CN114231635A - Marker and probe composition for lung cancer screening and application thereof - Google Patents

Abstract

The invention discloses a marker for screening lung cancer, a probe composition and application thereof, wherein the marker is selected from any one of 3 markers. The invention uses the marker, can sensitively and specifically detect the methylation state of the gene, thus being used for detecting free DNA of peripheral blood, and the composition is used for screening asymptomatic people in a non-invasive way, thus reducing the harm caused by invasive detection, and the composition has higher sensitivity and accuracy and can realize real-time monitoring.

Description

Marker and probe composition for lung cancer screening and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a marker and probe composition for lung cancer screening and application thereof.

Background

Lung cancer is one of the most prevalent and high mortality cancers worldwide. In China, the incidence and mortality of lung cancer are the top. Conventional means of screening for lung cancer have several protein markers in addition to low dose helical ct (ldct), such as: carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), neuron-specific enolase (NSE), and the like. However, these approaches are heterogeneous in sensitivity and specificity. Currently, DNA methylation has been shown to be tissue specific, useful for early cancer detection, and can be followed to the primary site of a tumor based on circulating tumor DNA (ctdna) methylation characteristics.

Disclosure of Invention

The invention aims to provide a marker for detecting lung cancer and a probe composition, which can be used for screening lung cancer, wherein the marker is used for screening asymptomatic people in a non-invasive mode and detecting prognosis of cancer patients, reduces harm caused by invasive detection, and has higher sensitivity and accuracy.

The specific technical scheme of the invention is as follows:

1. a marker for detecting lung cancer, wherein the marker is selected from one of the following: USP33, CHRNB2 and TMEM 219.

2. The marker according to the 1, wherein the nucleotide sequence of the marker is selected from one shown as SEQ ID NO 1-3, and 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, comprising a hypermethylated first probe composition for hybridizing to a bisulfite converted CG hypermethylated region and a hypomethylated second probe composition for hybridizing to a bisulfite converted CG hypomethylated region;

preferably, the first probe composition comprises n probes that hybridize to each nucleotide of the sense and antisense strands of the bisulfite converted CG hypermethylated region;

preferably, the second probe composition comprises m probes that hybridize to each nucleotide of the sense and antisense strands of the hypomethylated region of bisulfite converted CG;

preferably, n and m are each any integer from 1 to 10;

preferably, there is x between the nth-1 probe and the nth probe₁Nucleotide overlap, preferably, x₁Is any integer of 0 to 100;

preferably, there is x between the m-1 st probe and the m-th probe₂Nucleotide overlap, preferably, x₂Is any integer of 0 to 100;

further preferably, the first probe composition comprises one or two nucleotide sequences shown as SEQ ID NO. 4-9, and the second probe composition comprises one or two nucleotide sequences shown as SEQ ID NO. 10-15.

5. Use of a marker for the preparation of a kit for the detection of lung cancer, wherein the marker is selected from one of the following: USP33, CHRNB2 and TMEM 219.

6. The use according to claim 5, wherein the nucleotide sequence of the marker is selected from one of the group consisting of SEQ ID NO 1-3, preferably the marker is a methylated marker;

preferably, the probe composition is used to target a post-methylation marker of lung cancer;

preferably, the probe composition is the probe composition described in item 3 or 4.

7. A composition for lung cancer detection, comprising a nucleic acid for detecting methylation of any one of the following markers selected from the group consisting of: USP33, CHRNB2 and TMEM 219.

8. The composition of claim 7, wherein the nucleotide sequence of the marker is selected from the group consisting of SEQ ID NOS 1-3.

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 a fragment of at least 15 nucleotides in a target sequence of the marker, said fragment comprising at least one CpG dinucleotide sequence;

preferably, the composition further comprises an agent that converts unmethylated cytosine bases 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:

a blocker that preferentially binds to a target sequence in a non-methylated state.

10. A kit comprising a marker of item 1 or 2 or a probe composition of item 3 or 4 or a composition of any one of items 7-9.

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 invention utilizes epigenome and bioinformatics technology, searches a plurality of methylation genes related to lung cancer by analyzing genome methylation data of the lung cancer, determines a target sequence of methylation abnormality of the methylation gene of the lung cancer, and can sensitively and specifically detect the methylation state of the methylation gene through the target sequence of the methylation gene, thereby being used for detecting free DNA of peripheral blood.

The composition is used for screening asymptomatic people in a non-invasive mode, reduces harm caused by invasive detection, has higher sensitivity and accuracy, and can realize real-time monitoring.

Detailed Description

The present invention will be described in detail below. While specific embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as 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 invention to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, however, the description is given for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.

The invention provides a marker for detecting lung cancer, which is selected from one of the following: USP33, CHRNB2 and TMEM 219.

In a specific embodiment, the nucleotide sequence of the marker is selected from one of the markers shown in SEQ ID NO 1-3, preferably, the marker is a methylated marker.

Wherein the nucleotide sequence of USP33 is shown as SEQ ID NO 1; the nucleotide sequence of CHRNB2 is shown as SEQ ID NO. 2; the nucleotide sequence of TMEM219 is shown in SEQ ID NO. 3.

Wherein the sequences of the markers are sequences that have not been converted to bisulfite.

The invention provides a probe composition comprising a probe that targets methylation of the marker.

The methylation refers to the methylation process of the 5 th carbon atom on cytosine in CpG dinucleotide, and is an important epigenetic mechanism which can be inherited to new filial generation DNA along with the replication process of DNA under the action of DNA methyltransferase as a stable modification state. Aberrant methylation includes hypermethylation of cancer suppressor genes and DNA repair genes, hypomethylation of repeat DNA, loss of imprinting of certain genes, which is associated with the development of a variety of tumors.

