Method and kit for screening colorectal tumor by detecting methylation status of pknox2 gene region

Abstract

Disclosed herein are methods and kits for diagnosing colorectal neoplasia, screening for colorectal neoplasia formation or predisposition, or monitoring progression or prognosis of colorectal neoplasia in an individual. Specifically, the present disclosure diagnoses colorectal neoplasia, screens for colorectal neoplasia formation or predisposition, or monitors progression or prognosis of colorectal neoplasia by detecting the methylation status of a PKNOX2 gene region in a biological sample.

Description

Technical Field

[0001] This application relates to the field of tumor screening, specifically to methods and kits for screening colorectal cancer by detecting the methylation status of target genes in biological samples. Background Technology

[0002] Colorectal cancer is the third most common malignant tumor worldwide and also a common malignant tumor in my country. Based on the stage of disease progression, colorectal cancer is divided into stages I-IV. For patients with stage IV colorectal cancer, the overall survival rate is very low, with a 5-year survival rate of less than 10%. In contrast, patients with stage I cancer can achieve a survival rate of over 90%. Therefore, early diagnosis and treatment are crucial to improving the overall prognosis of colorectal cancer.

[0003] Currently, colorectal cancer screening technologies in China mainly include fecal occult blood testing, tumor marker detection, and colonoscopy. While colonoscopy remains the fundamental method for diagnosing colorectal cancer, its high cost, poor adherence, and potential invasiveness make it difficult to widely apply to screening asymptomatic individuals. Although fecal occult blood testing and tumor marker (e.g., peripheral blood carcinoembryonic antigen) testing are readily accepted, their relatively poor sensitivity and specificity also pose challenges to the early diagnosis and treatment of colorectal cancer.

[0004] In recent years, epigenetic and genetic alterations in intestinal epithelial cells have been found to potentially aid in the diagnosis of colorectal cancer. Currently, several commercially available kits are available for detecting Septin9 gene methylation. Kits that comprehensively detect BMP3 and NDRG4 gene methylation, as well as KRAS and β-actin gene mutations, are also available in the United States. However, some existing kits suffer from limitations in their detection efficiency for colorectal cancer, requiring further improvement. Researchers are also exploring other gene detection methods, but the achievable tumor diagnostic sensitivity and specificity vary considerably, and none meet the requirements for large-scale screening.

[0005] Therefore, colorectal cancer screening methods and kits with higher sensitivity and specificity are needed. Invention Summary

[0006] In one aspect, this application provides a method for diagnosing colorectal cancer in an individual, screening for colorectal cancer formation or a predisposition to formation, or monitoring colorectal cancer progression or prognosis. The method includes detecting the methylation status of a PKNOX2 gene region in a biological sample from the individual and comparing the detected methylation status of the PKNOX2 gene region with the normal methylation status of the PKNOX2 gene region. The change in the methylation status of the PKNOX2 gene region detected in the biological sample from the individual, relative to the normal methylation status of the PKNOX2 gene region, indicates that the individual has colorectal cancer, or that the individual has a predisposition to colorectal cancer formation or formation, or that the individual has a predisposition to colorectal cancer development or progression, or that the individual has a poor prognosis or a predisposition to colorectal cancer.

[0007] In some implementations, a higher methylation state of the PKNOX2 gene region detected in a biological sample from the individual, relative to the normal methylation state of the PKNOX2 gene region, indicates that the individual has colorectal cancer, or that the individual has a tendency to form or develop colorectal cancer, or that the individual has a tendency to develop or progress colorectal cancer, or that the individual has a poor prognosis or a tendency to have a poor prognosis for colorectal cancer.

[0008] In another aspect, this application provides a method for monitoring an individual's response to colorectal cancer treatment, the method comprising detecting the methylation status of a PKNOX2 gene region in biological samples from the individual before and after the individual receives colorectal cancer treatment, wherein a change in the methylation status of the PKNOX2 gene region after the individual receives colorectal cancer treatment, relative to the methylation status of the PKNOX2 gene region before receiving colorectal cancer treatment, indicates that the individual has responded to colorectal cancer treatment.

[0009] In some implementations, a lower methylation state of the PKNOX2 gene region after colorectal cancer treatment in an individual, relative to the methylation state of the PKNOX2 gene region before receiving colorectal cancer treatment, indicates that the individual has responded to colorectal cancer treatment.

