1.4.4.4. The supernatant was removed, 200. Mu.l of 80% ethanol solution was added to the PCR tube, and after standing for 30s, the supernatant was thoroughly removed (it was recommended to remove the bottom residual ethanol solution using a 10. Mu.l pipette).
1.4.4.5. Standing at room temperature for 3-5min to volatilize residual ethanol thoroughly.
1.4.4.6. Adding 22 μl of nuclease-free water, removing the PCR tube from the magnetic rack, gently sucking and beating the resuspended magnetic beads, avoiding generating bubbles, and standing at room temperature for 2min.
1.4.4.7. The PCR tube was placed on a magnetic rack for 2min to clarify the solution.
1.4.4.8. Mu.l of the supernatant was pipetted into a new PCR tube.
1.5 bisulfite treatment and purification:
1.5.1. the desired reagent was taken out in advance and dissolved. The reagents were added according to the following table:
table 10
Component (A) High concentration sample (1 ng-2. Mu.g) volume Low concentration sample (1-500 ng) volume
Linker ligation of purified products 20μl 40μl
Bisulfite solution 85μl 85μl
DNA protection buffer 35μl 15μl
Total volume of 140μl 140μl
The DNA protection buffer was added to the liquid to turn blue. Gently blotted and mixed, and then split into two tubes for PCR.
1.5.3. The following procedure was set up and run: the lid was heated to 105 ℃.
TABLE 11
Temperature (temperature) Time
95℃ 5min
60℃ 10min
95℃ 5min
60℃ 10min
4℃ ∞
1.5.4. The same sample from both tubes was combined into the same clean 1.5ml centrifuge tube by brief centrifugation.
1.5.5. To each sample, 310. Mu.l of buffer BL (sample size less than 100ng of 1. Mu.l of carrier RNA (1. Mu.g/. Mu.l) was added), vortexed, and briefly centrifuged.
1.5.6. 250 μl of absolute ethanol was added to each sample, vortexed and mixed for 15s, centrifuged briefly, and the mixture was added to the prepared corresponding column.
1.5.7. Standing for 1min, centrifuging for 1min, transferring the liquid in the collecting pipe into a centrifugal column again, centrifuging for 1min, and discarding the liquid in the centrifugal pipe.
1.5.8. Add 500. Mu.l buffer BW (note whether absolute ethanol was added) centrifuge for 1min and discard the waste.
1.5.9. Add 500. Mu.l buffer BD (note whether absolute ethanol was added) cover the tube and leave it for 15min at room temperature. Centrifuging for 1min, and discarding the centrifuged liquid.
1.5.10. Mu.l of buffer BW (note whether absolute ethanol was added) was added, centrifuged for 1min, the detached liquid was discarded, and repeated 2 times.
1.5.11. 250 μl of absolute ethanol was added, centrifuged for 1min, the column was placed in a new 2ml collection tube and all remaining liquid was discarded.
1.5.12. The column was placed in a clean 1.5ml centrifuge tube, 20. Mu.l of nuclease-free water was added to the center of the column membrane, the lid was gently covered, the column was placed at room temperature for 1min, and the column was centrifuged for 1min.
1.5.13. The liquid in the collection tube was re-transferred to a centrifuge column, left at room temperature for 1min, and centrifuged for 1min.
1.6. Pre-amplification and purification before hybridization:
1.6.1. preparing a reaction system according to the following table, blowing, mixing uniformly and centrifuging briefly:
table 12
1.6.2. The following procedure was set and the PCR procedure was started: thermal cover 105 DEG C
TABLE 13
The number of PCR cycles was adjusted according to the amount of DNA to be added, and the reference data were as follows:
TABLE 14
1.6.4. 50 mu l AgencourtAMPureXP magnetic beads are added into a PCR tube after the reaction is finished, and the mixture is blown and evenly mixed by a pipette to avoid generating bubbles (AgencourtAMPure XP is evenly mixed and balanced at room temperature in advance).
1.6.5. Incubating for 5-15min at room temperature, and placing the PCR tube on a magnetic rack for 3min to clarify the solution.
1.6.6. The supernatant was removed, the PCR tube was placed on a magnetic rack, 200. Mu.l of 80% ethanol solution was added to the PCR tube, and the mixture was allowed to stand for 30 seconds.
1.6.7. The supernatant was removed, 200. Mu.l of 80% ethanol solution was added to the PCR tube, and after standing for 30s, the supernatant was thoroughly removed (it was recommended to remove the bottom residual ethanol solution using a 10. Mu.l pipette).