Methylation according to the present invention can be the level of methylation, the degree of methylation or the state of methylation and one skilled in the art can use quantitative determination methods to determine methylation when analyzing methylation of such target sequences.

The probe is single-stranded or double-stranded DNA with the length of dozens to hundreds or even thousands of base pairs, and can be combined (hybridized) with complementary non-labeled single-stranded DNA or RNA in a sample to be detected by hydrogen bonds to form a double-stranded complex (hybrid) by utilizing the denaturation and renaturation of molecules and the high accuracy of base complementary pairing. After washing off the probe which is not coupled, the result of hybridization reaction can be detected by a detection system such as autoradiography or enzyme-linked reaction. In the present application, the region to which the probe complementarily binds or hybridizes is a specific target region, and a plurality of probe sets synthesize a probe composition.

In a specific embodiment, the probe composition comprises a hypermethylated first probe composition for hybridizing to a bisulfite converted CG hypermethylated region and a hypomethylated second probe composition for hybridizing to a bisulfite converted CG hypomethylated region.

Hypermethylation refers to the fact that the base C becomes the base T after bisulfite conversion of the marker, but if the base is CG, the base C remains unchanged;

the hypomethylation refers to that all bases CG are not methylated and all bases C are changed into bases T after the marker is converted by bisulfite.

The sequence of the marker converted to bisulfite varies from one person to another, and an extreme case of each marker is shown here, where all the CGs of the segment are hypermethylated, and the hypermethylated sequences of their complementary strands:

the sequence of an extreme case of SEQ ID NO. 1 is shown as SEQ ID NO. 16;

the complementary strand of the extreme sequence is shown as SEQ ID NO. 17;

the sequence of one extreme of SEQ ID NO. 2 is shown as SEQ ID NO. 18;

the complementary strand of the extreme sequence is shown as SEQ ID NO. 19;

an extreme sequence of SEQ ID NO. 3 is shown as SEQ ID NO. 20;

the complementary strand of the extreme sequence is shown in SEQ ID NO: 21.

Similarly, since each individual has a different methylation state, an extreme case is shown here, where all CGs are in hypomethylated state, and the sequence of the hypomethylated state of their complementary strands is also shown:

the sequence of an extreme case of SEQ ID NO. 1 is shown as SEQ ID NO. 22;

the complementary strand of the extreme sequence is shown as SEQ ID NO. 23;

the sequence of one extreme of SEQ ID NO. 2 is shown as SEQ ID NO. 24;

the complementary strand of the extreme sequence is shown as SEQ ID NO. 25;

the sequence of one extreme of SEQ ID NO. 3 is shown as SEQ ID NO. 26;

the complementary strand of the extreme sequence is shown in SEQ ID NO: 27.

In a specific embodiment, the first probe composition comprises n probes that hybridize to each nucleotide of the sense and antisense strands of the bisulfite converted CG hypermethylated region.

The second probe composition comprises m probes that hybridize to each nucleotide of the sense and antisense strands of the bisulfite converted CG hypomethylated region.

The number of probes in the first probe composition and the second probe composition is not limited in the present invention, and can be selected by one skilled in the art according to the need, for example, m and n can be any integer from 1 to 10, and m and n can be the same or different.

For example, m and n may be any integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and preferably, m ═ n ═ 2.

In a specific embodiment, there is x between the nth-1 probe and the nth probe₁Nucleotide overlap, preferably, x₁Is any integer of 0 to 100;

preferably, there is x between the m-1 st probe and the m-th probe₂Nucleotide overlap, preferably, x₂Is any integer of 0 to 100.

Wherein x is₁And x₂May be the same or different when x is₁When the number is 0, it indicates that the tail of the n-1 th probe is connected to the head of the nth probe, and similarly, when x is₂A value of 0 indicates that the tail of the m-1 th probe is connected to the head of the m-th probe.

According to the invention, the probe composition is hybridized with the marker converted by bisulfite, wherein the hypermethylated first probe composition is hybridized with the CG hypermethylated region, and the hypomethylated second probe composition is hybridized with the CG hypomethylated region, so that the methylation level of a target sequence can be efficiently and accurately detected, and the method can be further used for lung cancer screening.

In a specific embodiment, the hypermethylated first probe composition comprises one or both of SEQ ID NOS 4-9.

The hypomethylated second probe composition comprises one or two of SEQ ID NO 10-15.

Wherein the first probe composition for hybridizing to a methylated sequence of USP33 comprises the nucleotide sequence set forth in SEQ ID NO 4-5;

the first probe composition for hybridizing to the CHRNB2 methylated sequence comprises the nucleotide sequence set forth as SEQ ID NO 6-7;

the first probe composition for hybridization to the TMEM219 methylation sequence includes the nucleotide sequences set forth in SEQ ID NOs 8-9;

a second probe composition for hybridization to a methylated sequence of USP33 includes the nucleotide sequences shown in SEQ ID NOs 10-11;

the second probe composition for hybridizing to the CHRNB2 methylated sequence comprises the nucleotide sequence set forth as SEQ ID NO 12-13;

the second probe composition used for hybridization with the methylated sequence of TMEM219 includes the nucleotide sequences set forth in SEQ ID NOS: 14-15.

The invention provides an application of a marker in preparing a kit for detecting lung cancer, wherein the marker is selected from one of the following: USP33, CHRNB2 and TMEM 219.