[0010] In some embodiments, the PKNOX2 gene region includes: a) the region defined by Hg19 coordinates chr11:125034583-125303285 and its upstream and downstream 5kb; or b) the region corresponding to the region listed in a) above after bisulfite conversion; or c) the region corresponding to the region listed in a) above after treatment with a methylation-sensitive restriction enzyme (MSRE).

[0011] In some embodiments, the PKNOX2 gene region is selected from the following group: a) the region defined by Hg19 coordinates chr11:125034583-125303285 and its upstream and downstream 5kb; or b) the corresponding region of the region listed in a) above after bisulfite conversion; or c) the corresponding region of the region listed in a) above after treatment with a methylation-sensitive restriction enzyme (MSRE).

[0012] In some embodiments, detecting the methylation status of the PKNOX2 gene region includes determining the methylation status of cytosine residues at one or more CpG sites in the PKNOX2 gene region in a biological sample from the individual. In some embodiments, the methylation status of the PKNOX2 gene region includes the methylation status of a target DNA region comprising sequences with Hg19 coordinates selected from the group consisting of: chr11:125033654-125038552, chr11:125132945-125133979, chr11:125285780-125286198, and chr11:125036431-125036547, and 200 bp upstream and downstream regions of said region, and any combination thereof.

[0013] In some embodiments, the biological sample is selected from histological sections, tissue biopsies, paraffin-embedded tissue, surgically removed samples, isolated cells, body fluids, colonic effluent, and any combination thereof. In some embodiments, the body fluid is selected from the group consisting of whole blood, serum, plasma, urine, saliva, mucus, peritoneal fluid, pleural fluid, pleural effusion, synovial fluid, cerebrospinal fluid, thoracentesis fluid, ascites, and any combination thereof. In some embodiments, the colonic effluent is selected from feces and enema washing samples.

[0014] In some implementations, prior to detecting the methylation status of the PKNOX2 gene region in a biological sample from said individual, the following steps are further included:

[0015] (a) Obtaining a biological sample containing DNA from the individual;

[0016] (b) The DNA in the biological sample obtained in step (a) is treated with a reagent that can distinguish between methylated and unmethylated CpG sites in the DNA, thereby obtaining the treated DNA.

[0017] In some embodiments, the DNA includes genomic DNA or cell-free extracellular DNA. In some embodiments, the cell-free extracellular DNA includes circulating tumor DNA.

[0018] In some embodiments, the reagent in step (b) is a bisulfite reagent or a methylation-sensitive restriction enzyme (MSRE). In some embodiments, the bisulfite reagent is selected from the group consisting of: ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, bisulfite ion, and any combination thereof. In some embodiments, the MSRE is selected from the group consisting of: HpaII enzyme, SalI enzyme, Enzymes, ScrFI enzyme, BbeI enzyme, NotI enzyme, SmaI enzyme, XmaI enzyme, MboI enzyme, BstBI enzyme, ClaI enzyme, MluI enzyme, NaeI enzyme, NarI enzyme, PvuI enzyme, SacII enzyme, HhaI enzyme, and any combination thereof.

[0019] In some implementations, the detection uses amplification-based methods, hybridization-based methods, sequencing-based methods, or restriction enzyme digestion-based methods.

[0020] In some embodiments, detecting the methylation status of the PKNOX2 gene region includes amplifying the treated DNA using an amplification enzyme and one or more sets of primers to produce at least one amplification product or to prevent amplification of the treated DNA. Optionally, the treated DNA comprises the nucleotide sequences shown in SEQ ID NOs:4-9, 11-12 and any combination thereof, or a nucleotide sequence selected from SEQ ID NOs:4-9, 11-12. In some embodiments, the amplification enzyme comprises a thermostable DNA polymerase or a polymerase lacking 5'-3' exonuclease activity.

[0021] In some embodiments, the amplification is performed in the presence of a detection reagent or a blocking reagent. In some embodiments, the detection reagent comprises a detectable marker-tagged oligonucleotide probe, and / or the blocking reagent comprises a blocking oligonucleotide that cannot be elongated by polymerase. In some embodiments, the oligonucleotide probe contains a sequence capable of hybridizing with the amplification product, and / or the blocking oligonucleotide contains a sequence capable of hybridizing with the amplification product in a methylation-specific manner.