1.6.8. Standing at room temperature for 5min to volatilize residual ethanol thoroughly.
1.6.9. Add 30. Mu.l of nuclease free water, remove the centrifuge tube from the magnetic rack and gently pipette the resuspended beads using a pipette.
1.6.10. Standing at room temperature for 2min, and placing 200 μl PCR tube on a magnetic rack for 2min to clarify the solution.
1.6.11. The supernatant was transferred to a new 200. Mu.l PCR tube (placed on an ice box) with a pipette, and the reaction tube was marked with a sample number, and prepared for the next reaction.
1.6.12. 1 μl of the sample was used for library concentration determination using Qubit, and library concentration was recorded.
1.6.13. 1 μl of the sample was used for library fragment length measurement using Agilent2100, the library length being approximately between 270bp-320 bp.
1.7. Hybridization of sample to probe:
1.7.1. sample libraries were mixed with various Hyb blockers, labeled B, according to the following system:
TABLE 15
Component (A) Volume of
Pre-amplification product 750ng of corresponding volume
Hyb human blockers 5μl
Joint blocking material 6μl
Reinforcing agent 5μl
1.7.2. The prepared mixture of the sample and the Hyb blocker is put into a vacuum concentration centrifuge, a PCR tube cover is opened, the centrifuge is started, a vacuum pump switch is opened, and concentration is started.
1.7.3. The drained sample was redissolved in about 9 μl of nuclease-free water, and mixed gently by pipetting, briefly centrifuged and placed on ice for use, labeled B.
1.7.4. And (3) placing the Hyb buffer solution in a room temperature for melting, wherein precipitation appears after melting, placing the mixture in a water bath at 65 ℃ for preheating after uniformly mixing, placing 20 mu l of the Hyb buffer solution (without precipitation and turbidity) in a new 200 mu l PCR tube after complete dissolution, covering a tube cover, marking as A, and continuously placing the tube cover in the water bath at 65 ℃ for incubation for later use.
1.7.5. The methylation probe sequence described before was synthesized by Ai Jitai c biotechnology (beijing) limited:
1.7.6. mu.l of the RNase-blocking material and 2. Mu.l of the probe composition were placed in a 200. Mu.l PCR tube, gently blotted and mixed, centrifuged briefly and placed on ice for use, labeled C.
1.7.7. Setting parameters of a PCR instrument, and heating the cover to 100 ℃,95 ℃ for 5min; and (5) maintaining at 65 ℃.
1.7.8. The PCR tube B was placed on a PCR instrument and the procedure was run.
When the temperature of the PCR instrument is reduced to 65 ℃, the PCR tube A is placed on the PCR instrument for incubation, and a thermal cover of the PCR instrument is covered.
After 1.7.10.5min, C was placed on PCR for incubation and covered with the thermal cover of the PCR instrument.
1.7.11. Placing the PCR tube C into a PCR instrument for 2min, adjusting the liquid transfer device to 13 μl, sucking 13 μl of Hyb buffer solution from the PCR tube A, transferring to the PCR tube C, sucking all samples in the PCR tube B, transferring to the PCR tube C, gently sucking for 10 times, mixing thoroughly, avoiding generating a large amount of bubbles, sealing the tube cover, covering the thermal cover of the PCR instrument, and incubating overnight at 65deg.C (16-24 h).
1.8. Capturing a target region DNA library:
1.8.1. preparation of Capture magnetic beads
1.8.1.1. The beads (DynabeadsMyOne Streptavidin T1) were removed from 4 ℃, resuspended by vortexing.
1.8.1.2. 50 μl of magnetic beads were placed in a new PCR tube, placed on a magnetic rack for 1min to clarify the solution, and the supernatant was removed.
1.8.1.3. The PCR tube was removed from the magnetic rack, 200. Mu.L of binding buffer was added and gently pipetted several times to mix well and resuspend the beads.
1.8.1.4. Placing on a magnetic rack for 1min, and removing the supernatant.
1.8.1.5. Repeating the steps 3-4 twice, and washing the magnetic beads for 3 times.
1.8.1.6. The PCR tube was removed from the magnetic rack and 200. Mu.L of binding buffer was added to gently pipette 6 times to resuspend the beads for use.