In a specific embodiment, the nucleotide sequence of the marker is selected from one of the markers as shown in SEQ ID NO 1-3, preferably the marker is a methylated marker.

The invention provides application of a probe composition in preparing a kit for detecting lung cancer, wherein the probe composition is used for targeting a marker after lung cancer methylation.

In a specific embodiment, the probe composition is the probe composition described above.

The present invention provides a composition for lung cancer detection, the composition comprising a nucleic acid for detecting methylation of any one of the following markers selected from the group consisting of: USP33, CHRNB2 and TMEM219, preferably, the nucleotide sequence of the marker is selected from one shown in SEQ ID NO 1-3.

In a specific embodiment, the nucleic acid comprises the probe composition described above.

In a specific 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 the NDA of the 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 of the sequence after bisulfite conversion of the target sequence of the marker, said fragment comprising at least one CpG dinucleotide sequence.

In a specific embodiment, the nucleic acid further comprises:

a probe that hybridizes under moderate stringency or stringent conditions to a fragment of at least 15 nucleotides in a target sequence of the marker, said fragment comprising at least one CpG dinucleotide sequence.

In a specific embodiment, the composition further comprises an agent that converts unmethylated cytosine bases at position 5 of the target sequence of the marker to uracil, e.g., the agent can be bisulfite and the like; preferably, the nucleic acid for detecting methylation of a target sequence of a marker further comprises:

a blocker that preferentially binds to a target sequence in a non-methylated 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 primer or the reverse primer have an overlapping region of more than or equal to 5 nucleotides, the blocker is complementary with the forward primer or the reverse primer and the same strand of the target gene target sequence DNA, the melting temperature of the blocker is higher than (including) 5 ℃ of the forward primer or the reverse primer, 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 methylated after bisulfite conversion. Therefore, when the genomic DNA of the biological sample to be detected is a mixture of methylated and unmethylated states, particularly in the case where the amount of methylated DNA is much smaller than that of unmethylated DNA, bisulfite-converted unmethylated DNA preferentially binds to the blocking agent and thus binds to the PCR obligation and thus the DNA template, and thus PCR amplification does not occur, whereas methylated DNA does not bind to the blocking agent and thus PCR amplification occurs with the primer set, and then the fragments obtained by amplification are detected directly or indirectly.

The present invention provides a kit comprising the above marker or the above probe composition or the above composition.

In a specific embodiment, the kit further comprises a container for holding a biological sample of the subject.

In a specific embodiment, the kit further comprises instructions for using and interpreting the results of the assay.

The biological sample may be, for example, peripheral blood whole blood, plasma or serum.

The present invention is not limited to the method for detecting the methylation level of a target sequence by using the above-mentioned kit, and those skilled in the art can select the method according to the need, for example, the present invention provides a method for detecting the methylation level of a marker target sequence by using the above-mentioned kit, which comprises the following steps:

collecting a subject sample;

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 subjected to the pre-PCR amplification by using the probe composition;

amplifying the product obtained after hybridization capture by utilizing PCR;

performing high-throughput second-generation sequencing on a product obtained after hybridization and capture after PCR amplification;

analyzing the sequencing data to determine the methylation level of the sample;

calculating a threshold for each marker based on the methylation status of the existing sample, interpreting the patient's disease status based on the methylation level of a certain marker in the sample, and determining a cancer sample if the methylation level of a certain marker in the sample exceeds the threshold and a healthy human sample if the methylation level is below the threshold.

For another example, the present invention provides a method for detecting the methylation level of a target sequence of a marker using the above-described kit, comprising the steps of:

(1) drawing peripheral blood from the 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 5-unmethylated cytosine base to uracil or another base, i.e., the 5-unmethylated cytosine base of the target sequence of the marker is converted to uracil or another base, and the converted base is different from the 5-unmethylated cytosine base in hybridization properties and is detectable;

(4) contacting the free DNA treated in step (3) with a DNA polymerase and a primer for the target sequence of the marker such that the target sequence of the treated marker is amplified to produce an amplification product or is not amplified; the target sequence of the processed marker, if subjected to DNA polymerization, produces an amplification product; the target sequence of the treated marker is not amplified if no DNA polymerization reaction occurs;

(5) detecting the amplification 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 invention provides a chip comprising the above-mentioned marker or the above-mentioned probe composition or the above-mentioned composition.

The chip is also called gene chip, and the sequencing principle 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 the probes with target nucleotides with known sequences are fixed on the surface of a substrate. When the nucleic acid sequence with fluorescent label in the solution is complementary matched with the nucleic acid probe in the corresponding position on the gene chip, the probe position with the strongest fluorescence intensity is determined to obtain a group of probe sequences with completely complementary sequences.

The chip is prepared by mainly using a glass sheet or a silicon sheet as a carrier and arranging oligonucleotide fragments or cDNA as probes on the carrier in sequence by adopting an in-situ synthesis and microarray method.

The chip is based on signal detection of DNA sequence hybridization after bisulfite treatment, wherein the bisulfite treatment is to change non-methylated cytosine into uracil, while the methylated cytosine is kept unchanged, then the uracil is converted into thymine, and finally the chip hybridization is carried out; and finally, judging the type of the added base according to the fluorescence color, and further determining whether the site is methylated.

The invention provides a method for screening lung cancer, which comprises the following steps:

detecting the methylation level of the marker, and

determining a subject's risk of having lung cancer based on the methylation level, the marker being selected from one of: USP33, CHRNB2 and TMEM 219.