[0022] In some embodiments, the primer is a methylation-specific primer. In some embodiments, the primer comprises a sequence substantially complementary to or substantially identical to at least nine consecutive nucleotides of the PKNOX2 gene region sequence in the treated DNA, wherein said consecutive nucleotides comprise at least one CpG, TpG, or CpA dinucleotide, or wherein said amplification product comprises at least one CpG, TpG, or CpA dinucleotide. In some embodiments, the primer comprises a sequence substantially complementary to or substantially identical to at least nine consecutive nucleotides of the sequence selected from SEQ ID NOs:1-12. In some embodiments, the primer is selected from the group consisting of:

[0023] GTTTTAGGAGTTATTTGGGTTTGC(SEQ ID NO:13) PKNOX2 upstream primer sequence ACTATAACACCTCGCTACTAACGCT(SEQ ID NO:14) PKNOX2 downstream primer sequence

[0024] And / or, the oligonucleotide probe is selected from the group consisting of:

[0025] CGGTGGTTCGTAGGGGTCGCG (SEQ ID NO:15) PKNOX2 probe sequence

[0026] In some embodiments, the methylation status of the PKNOX2 gene region is determined based on the presence and properties of the amplification product. In some embodiments, detecting or determining the methylation status of the PKNOX2 gene region includes using polymerase chain reaction (e.g., real-time polymerase chain reaction, digital polymerase chain reaction), nucleic acid sequencing, quality-based separation (e.g., electrophoresis, mass spectrometry), or target capture (e.g., microarray). In some embodiments, the determination includes sequencing the amplification product.

[0027] In some implementations, the normal methylation state of the PKNOX2 gene region represents the methylation state of the PKNOX2 gene region in individuals who do not have colorectal cancer, or individuals who do not have colorectal cancer formation or a tendency to form colorectal cancer, or individuals who do not have colorectal cancer development or a tendency to develop colorectal cancer, or individuals who have a good or favorable prognosis for colorectal cancer.

[0028] In some embodiments, the colorectal tumor is a colorectal tumor. In some embodiments, the colorectal tumor is colorectal cancer, colorectal adenoma, or sessile serrated polyp. In some embodiments, the colorectal tumor is precancerous.

[0029] In some implementations, the individual is a person.

[0030] In another aspect, this application provides an oligonucleotide for use as a detection tool, comprising at least nine consecutive nucleotides of the PKNOX2 gene region or its complementary sequence, or consisting of at least nine consecutive nucleotides of the PKNOX2 gene region or its complementary sequence.

[0031] In another aspect, this application provides an oligonucleotide for use as a detection tool, comprising at least nine consecutive nucleotides of a treated DNA sequence of the PKNOX2 gene region or its complementary sequence thereof, or consisting of at least nine consecutive nucleotides of a treated DNA sequence of the PKNOX2 gene region or its complementary sequence thereof, said treatment being adapted to convert at least one unmethylated cytosine residue in the PKNOX2 gene region into a uracil residue, a thymine residue, or other residue that is detectably different from cytosine in hybridization.

[0032] In another aspect, this application provides a kit for diagnosing colorectal cancer, screening for colorectal cancer formation or a predisposition to formation, or monitoring colorectal cancer progression or prognosis, comprising a first reagent containing one or more oligonucleotides as described in this application. In some embodiments, the oligonucleotide comprises a sequence substantially complementary to or substantially identical to at least nine consecutive nucleotides selected from sequences of SEQ ID NOs:1-12. In some embodiments, the oligonucleotide is selected from the group consisting of:

[0033] GTTTTAGGAGTTATTTGGGTTTGC(SEQ ID NO:13) PKNOX2 upstream primer sequence ACTATAACACCTCGCTACTAACGCT(SEQ ID NO:14) PKNOX2 downstream primer sequence CGGTGGTTCGTAGGGGTCGCG (SEQ ID NO:15) PKNOX2 probe sequence

[0034] In some embodiments, the kit described in this application further includes a second reagent capable of distinguishing between methylated and unmethylated CpG sites in DNA. In some embodiments, the second reagent is a bisulfite reagent or a methylation-sensitive restriction enzyme (MSRE). In some embodiments, the bisulfite reagent is selected from the group consisting of: ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, bisulfite ions, and any combination thereof. In some embodiments, the MSRE is selected from the group consisting of: HpaII enzyme, SalI enzyme, ... Enzymes, ScrFI enzyme, BbeI enzyme, NotI enzyme, SmaI enzyme, XmaI enzyme, MboI enzyme, BstBI enzyme, ClaI enzyme, MluI enzyme, NaeI enzyme, NarI enzyme, PvuI enzyme, SacII enzyme, HhaI enzyme, and any combination thereof.