1.8.2. Capturing a target DNA library
1.8.2.1. The hybridization product PCR tube C is kept on a PCR instrument, 200 mu L of prepared capture magnetic beads are added into the hybridization product PCR tube C, the hybridization product PCR tube C is sucked and beaten for 6 times by a pipette for uniform mixing, and the hybridization product PCR tube C is placed on a rotary mixer for 30min at room temperature (the rotating speed is preferably not more than 10 revolutions per minute).
1.8.2.2. The PCR tube was placed on a magnetic rack for 2min to clarify the solution and the supernatant was removed.
1.8.2.3. 200 mu L of washing buffer 1 is added into the PCR tube C, gently sucked and beaten for 6 times of uniform mixing, placed on a rotary mixer for cleaning for 15min (the rotating speed is preferably not more than 10 revolutions per minute), then centrifuged briefly, the PCR tube is placed on a magnetic rack for 2min to clarify the solution, and the supernatant is removed.
1.8.2.4. 200 μl of washing buffer 2 preheated at 65deg.C is added, gently sucked and beaten for 6 times, mixed well, placed on a mixing instrument, incubated at 65deg.C for 10min, and washed at 800 rpm.
1.8.2.5. The PCR tube was placed on a magnetic rack for 2min after brief centrifugation and the supernatant removed. The washing with wash buffer 2 was repeated 2 more times for a total of 3 times. The wash buffer 2 was removed thoroughly last time.
The PCR tube was placed on a magnetic rack, 200. Mu.l of 80% ethanol was added to the PCR tube, and after standing for 30 seconds, the ethanol solution was thoroughly removed and dried at room temperature for 2 minutes.
1.8.2.7. Adding 30 mu L nuclease-free water into the PCR tube, taking the PCR tube off the magnetic rack, and lightly sucking and beating the magnetic beads for 6 times for later use.
1.9. Post-capture amplification and purification
1.9.1. Preparing a reaction system according to the following table, enriching a capture library, lightly blowing and uniformly mixing, and then briefly centrifuging:
table 16
1.9.2. The following procedure was set, the samples were placed in a PCR instrument, and the procedure was run: the lid was heated to 105 ℃.
TABLE 17
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After the PCR was completed, 55. Mu. l AgencourtAMPure XP beads were added to the sample, and the mixture was gently pipetted and stirred.
1.9.4. Incubation was performed for 5min at room temperature, and the PCR tube was placed on a magnetic rack for 3min to clarify the solution.
1.9.5. The supernatant was removed, the PCR tube was placed on a magnetic rack, 200. Mu.l of 80% absolute ethanol was added, and the mixture was allowed to stand for 30 seconds.
1.9.6. The supernatant was removed, 200. Mu.l of 80% absolute ethanol was added to the PCR tube, and the supernatant was thoroughly removed after standing for 30.
1.9.7. Standing at room temperature for 5min to volatilize residual ethanol thoroughly.
1.9.8. Add 25. Mu.l of nuclease-free water, remove the PCR tube from the magnetic rack, gently blow mix and re-suspend the beads and leave for 2min at room temperature.
1.9.9. The PCR tube was placed on a magnetic rack for 2min to clarify the solution.
1.9.10. Mu.l of the supernatant was pipetted into a 1.5ml centrifuge tube and labeled with sample information.
1.9.11. 1 μl of library was quantified using Qubit and library concentrations were recorded.
1.9.12. 1 μl of sample was taken and used for library fragment length determination using Agilent 2100.
1.9.13. Sequencing was performed using Illumina high throughput sequencing platform.
1.10. Methylation letter analysis flow. The method is approximately as follows: checking sequencing quality by using fastp quality control software, removing low-quality reads, comparing the quality-controlled clean data to a reference genome by using bismar_methyl_extraction software, and extracting corresponding methylation sites by using bismar_methyl_extraction software. Finally, the methylation level of each marker was calculated.