Examples

The invention is described generally and/or specifically for the materials used in the tests and the test methods, in the following examples,% means wt%, i.e. percent by weight, unless otherwise specified. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

Example 1 screening markers

1) Sample collection: downloading 450k methylated chip cancer tissue data in TCGA, relating to 7769 cancer tissue samples of 26 tumors, the traditional Chinese medicine composition comprises adrenocortical carcinoma (80), urinary bladder urothelial carcinoma (409), acute myeloid leukemia (140), brain low-grade glioma (654), breast cancer (740), cervical cancer (286), colorectal cancer (348), esophageal cancer (183), uveal melanoma (80), head and neck squamous cell carcinoma (527), kidney cancer (660), liver cancer (377), lung adenocarcinoma (425), lung squamous carcinoma (372), diffuse large B-cell lymphoma (29), ovarian serous cystadenocarcinoma (10), pancreatic cancer (184), mesothelioma (116), prostate cancer (488), skin melanoma (104), sarcoma (117), gastric cancer (397), testicular cancer (134), thymus cancer (94), thyroid cancer (506) and endometrial cancer (309). For healthy population, plasma from 38 healthy persons was collected in the Boercheng and subjected to Whole Genome methylation Sequencing (WGBS).

2) Candidate marker screening: for healthy plasma samples, the third quartile (Q3), also called the "larger quartile", of the β value of each probe corresponding to the 450K corresponding region was calculated, and the site with Q3<0.02 was screened for List 1. For 450K chip tissue data, the first quartile (Q1) of each probe beta value corresponding to 450K corresponding region was calculated, Q1 is also called "smaller quartile", and sites with Q1>0.1 were screened, resulting in List 2. Taking the intersection of List1 and List2 yielded 65739 differentially methylated regions.

3) And (3) selecting a marker: among the above markers, 29 markers were selected from markers specific to lung cancer. Meanwhile, the difference of the methylation levels of 450k chip lung cancer (including lung adenocarcinoma and lung squamous carcinoma) tissues (797) and paracarcinoma tissues (73) in TCGA is required to be more than 0.2, and finally 17 differential methylation regions are obtained.

4) And (3) marker verification: probe capture is designed for the 17 differential methylation regions, and verification is performed by using borchenne plasma sample data (the number of lung cancer samples is 24, and the number of healthy people samples is 38), so that 3 markers capable of distinguishing lung cancer from healthy people are finally obtained. The sequences are respectively shown in SEQ ID NO 1-3.

Customizing a probe composition (panel) based on the resulting target sequence region, wherein the first probe composition comprises two probes per marker and a hypermethylated first probe composition and a hypomethylated second probe composition, wherein the first probe composition comprises the nucleotide sequences shown as SEQ ID NO 4-5 for SEQ ID NO 1;

for SEQ ID NO. 2, the first probe composition comprises the nucleotide sequences shown as SEQ ID NO. 6-7; for SEQ ID NO. 3, the first probe composition includes the nucleotide sequences shown in SEQ ID NO. 8-9.

The second probe composition comprises two probes, wherein, for SEQ ID NO:1, the second probe composition comprises nucleotide sequences shown as SEQ ID NO: 10-11; for SEQ ID NO. 2, the second probe composition comprises the nucleotide sequences shown as SEQ ID NO. 12-13; for SEQ ID NO. 3, the second probe composition includes the nucleotide sequences shown in SEQ ID NO. 14-15.

Then, the test sample is verified in a plasma sample, and the test method is as follows:

extraction and purification of cfDNA

1.1.1. Plasma sample preparation:

the blood samples were centrifuged at 2000g for 10min at 4 ℃ and the plasma 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 otherwise used in this 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 the binding solution/bead mixture were thoroughly mixed.

1.1.2.3. Binding was performed on a spin mixer for 10min sufficient to bind cfDNA to the magnetic beads.

1.1.2.4. The binding tube was placed on a magnetic stand for 5min until the solution became clear and the magnetic beads were completely adsorbed on the magnetic stand.

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 machine

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-adsorbing 1.5ml centrifuge tube. The bonded tube is retained.

1.1.3.3. The centrifuge tube containing the bead resuspension was placed on a magnetic rack for 20 s.

1.1.3.4. The separated supernatant was aspirated to wash the binding tubes, and the washed residual beads were collected again in a resuspension, discarding the lysis/binding tubes.

1.1.3.5. The tube was placed on a magnetic rack for 2min until the solution became clear, the beads were collected in the magnetic rack, and the supernatant was removed with a 1ml pipette.

1.1.3.6. The tube was left on the magnetic rack and the remaining liquid was removed as much as possible with a 200 μ L pipette.

1.1.3.7. The tube was removed from the magnetic stand, 1ml of wash solution was added, and vortexed for 30 s.

1.1.3.8. Place on magnetic rack for 2min until the solution cleared, 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 magnetic rack and the residual liquid was removed thoroughly with a 200 μ L pipette.

1.1.3.10. The tube was removed from the magnetic stand, 1ml of 80% ethanol was added, and vortexed for 30 s.

1.1.3.11. The solution became clear by placing on a magnetic rack for 2min and the supernatant was removed with a 1ml pipette.

1.1.3.12. The tube was left on the magnetic rack and the remaining liquid was removed with a 200 μ L pipette.

1.1.3.13. Repeat the 1.1.3.10-1.1.3.12 steps with 80% ethanol once to remove the supernatant as much as possible.

1.1.3.14. The tube was left on the magnetic stand 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 as in the table below.

TABLE 3

1.1.4.2. Vortex for 5min, place on magnetic rack for 2min, the solution becomes clear, and the cfDNA in the supernatant is aspirated.