[0035] In some embodiments, the first reagent and the second reagent are packaged in a single container or in separate containers. In some embodiments, the kit described in this application further includes a container suitable for holding biological samples from the individual. In some embodiments, the kit described in this application further includes instructions for use and / or an interpretation of the kit's test results.

[0036] In another aspect, this application provides the use of reagents for detecting the methylation status of a PKNOX2 gene region in the preparation of kits for methods of diagnosing colorectal cancer in an individual, screening for colorectal cancer formation or a predisposition to form, or monitoring colorectal cancer progression or prognosis, wherein the method includes detecting the methylation status of a PKNOX2 gene region in a biological sample from the individual and comparing the detected methylation status of the PKNOX2 gene region with the normal methylation status of the PKNOX2 gene region, wherein a change in the methylation status of the PKNOX2 gene region detected in the biological sample from the individual, relative to the normal methylation status of the PKNOX2 gene region, indicates that the individual has colorectal cancer, or that the individual has a predisposition to colorectal cancer formation or a predisposition to colorectal cancer development or progression, or that the individual has a poor prognosis or a predisposition to colorectal cancer.

[0037] In some implementations, a higher methylation state of the PKNOX2 gene region detected in a biological sample from the individual, relative to the normal methylation state of the PKNOX2 gene region, indicates that the individual has colorectal cancer, or that the individual has a tendency to form or develop colorectal cancer, or that the individual has a tendency to develop or progress colorectal cancer, or that the individual has a poor prognosis or a tendency to have a poor prognosis for colorectal cancer.

[0038] In another aspect, this application provides the use of reagents for detecting the methylation status of a PKNOX2 gene region in a kit for preparing a method for monitoring the individual's response to colorectal cancer treatment, wherein the method includes detecting the methylation status of a PKNOX2 gene region in a biological sample from the individual, wherein a change in the methylation status of the PKNOX2 gene region after receiving colorectal cancer treatment in the individual, relative to the methylation status of the PKNOX2 gene region before receiving colorectal cancer treatment, indicates that the individual has responded to colorectal cancer treatment.

[0039] In some implementations, a lower methylation state of the PKNOX2 gene region after colorectal cancer treatment in an individual, relative to the methylation state of the PKNOX2 gene region before receiving colorectal cancer treatment, indicates that the individual has responded to colorectal cancer treatment.

[0040] In some embodiments, the reagent for detecting the methylation status of the PKNOX2 gene region comprises one or more groups of oligonucleotides, said oligonucleotides containing a sequence substantially complementary to or substantially identical to at least nine consecutive nucleotides selected from sequences of SEQ ID NOs:1-12. In some embodiments, said oligonucleotides are selected from the group consisting of SEQ ID NOs:13, 14, and 15.

[0041] In another aspect, this application provides the use of at least one reagent that distinguishes between methylated and unmethylated CpG sites in a target DNA region in the preparation of a kit for methods of diagnosing colorectal cancer in an individual, screening for colorectal cancer formation or a predisposition to formation, or monitoring colorectal cancer progression or prognosis, wherein the method comprises contacting DNA isolated from a biological sample of the individual with the at least one reagent and one or more oligonucleotides that hybridize with the target DNA region under stringent, moderately stringent, or highly stringent conditions, wherein the target DNA region comprises at least nine consecutive nucleotides of a PKNOX2 gene region or its complementary sequence, wherein the consecutive nucleotides comprise at least one CpG site.

[0042] In another aspect, this application provides the use of at least one reagent that distinguishes methylated and unmethylated CpG sites in a target DNA region in the preparation of a kit for monitoring the individual's response to colorectal cancer treatment, wherein the method comprises contacting DNA isolated from a biological sample of the individual with the at least one reagent and one or more oligonucleotides that hybridize with the target DNA region under stringent, moderately stringent, or highly stringent conditions, wherein the target DNA region comprises at least nine consecutive nucleotides of a PKNOX2 gene region or its complementary sequence, wherein the consecutive nucleotides comprise at least one CpG site. In some embodiments, the reagent is a bisulfite reagent or a methylation-sensitive restriction enzyme (MSRE).

[0043] In some embodiments, the kit described in this application includes (a) a bisulfite reagent; and (b) one or more sets of primers containing a sequence that is substantially complementary to or substantially identical to at least nine consecutive nucleotides of the PKNOX2 gene region sequence in bisulfite-treated DNA.