1.11 based on collection of 17 samples clinically diagnosed as renal cancer from Beijing area and 32 healthy human samples collected from Beijing area, methylation levels of the screened 1 methylation biomarkers were calculated using the methylation pooling method described in example 1, and threshold (hereinafter referred to as site or marker) and separately differentiated AUC values of the 1 methylation biomarkers were calculated from methylation levels of the 1 methylation biomarkers in renal cancer sample and normal human sample data sets as shown in Table 1;
the methylation level threshold calculation method comprises the following steps: drawing an ROC curve according to the data set (comprising the type and methylation level of each sample), wherein the confusion matrix corresponding to the optimal threshold point on the ROC curve is the basis for calculating indexes such as sensitivity, specificity and accuracy. Typically we will choose by a boulder index (you index). The about index, also called the correct index, refers to the sum of sensitivity and specificity minus 1: youden index=sensitivity+specificity-1. The value of about dengue index range is between 0 and 1, which represents the total ability of the classification model to find true patients and non-patients. The larger the about log index, the better the classification model performance:
table 18 data on the specific representation of 28 methylation markers
Example 2
Collecting 3 kidney cancer samples (clinically diagnosed as kidney cancer) from Beijing area and 3 healthy human samples (S1-3 healthy human samples and S4-6 healthy human samples) from Beijing area, and collecting peripheral blood according to the method of example 1 by adopting the methylation marker detection method; establishing a library, and sequencing through an Illumina platform; sequencing data is subjected to the biological information analysis flow to obtain the methylation level of each marker, the disease condition of the patient is predicted according to the threshold value of each marker, if the disease condition exceeds the threshold value, the disease condition is a cancer sample, and if the disease condition is lower than the threshold value, the disease condition is a healthy human sample, and the specific results are shown in the following table:
wherein, the interpretation result, 0, represents the classification as normal, i.e. healthy; 1 represents a classification as abnormal, i.e. tumor.
Table 19 methylation values and interpretation results for samples
In summary, the inventors of the present application have obtained a methylation gene associated with kidney cancer and determined a target sequence of methylation abnormality of the methylation gene of kidney cancer, and, through the target sequence of the methylation gene, the methylation state of the gene can be sensitively and specifically detected, so that the methylation state can be used for detecting free DNA of peripheral blood, and the composition of the present application can realize real-time monitoring, and has higher sensitivity and accuracy.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present application still fall within the protection scope of the technical solution of the present application.
The sequence table used in the application is shown in table 20:
table 20
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Claims (9)

1. The marker for detecting the kidney cancer is characterized in that a marker corresponding gene is MCF2L, the marker is a methylated marker, and the nucleotide sequence of the marker is shown as SEQ ID NO. 1.
2. A probe composition comprising a probe that targets methylation of the marker of claim 1, the probe composition comprising a hypermethylated first probe composition for hybridization to a hypermethylated region converted by bisulfite and a hypomethylated second probe composition for hybridization to a hypomethylated region converted by bisulfite;
the first probe composition comprises probes shown as SEQ ID NOS.2-3, and the second probe composition comprises probes shown as SEQ ID NOS.4-5.
3. The application of the marker in preparation of a kit for detecting kidney cancer is characterized in that the corresponding gene of the marker is MCF2L, the marker is a methylated marker, and the nucleotide sequence of the marker is shown as SEQ ID NO. 1.
4. A composition for use in the detection of renal cancer, comprising a nucleic acid for detecting methylation of the marker corresponding gene MCF2L of claim 1, said nucleic acid comprising the probe composition of claim 2.
5. The composition of claim 4, wherein the nucleic acid comprises:
a primer that is a fragment of at least 9 nucleotides in a target sequence of the marker, the fragment comprising at least one CpG dinucleotide sequence.
6. The composition of claim 4, further comprising an agent that converts an unmethylated cytosine base at position 5 of the target sequence of the marker to uracil.
7. The composition of claim 4, wherein the nucleic acid for detecting methylation of a marker-corresponding gene further comprises:
blocking agents that preferentially bind to target sequences in the unmethylated state.
8. A kit comprising the probe composition of claim 2 or the composition of any one of claims 5-7.
9. A chip comprising the marker of claim 1 or the probe composition of claim 2.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008066878A2 (en) * 2006-11-29 2008-06-05 University Of Vermont And State Agricultural College Methods and products for diagnosing cancer
CN107727865A (en) * 2016-08-11 2018-02-23 博尔诚（北京）科技有限公司 The systemic detection method of tumor markers and its application
KR101860238B1 (en) * 2016-12-30 2018-05-23 충북대학교 산학협력단 Use of ZFP28, FAM155A and DPP6 for Prognostic Marker Diagnosis of renal cell carcinoma

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008066878A2 (en) * 2006-11-29 2008-06-05 University Of Vermont And State Agricultural College Methods and products for diagnosing cancer
CN107727865A (en) * 2016-08-11 2018-02-23 博尔诚（北京）科技有限公司 The systemic detection method of tumor markers and its application
KR101860238B1 (en) * 2016-12-30 2018-05-23 충북대학교 산학협력단 Use of ZFP28, FAM155A and DPP6 for Prognostic Marker Diagnosis of renal cell carcinoma

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