1.1.4.3. Purified cfDNA was used immediately, or the supernatant was transferred to a new centrifuge tube and stored at-20 ℃.

gDNA disruption and purification:

1.2.1. according to the Qubit concentration, 2. mu.g of gDNA was taken, supplemented to 125. mu.l with water, added to a covaris 130. mu.l stoptube, and the program was set: 50W, 20%, 200 cycles, 250 s.

1.2.2. After the interruption, 1 μ l of sample is taken for fragment detection by using Agilent2100, and the main peak of the sample detection after normal interruption is about 150bp-200 bp.

For cfDNA samples, Agilent2100 performed fragment detection and the qubits were directly quibit for subsequent experiments.

1.3. End repair, 3' end addition of "a":

1.3.1. taking 20ng of the broken gDNA or cfDNA into a PCR tube, supplementing 50 mu l of the broken gDNA or cfDNA with nuclease-free water, adding the following reagents, and mixing by vortex:

TABLE 4

Components	Volume of
		gDNA/cfDNA	50μl
Stop repair and A tailing buffer	7μl
		Stop repair and A tailing enzyme mixture	3μl
Total volume	60μl

1.3.2. The following program was set up to perform the reaction on a PCR instrument: the hot lid temperature was 85 ℃.

TABLE 5

Temperature of	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

50. mu.l of fragmented DNA for ER and AT reactions	Joint concentration
		1μg	10μM
500ng	10μM
		250ng	10μM
100ng	10μM
		50ng	10μM
25ng	10μM
		10ng	3μM
5ng	5μM
		2.5ng	2.5μM
1ng	625nM

1.4.2. The following reagents were prepared according to the following table, gently pipetted and mixed, and briefly centrifuged:

TABLE 7

Components	Volume of
		End repair, addition of "A" reaction product	60μl
Joint	5μl
		Nuclease-free water	5μl
Ligation buffer	30μl
		DNA ligase	10μl
Total volume	110μl

1.4.3. The following program was set up to perform the reaction on a PCR instrument: without a heat cover.

TABLE 8

Temperature of	Time
		20℃	30min
4℃	∞

1.4.4. Purified magnetic beads were added for the experiment according to the following system (Agencourt AMPure XP beads were brought to room temperature in advance, shaken and mixed well for use):

TABLE 9

Components	Volume of
		Joint ligation product	110μl
Agencourt AMPure XP bead	110μl
		Total volume	220μl

1.4.4.1. Gently suck and mix for 6 times.

1.4.4.2. And (3) standing and incubating for 5-15min at room temperature, and placing the PCR tube on a magnetic frame for 3min to clarify the solution.

1.4.4.3. The supernatant was removed, the PCR tube was placed on a magnetic stand, 200. mu.l of 80% ethanol solution was added to the PCR tube, and the tube 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 the supernatant was removed thoroughly after standing for 30s (it was recommended to remove the residual ethanol solution at the bottom using a 10. mu.l pipette).

1.4.4.5. Standing at room temperature for 3-5min to completely volatilize residual ethanol.

1.4.4.6. Adding 22. mu.l of nuclease-free water, taking down the PCR tube from the magnetic frame, gently sucking and beating the resuspended magnetic beads to avoid generating bubbles, and standing at room temperature for 2 min.

1.4.4.7. The PCR tube was placed on a magnetic stand for 2min to clarify the solution.

1.4.4.8. Pipette 20. mu.l of the supernatant and transfer to a new PCR tube.

1.5 bisulfite treatment and purification:

1.5.1. the required reagents were taken out beforehand and dissolved. The reagents were added according to the following table:

watch 10

Components	High concentration sample (1 ng-2. mu.g) volume	Volume of low concentration sample (1-500ng)
			Linker ligation of purified products	20μl	40μl
Bisulfite solution	85μl	85μl
			DNA protection buffer	35μl	15μl
Total volume	140μl	140μl

1.5.2.DNA protection buffer added to the liquid turned blue. Mix by gentle pipetting and then divide into two tubes and place on the PCR instrument.

1.5.3. The following programs are set and run: hot lid 105 ℃.

TABLE 11

Temperature of	Time
		95℃	5min
60℃	10min
		95℃	5min
60℃	10min
		4℃	∞

1.5.4. Brief centrifugation pooled two identical samples into the same clean 1.5ml centrifuge tube.

1.5.5. Mu.l of buffer BL (sample size less than 100ng with 1. mu.l of vector RNA (1. mu.g/. mu.l)) was added to each sample, vortexed, and briefly centrifuged.

1.5.6. Add 250. mu.l of absolute ethanol to each sample, vortex and mix for 15s, centrifuge briefly, and add the mixture to the corresponding spin column ready.

1.5.7. Standing for 1min, centrifuging for 1min, transferring the liquid in the collecting tube to the centrifugal column again, centrifuging for 1min, and discarding the liquid in the centrifugal tube.

1.5.8. Add 500. mu.l buffer BW (note whether absolute ethanol is added or not), centrifuge for 1min, discard waste.

1.5.9. Add 500. mu.l buffer BD (note whether absolute ethanol was added), cover the tube, and let stand at room temperature for 15 min. Centrifuging for 1min, and discarding the centrifuged liquid.

1.5.10. Add 500. mu.l buffer BW (note whether absolute ethanol is added), centrifuge for 1min, discard the liquid from the centrifuge, repeat once for 2 times.

1.5.11. Add 250. mu.l of absolute ethanol, centrifuge for 1min, place the column in a new 2ml collection tube and discard all remaining liquid.