[0044] In another aspect, this application provides a kit suitable for carrying out the methods described herein, comprising (a) a methylation-sensitive restriction enzyme; and (b) one or more sets of primers containing a sequence substantially complementary to or substantially identical to at least nine consecutive nucleotides of a PKNOX2 gene region sequence in DNA treated with the methylation-sensitive restriction enzyme. In some embodiments, the kit further comprises a DNA polymerase, optionally a thermostable DNA polymerase or a polymerase lacking 5'-3' exonuclease activity.

[0045] In another aspect, this application provides a method for diagnosing colorectal cancer in an individual, screening for colorectal cancer formation or a predisposition to formation, or monitoring colorectal cancer progression or prognosis, comprising:

[0046] (a) Obtaining a biological sample containing DNA from the individual;

[0047] (b) The DNA in the biological sample obtained in step (a) is treated with a reagent that can distinguish between methylated CpG sites and unmethylated CpG sites in the DNA, thereby obtaining the treated DNA;

[0048] (c) Contact the DNA treated in step (b) with an amplification enzyme and one or more sets of primers suitable for amplifying a target DNA region, the target DNA region comprising at least nine consecutive nucleotides of a PKNOX2 gene region or its complementary sequence, wherein the consecutive nucleotides comprise at least one CpG site.

[0049] (d) Determine the methylation status of the target DNA region based on the presence and properties of the amplification products;

[0050] (e) Compare the methylation state of the target DNA region determined in step (d) with the normal methylation state of the target DNA region.

[0051] The change in the methylation state of the target DNA region determined in step (d), relative to the normal methylation state of the target DNA region, indicates that the individual has colorectal cancer, or the individual has a tendency to form or develop colorectal cancer, or the individual has a tendency to develop or progress colorectal cancer, or the individual has a poor prognosis or a tendency to have a poor prognosis for colorectal cancer.

[0052] In some implementations, a higher methylation state of the target DNA region determined in step (d) relative to the normal methylation state of the target DNA region indicates that the individual has colorectal cancer, or the individual has a tendency to form or develop colorectal cancer, or the individual has a tendency to develop or progress colorectal cancer, or the individual has a poor prognosis or a tendency to have a poor prognosis for colorectal cancer.

[0053] In another aspect, this application provides a kit suitable for carrying out the methods described in this application, comprising:

[0054] (a) Bisulfite reagent;

[0055] (b) A container suitable for containing the reagents and biological samples from the individual;

[0056] (c) One or more sets of primers comprising a sequence substantially complementary to or substantially identical to at least nine consecutive nucleotides of the PKNOX2 gene region sequence in bisulfite-treated DNA; and optionally,

[0057] (d) Instructions for use and / or interpretation of the test results from the kit.

[0058] In another aspect, this application provides a kit suitable for carrying out the methods described in this application, comprising:

[0059] (a) Methylation-sensitive restriction enzyme reagent;

[0060] (b) A container suitable for containing the reagents and biological samples from the individual;

[0061] (c) One or more sets of primers comprising a sequence substantially complementary to or substantially identical to at least nine consecutive nucleotides of the PKNOX2 gene region sequence in DNA treated with a methylation-sensitive restriction enzyme; and optionally,

[0062] (d) Instructions for use and / or interpretation of the test results from the kit. Attached Figure Description

[0063] Figure 1 Shows the target DNA region within the PKNOX2 gene region ( Figure 1 A) and internal reference gene ACTB ( Figure 1 B) Validation of methylation-specific primers. The vertical axis represents the ΔRn value, which is obtained by subtracting the baseline fluorescence intensity value from the fluorescence intensity value detected at a specific number of cycles. The horizontal axis represents the number of cycles. For example... Figure 1 As shown in Figure A, the Ct value decreases with increasing percentage of methylated DNA transformed in the DNA mixture, indicating that the primers used to amplify the target region of the PKNOX2 gene are methylation-specific. Figure 1 As shown in B, the curves for each DNA mixture overlap, indicating that although the percentage of methylated DNA transformed in the DNA mixture increases, the Ct value remains unchanged. This is also consistent with the fact that the primers used to amplify the internal reference gene ACTB are non-methylation-specific primers.

[0064] Figure 2A This study compares the methylation abundance of the PKNOX2 gene region in blood cells and different tissue samples (adjacent tissue, high-grade adenoma tissue, and colorectal cancer tissue). The internal control gene ACTB was used as a reference. Figure 2B This chart compares the methylation abundance of the internal reference gene ACTB in blood cells and different tissue samples (adjacent tissue, high-grade adenoma tissue, and colorectal cancer tissue). The vertical axis represents the Ct value, and the horizontal axis represents the various sample types (blood cells, adjacent tissue, high-grade adenoma tissue, and colorectal cancer tissue). The Ct value is inversely correlated with methylation abundance; that is, the higher the Ct value, the lower the methylation abundance. Therefore, from... Figure 2A It can be seen that the methylation abundance of the PKNOX2 gene region in blood cells is much lower than that in tissue samples, and it is lower in adjacent normal tissues than in high-grade adenomas and colorectal cancer tissues, indicating its potential for blood screening.

[0065] Figure 3 This chart compares the methylation abundance of the PKNOX2 gene target region in plasma samples from individuals with negative colonoscopy findings and those with colorectal cancer. The vertical axis represents the Ct value, and the horizontal axis represents the plasma samples from individuals with negative colonoscopy findings and those with colorectal cancer. The Ct value is inversely correlated with the methylation level; that is, the lower the Ct value, the higher the methylation level. Figure 3 It can be seen that the methylation level of the PKNOX2 gene region in the plasma samples of individuals with colorectal cancer is significantly higher than that in the plasma samples of individuals with negative colonoscopy.

[0066] Figure 4 This shows the sequence of an exemplary target DNA region, primer sequence, and probe sequence within the PKNOX2 gene region. Invention Details

[0068] While this application discloses various aspects and embodiments thereof, those skilled in the art can make various equivalent changes or modifications without departing from the spirit and scope of this application. The various aspects and embodiments disclosed herein are exemplary and not intended to limit the scope of this application; the actual scope of protection of this application is determined by the claims. Unless otherwise stated, all technical and scientific terms used in this application have the meanings commonly understood by those skilled in the art. All references, patents, and patent applications cited in this application are incorporated herein by reference.

[0069] 1. Basic Terminology

[0070] In the specification and claims of this application, unless otherwise stated, the terms "comprising" or "including" mean containing the listed values, steps or ingredients, but do not exclude the inclusion of other values, steps or ingredients.

[0071] In this application, the term "individual" includes both human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. "Individual" can also be livestock, such as cattle, pigs, sheep, poultry, and horses; or rodents, such as rats and mice; or primates, such as apes and monkeys; or domesticated animals, such as dogs and cats. In some embodiments, the individual is a human.

[0072] In this application, the term "large intestine" refers to the terminal region beginning in the ileum, which includes the following anatomical regions: cecum, ascending colon, transverse colon, descending colon, sigmoid colon, rectum, splenic flexure, and hepatic flexure.

[0073] In this application, the term "tumor" or "carcinoma" refers to a disease characterized by abnormal cell proliferation. Tumors can be benign (e.g., adenoma), potentially malignant, or malignant (often referred to as carcinoma), wherein malignant tumors are characterized by the malignant proliferation of tumor cells, i.e., the growth of tumor cells is unregulated and lacks differentiation, and they have the potential or ability to invade local tissues and metastasize. As used in this application, colorectal tumor refers to a tumor in the large intestine.

[0074] In this application, the term "diagnosis" refers to a medical judgment of an individual's health status. As used in this application, diagnosing colorectal cancer in an individual means determining whether the individual has colorectal cancer (including colorectal cancer at any stage of progression).

[0075] In this application, the term "screening" refers to the analysis of one or more biological samples from an individual to determine whether the individual has a specific disease and / or to determine its stage of progression. Screening is an important means of detecting early-stage cancer and precancerous lesions, and is commonly used to identify individuals from an outwardly healthy population who have or may have a specific tumor (such as colorectal cancer).

[0076] In this application, the term "formation" as used in describing colorectal tumors refers to the abnormal proliferation of one or more cells in an individual's large intestine. This can refer to a colorectal tumor in its very early stages, with a small number of abnormally proliferating large intestine cells, or it can refer to a colorectal tumor that has developed into a mass containing abnormally proliferating cells.

[0077] In this application, the term "proneness" refers to a state that an individual has not yet exhibited but is very likely to develop in the future.

[0078] In this application, the term "monitoring colorectal cancer progression" refers to monitoring the progression stage of colorectal cancer in an individual diagnosed with colorectal cancer.

[0079] In this application, the term "prognosis" refers to a prediction or expectation of the future course or outcome of a disease or condition. The term also refers to a prediction of the likelihood of clinical benefit from treatment. In some embodiments, statistical algorithms are used to provide a prognosis for an individual. For example, a prognosis may be surgery, progression of a clinical subtype of cancer, progression of one or more clinical factors, or recovery from the disease. A prognosis can be poor (e.g., possible recurrence or development of drug resistance) or good.