1.5.12. Placing the column in a clean 1.5ml centrifuge tube, adding 20 μ l nuclease-free water to the center of the column membrane, lightly covering the tube cover, standing at room temperature for 1min, and centrifuging for 1 min.

1.5.13. And transferring the liquid in the collecting pipe to a centrifugal column again, standing at room temperature for 1min, and centrifuging for 1 min.

1.6. Pre-amplification and purification before hybridization:

1.6.1. preparing a reaction system according to the following table, uniformly mixing by blowing, and centrifuging for a short time:

TABLE 12

1.6.2. The following program was set up and the PCR program was started: 105 deg.C thermal cover

Watch 13

1.6.3 PCR cycle numbers were adjusted depending on the amount of DNA dosed, reference data are as follows:

TABLE 14

1.6.4. And adding 50 mu l of Agencour AMPure XP magnetic beads into the PCR tube after the reaction is finished, and blowing and uniformly mixing the mixture by using a pipettor to avoid generating bubbles (the Agencour AMPure XP is uniformly mixed and balanced at room temperature in advance).

1.6.5. Incubate at room temperature for 5-15min, and place the PCR tube on a magnetic frame for 3min to clarify the solution.

1.6.6. The supernatant was removed, the PCR tube was placed on a magnetic stand, 200. mu.l of 80% ethanol solution was added to the PCR tube, and the tube 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 the supernatant was removed thoroughly after standing for 30s (it was recommended to remove the residual ethanol solution at the bottom using a 10. mu.l pipette).

1.6.8. Standing at room temperature for 5min to completely volatilize residual ethanol.

1.6.9. Add 30. mu.l of nuclease-free water, remove the centrifuge tube from the magnetic rack, and gently pipette and resuspend the magnetic beads.

1.6.10. After standing at room temperature for 2min, 200. mu.l of PCR tube was placed on a magnetic stand for 2min to clarify the solution.

1.6.11. The supernatant was transferred to a new 200. mu.l PCR tube (on an ice box) using a pipette, and the reaction tube was labeled with a sample number and ready for the next reaction.

1.6.12. A1. mu.l sample was taken for library concentration determination using the Qubit and library concentration was recorded.

1.6.13. A1. mu.l sample was taken and the library fragment length was determined using Agilent2100, with a library length of approximately 270bp to 320 bp.

1.7. Sample and probe hybridization:

1.7.1. the sample library was mixed well with various Hyb blockers, labeled B, according to the following system:

watch 15

Components	Volume of
		Pre-amplification product	750ng corresponding volume
Hyb human blockers	5μl
		Linker blocker	6μl
Reinforcing agent	5μl

1.7.2. And (3) putting the prepared mixture of the sample and the Hyb blocker into a vacuum concentration centrifuge, opening a PCR tube cover, starting the centrifuge, opening a switch of a vacuum pump, and starting concentration.

1.7.3. The drained sample was redissolved in about 9. mu.l nuclease-free water in a total volume of 10. mu.l, gently pipetted and mixed, centrifuged briefly and placed on ice for use, labeled B.

1.7.4. Melting Hyb buffer solution at room temperature, allowing precipitate to appear after melting, mixing, preheating in a 65 deg.C water bath, dissolving completely (no precipitate and turbid substance), placing 20 μ l Hyb buffer solution in a new 200 μ l PCR tube, covering the tube cover, labeling with A, and incubating in a 65 deg.C water bath for further use.

1.7.5. The methylation probe sequence described previously was synthesized by agutazone biotechnology (beijing) ltd:

1.7.6. mu.l of RNase blocker and 2. mu.l of probe composition were placed in a 200. mu.l PCR tube, gently pipetted and mixed, centrifuged briefly and placed on ice until needed, labeled C.

1.7.7. Setting PCR instrument parameters, hot cover 100 deg.C, 95 deg.C, 5 min; and keeping at 65 ℃.

1.7.8. Place PCR tube B on the PCR instrument and run the above program.

And 1.7.9. when the temperature of the PCR instrument is reduced to 65 ℃, placing the PCR tube A on the PCR instrument for incubation, and covering a hot cover of the PCR instrument.

1.7.10.5min later, C was placed on the PCR and incubated, and the lid was closed to the PCR instrument.

1.7.11. And (3) placing the PCR tube C into a PCR instrument for 2min, adjusting a pipettor to 13 mu l, sucking 13 mu l of Hyb buffer solution from the PCR tube A, transferring the Hyb buffer solution into the PCR tube C, sucking all samples in the PCR tube B, transferring the samples into the PCR tube C, slightly sucking and beating for 10 times, fully mixing the samples uniformly to avoid generating a large amount of bubbles, sealing a tube cover, covering a hot cover of the PCR instrument, and incubating at 65 ℃ overnight (16-24 h).

1.8. Capture target region DNA library:

1.8.1. preparation of the Capture magnetic beads

1.8.1.1. The magnetic beads (Dynabeads MyOne Streptavidin T1 magnetic beads) were removed from 4 ℃ and resuspended by vortexing.

1.8.1.2. 50 μ l of the 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 frame, 200. mu.L of binding buffer was added and gently pipetted several times to mix well, and the magnetic beads were resuspended.

1.8.1.4. Placing on a magnetic frame for 1min, and removing the supernatant.

1.8.1.5. Repeating the steps 3-4 twice, and cleaning the magnetic beads 3 times in total.

1.8.1.6. The PCR tube was removed from the magnetic frame and 200. mu.L of binding buffer was added and the resuspended beads were gently pipetted 6 times for use.