[0080] In this application, the term "biological sample" refers to any sample containing biological material obtained from an individual, including humans and non-human animals. A biological sample can be a sample obtained directly from the individual or a processed sample, and can be freshly harvested or stored (e.g., frozen). Biological samples include, but are not limited to, body fluids (e.g., whole blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, sweat, semen, feces, sputum, saliva, tears, mucus, amniotic fluid, effusion, bone marrow samples, ascites, pleural fluid, spinal fluid, lymph, and eye discharge), samples introduced into an individual and subsequently removed (e.g., pelvic lavage fluid, enema washing samples), extracts from nasal, throat, or genital swabs, tissue and organ samples, and processed samples derived from the above samples (e.g., cell suspensions from digested tissues, fecal extracts).

[0081] In this application, the term "PKNOX2 gene region" refers to a specific region of genomic DNA containing all the deoxynucleotide sequences required to produce all transcripts of the PKNOX2 gene, including transcriptional regulatory elements and promoter sequences that regulate PKNOX2 gene transcription. This DNA region can be identified by gene name or a set of chromosome coordinates. The human PKNOX2 gene is also known as PBX / Knotted 1Homeobox 2, Homeobox protein PKNOX2, Homeobox protein PREP2, or PREP2 gene. The PKNOX2 gene region that can be used in the methods described in this application includes the region at Hg19 coordinates chr11:125034583-125303285 and its upstream and downstream 5kb regions, or the region selected from the region at Hg19 coordinates chr11:125034583-125303285 and its upstream and downstream 5kb regions. The specific sequence of Hg19 coordinates chr11:125034583-125303285 and its upstream and downstream 5kb regions can be found in publicly available databases such as the UCSC Genome Browser, Ensemble, and the NCBI website. It is known that genes may have multiple variants among individuals, such as allelic variations or single nucleotide polymorphisms (SNPs, including insertions and deletions of varying sizes and simple sequence repetitions, such as dinucleotide and trinucleotide repeats). Therefore, the term "PKNOX2 gene region" should also be understood to include the PKNOX2 gene region corresponding to all variant sequences of the PKNOX2 gene. Furthermore, the term "PKNOX2 gene region" should also be understood to include the sequences of both the sense and antisense strands within this gene region.

[0082] Furthermore, the term "PKNOX2 gene region" broadly includes: (1) the original PKNOX2 gene region found in a biological sample or genomic DNA (especially the original PKNOX2 gene region with a specific methylation state); and (2) its processed sequence (e.g., a sequence treated with bisulfite or a methylation-sensitive restriction enzyme (MSRE)). The bisulfite-converted sequence differs from its original sequence in that one or more unmethylated cytosine residues in the original PKNOX2 gene region are converted to uracil, thymine, or other bases that are dissimilar to cytosine in hybridization. The MSRE-treated sequence differs from its original sequence in that the sequence is cleaved at one or more MSRE cleavage sites.

[0083] In this application, the term "methylation state" refers to the methylated or unmethylated state of one or more cytosine bases within a DNA sequence. In mammals, DNA methylation typically occurs at the cytosine site in CpG dinucleotides. CpGs can exist in clusters as CpG islands, which are present in the 5' regulatory regions of many genes, and their methylation state can affect gene transcription. Methylated cytosines can take various forms, such as 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). The methylation state of CpG sites within a DNA sequence includes unmethylation (CpGs on both DNA strands are unmethylated), full methylation (CpGs on both DNA strands are methylated), and hemimethylation (CpGs on only one DNA strand are methylated). Various methods for determining the methylation state or level of DNA are known to those skilled in the art. In this application, when analyzing the DNA methylation status in a biological sample, a quantitative assay can be used to determine the methylation level (e.g., percentage, fraction, ratio, or degree) of one or more methylation sites (e.g., CpG dinucleotides). Accordingly, the term "methylation status" should also be considered as a value reflecting the degree of methylation of one or more cytosines in the DNA of a biological sample.

[0084] In this application, the term "methylation status of the PKNOX2 gene region" refers to the methylated or unmethylated state of one or more cytosine bases within the PKNOX2 gene region. The methylation status of one or more sequences within the PKNOX2 gene region can represent the methylation status of the PKNOX2 gene region. For example, the methylation status of the PKNOX2 gene region can be determined by detecting the methylation status of one or more target DNA regions within the PKNOX2 gene region.