1.8.2. Capturing a target DNA library

1.8.2.1. Keeping the hybridization product PCR tube C on the PCR instrument, adding the prepared 200. mu.L of capture magnetic beads into the hybridization product PCR tube C, pipetting for 6 times, mixing, and placing on a rotary mixer for bonding at room temperature for 30min (preferably, the rotation speed is not more than 10 rpm).

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. Add 200. mu.L of Wash buffer 1(23.5ml nuclease-free Water, 1.25ml 20 XSSC, 250. mu.l 10% SDS) to PCR tube C, gently pipette 6 times and mix, wash on a spin mixer for 15min (preferably not more than 10 rpm), centrifuge briefly, place the PCR tube on a magnetic stand for 2min to clarify the solution, and remove the supernatant.

1.8.2.4. 200. mu.l of washing buffer 2(24.6ml of nuclease-free water, 125. mu.l of 20 XSSC, 250. mu.l of 10% SDS) preheated at 65 ℃ were added, gently pipetted 6 times and mixed, and the mixture was incubated on a mixer at 65 ℃ for 10min and washed at a rotation speed of 800 rpm.

1.8.2.5. Briefly, centrifuge, place PCR tube on magnetic rack for 2min, remove supernatant. The wash was repeated 2 more times with wash buffer 2 for a total of 3 times. The wash buffer 2 was removed completely for the last time.

1.8.2.6. the PCR tube was placed on a magnetic stand, 200. mu.l of 80% ethanol was added to the PCR tube, left to stand for 30s, the ethanol solution was removed completely, and the tube was air-dried at room temperature for 2 min.

1.8.2.7. Add 30. mu.L nuclease-free water to the PCR tube, remove the PCR tube from the magnetic frame, and gently pipette 6 times of resuspended beads for use.

1.9. Post capture amplification and purification

1.9.1. A reaction system is prepared according to the following table for enriching the capture library, and after the capture library is lightly blown, uniformly mixed, the capture library is centrifuged for a short time:

TABLE 16

1.9.2. The following program was set up, the samples were placed in a PCR instrument, and the program was run: hot lid 105 ℃.

TABLE 17

And 1.9.3. adding 55 mu.l of Agencourt AMPure XP magnetic beads into the sample after the PCR is finished, and gently sucking and mixing the mixture by using a pipettor.

1.9.4. Incubate at room temperature for 5min, and place the PCR tube on a magnetic frame for 3min to clarify the solution.

1.9.5. The supernatant was removed, the PCR tube was further placed on a magnetic stand, 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 completely removed after standing for 30 seconds.

1.9.7. Standing at room temperature for 5min to completely volatilize residual ethanol.

1.9.8. Add 25. mu.l nuclease-free water, remove the PCR tube from the magnetic frame, gently blow and mix the resuspended beads, and stand at room temperature for 2 min.

1.9.9. The PCR tube was placed on a magnetic stand for 2min to clarify the solution.

1.9.10. Pipette 23. mu.l of the supernatant into a 1.5ml centrifuge tube and label the sample information.

1.9.11. 1 μ l of the library was quantitated using a Qubit and the library concentration was recorded.

1.9.12. A1. mu.l sample was taken for library fragment length determination using Agilent 2100.

1.9.13. Sequencing was performed using the Illumina high throughput sequencing platform.

1.10. Methylation letter analysis process. Roughly as follows: checking sequencing quality by using fast quality control software, removing low-quality reads, comparing clean data after quality control to a reference genome by using Bismark comparison software, extracting corresponding methylation sites by using Bismar _ methylation _ extra software, and finally calculating the methylation level of each marker.

Example 2

Based on 24 samples clinically diagnosed as lung cancer collected from Beijing area and 38 healthy human samples collected from Beijing area, the methylation library construction method described in example 1 was used to calculate the methylation levels of the 3 screened methylated biomarkers, and the threshold values (hereinafter referred to as site or marker) and the independently differentiated AUC values were calculated from the methylation levels of the 3 methylated biomarkers in the lung cancer samples and the normal human sample data set, as shown in Table 18.

The methylation level threshold value is calculated by the following method: an ROC curve is drawn by using R packets pROC according to a data set (comprising the type and the methylation level of each sample), and a confusion matrix corresponding to an optimal threshold point on the ROC curve is a basis for calculating indexes such as sensitivity (sensitivity), specificity (specificity) and accuracy. Typically we will select by the joyden index (Youden index). Jotan index, also known as correct index, refers to the sum of sensitivity and specificity minus 1: youden index ═ Sensitivity + Specificity-1. The johnson index range value is between 0 and 1, and represents the total ability of the classification model to find real patients and non-patients. The larger the jotan index, the better the classification model performance, and the threshold, sensitivity and specificity of each marker are shown in table 18.

As can be seen from Table 18, the AUC values of the markers of the present invention are high.

TABLE 183 methylation marker data

SEQ ID	Threshold value	Specificity of	Sensitivity of the probe	AUC
					SEQ ID NO.1	0.11	0.94	0.89	0.93
SEQ ID NO.2	0.11	0.87	0.91	0.90
					SEQ ID NO.3	0.11	0.95	0.84	0.92

Example 3

6 human samples (healthy human sample at S1-3, lung cancer patient sample at S4-6) were collected by the methylation marker detection method of the present application as in example 1; establishing a library, and sequencing by an Illumina platform; the sequencing data is analyzed by the above-mentioned bioinformatics analysis procedure to obtain the methylation level of each marker, and according to the threshold value of each marker, the diseased status of the patient is predicted, if the threshold value is exceeded, the patient is a cancer sample, if the threshold value is lower, the patient is a healthy human sample, and the specific results are shown in table 19:

wherein 0 represents the classification as normal, i.e. healthy, in the interpretation result; 1 represents a classification as abnormal, i.e. tumor.

Methylation values and interpretation results for the samples of Table 19

In conclusion, the inventor of the invention obtains the methylation gene related to the lung cancer, determines the target sequence of methylation abnormality of the methylation gene of the lung cancer, and can sensitively and specifically detect the methylation state of the methylation gene through the target sequence of the methylation gene, so that the methylation gene can be used for detecting free DNA of peripheral blood, and the composition can realize real-time monitoring and has higher sensitivity and accuracy.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

The sequence listing is shown in table 20:

watch 20

Sequence listing

<110> Boercheng (Beijing) science and technology Limited

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<130> PE02016

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<213> Artificial sequence

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<210> 10

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<213> Artificial sequence

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<212> DNA

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<210> 13

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<213> Artificial sequence

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<210> 14

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<212> DNA

<213> Artificial sequence

<220>

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aacaccaacc tcacaactca ccaccaaaat aaaaacatac caaaaaaaca cccaaaaaaa 60

aaaccaccaa aaaaaaaaac ctccataaaa aaaataaaaa caaaaacaaa accaaaaaac 120

<210> 15

<211> 120

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 15

actccctaac ctcactccca tttccacccc tcccacaaaa attttccttt ctaataactc 60

cttccttaaa tatcccttta atacaccccc accctaacaa taaactacaa aactaacacc 120

<210> 16

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

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<210> 17

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 17

atatattttg tatagttagt ttgtagttag agtagggagt cggtttgggt tttcgtattt 60

<210> 18

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 18

cggaaaagtt ttcgtttgtt tatattagga taggtaagaa gttggttttt tttcgtagtt 60

<210> 19

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 19

ggttgcgagg agaaattagt tttttgttta ttttggtatg agtaggcgga ggttttttcg 60

<210> 20

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 20

ttttacggag gttttttttt ttggtggttt tttttttggg tgtttttttg gtacgttttt 60

<210> 21

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 21

gggggcgtat taaagggata tttaaggaag gagttattag aaaggaaaat tttcgtggga 60

<210> 22

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 22

aggtgtgggg atttaggttg gttttttatt ttgattgtaa attaattgta tagggtatgt 60

<210> 23

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 23

atatattttg tatagttagt ttgtagttag agtagggagt tggtttgggt ttttgtattt 60

<210> 24

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 24

tggaaaagtt tttgtttgtt tatattagga taggtaagaa gttggttttt ttttgtagtt 60

<210> 25

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 25

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<210> 26

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 26

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<210> 27

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

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gggggtgtat taaagggata tttaaggaag gagttattag aaaggaaaat ttttgtggga 60

Claims (11)

1. A marker for detecting lung cancer, wherein the marker is selected from one of the following: USP33, CHRNB2 and TMEM 219.

2. Marker according to claim 1, wherein the nucleotide sequence of the marker is selected from one of the group consisting of SEQ ID NO 1-3, preferably wherein the marker is a methylated marker.

3. A probe composition comprising a probe that targets methylation of the marker of claim 1 or 2.

4. The probe composition of claim 3, wherein the probe composition comprises a hypermethylated first probe composition for hybridizing to a bisulfite converted CG hypermethylated region and a hypomethylated second probe composition for hybridizing to a bisulfite converted CG hypomethylated region;

preferably, the first probe composition comprises n probes that hybridize to each nucleotide of the sense and antisense strands of the bisulfite converted CG hypermethylated region;

preferably, the second probe composition comprises m probes that hybridize to each nucleotide of the sense and antisense strands of the hypomethylated region of bisulfite converted CG;

preferably, n and m are each any integer from 1 to 10;

preferably, there is x between the nth-1 probe and the nth probe₁Nucleotide overlap, preferably, x₁Is any integer of 0 to 100;

preferably, there is x between the m-1 st probe and the m-th probe₂Nucleotide overlap, preferably, x₂Is any integer of 0 to 100;

further preferably, the first probe composition comprises one or two nucleotide sequences shown as SEQ ID NO. 4-9, and the second probe composition comprises one or two nucleotide sequences shown as SEQ ID NO. 10-15.

5. Use of a marker for the preparation of a kit for the detection of lung cancer, wherein the marker is selected from one of the following: USP33, CHRNB2 and TMEM 219.

6. Use according to claim 5, wherein the nucleotide sequence of the marker is selected from one of the group consisting of SEQ ID NO 1-3, preferably the marker is a methylated marker;

preferably, the probe composition is used to target a post-methylation marker of lung cancer;

preferably, the probe composition is the probe composition according to claim 3 or 4.

7. A composition for lung cancer detection, comprising a nucleic acid for detecting methylation of any one of the following markers selected from the group consisting of: USP33, CHRNB2 and TMEM 219.

8. The composition of claim 7, wherein the nucleotide sequence of the marker is selected from the group consisting of SEQ ID NOs 1-3.

9. The composition of claim 7 or 8, wherein the nucleic acid comprises the probe composition of claim 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 a fragment of at least 15 nucleotides in a target sequence of the marker, said fragment comprising at least one CpG dinucleotide sequence;

preferably, the composition further comprises an agent that converts unmethylated cytosine bases 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:

a blocker that preferentially binds to a target sequence in a non-methylated state.

10. A kit comprising a marker of claim 1 or 2 or a probe composition of claim 3 or 4 or a composition of any one of claims 7-9.

11. A chip comprising the marker of claim 1 or 2 or the probe composition of claim 3 or 4 or the composition of claim 7 or 8.