[0085] In this application, the term "target DNA region" refers to a DNA sequence within the PKNOX2 gene region that contains at least nine consecutive nucleotides of the PKNOX2 gene region and includes at least one methylation site (e.g., a CpG dinucleotide site). In this application, analyzing the methylation status of the target DNA region is particularly useful for diagnosing colorectal cancer in individuals, screening for colorectal cancer formation or a predisposition to formation, monitoring colorectal cancer progression or prognosis, or monitoring an individual's response to colorectal cancer treatment. In some embodiments, the target DNA region contains at least nine, ten, twelve, fifteen, eighteen, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, twenty, thirty, twenty ... In some implementations, the target DNA region may contain one or more CpG dinucleotide sites, such as at least one or at least two, three, four, five, eight or ten CpG dinucleotide sites, depending on the length of the target DNA region.

[0086] In some embodiments, the target DNA region contains a high density of CpG dinucleotides (or TpG dinucleotides or CpA dinucleotides derived from them). The degree of methylation of these CpG dinucleotides in the genomic sequence has been found to be associated with colorectal tumor formation (such as colorectal cancer). Without being theoretically limited, it is thought that diagnostic results can be obtained by analyzing the methylation status of these shorter, more clearly defined target DNA regions, and that analysis of these target DNA regions would provide more accurate diagnostic results compared to analysis of the methylation status across the entire PKNOX2 gene region; therefore, the target DNA regions within the PKNOX2 gene region are particularly useful for the methods provided in this invention. In some embodiments, the target DNA region includes sequences with Hg19 coordinates selected from the group consisting of: chr11:125033654-125038552, chr11:125132945-125133979, chr11:125285780-125286198, and chr11:125036431-125036547, and 200 bp upstream and downstream regions of said region, and any combination thereof. In some embodiments, the target DNA region includes any of the nucleotide sequences shown in SEQ ID NOs:1-3 or 10. In some embodiments, the target DNA region includes a processed sequence of the above-described genomic sequence (e.g., a sequence treated with bisulfite or MSRE). In some embodiments, the target DNA region comprises sequences selected from the group consisting of: SEQ ID NOs:4-9 and 11-12. Figure 4 The specific sequences of SEQ ID NOs:1-12 are shown.

[0087] In this application, the term "normal methylation status" refers to a threshold level representing the methylation status of the PKNOX2 gene region or target region in individuals who do not have colorectal cancer, or individuals who do not have colorectal cancer formation or a predisposition to colorectal cancer formation, or individuals who do not have colorectal cancer development or a predisposition to colorectal cancer with a good prognosis or a predisposition to colorectal cancer. Normal methylation status can be obtained by any suitable means known to those skilled in the art. For example, a set of biological samples from a healthy population can be obtained and their methylation status in the PKNOX2 gene region assessed to establish a standard value or range for comparison with the methylation status of future biological samples. It should be understood that the methylation status of the PKNOX2 gene region may be influenced by multiple characteristics such as age, sex, ethnicity, etc. Therefore, in some embodiments, multiple corresponding standard values ​​or ranges of normal methylation status can be established for multiple specific cohorts of populations.

[0088] In this application, the term "higher methylation state" refers to the presence of a higher level of methylated cytosine compared to a normal methylation state, such as a higher proportion of DNA molecules methylated at one or more CpG sites, or the presence of more methylated CpG sites.

[0089] In this application, the term "lower methylation state" refers to the presence of a lower level of methylated cytosine compared to a normal methylation state, such as a lower proportion of DNA molecules methylated at one or more CpG sites, or the presence of fewer methylated CpG sites.

[0090] In this application, the term "hybridization" refers to the process by which a base on one nucleic acid strand binds to a complementary base on another nucleic acid strand through base pairing. The hybridization reaction can be selective, allowing selection of a specific target sequence from a sample even when it is present at low concentrations. The stringency of the hybridization conditions (e.g., highly stringent, moderately stringent, stringent) can be adjusted by, for example, the concentration of salt or formamide in the pre-hybridization solution and the hybridization solution, or the hybridization temperature; for example, stringency can be increased by decreasing the salt concentration, increasing the formamide concentration, or increasing the hybridization temperature. Generally, stringent conditions include hybridization at a temperature of about 25°C to about 42°C in at least about 0% to at least about 15% v / v formamide and at least about 1M to at least about 2M salt, followed by washing in at least about 1M to at least about 2M salt; moderately stringent conditions include hybridization at a temperature of about 25°C to about 65°C in at least about 16% to at least about 30% v / v formamide and at least about 0.5M to at least about 0.9M salt, followed by washing in at least about 0.5M to at least about 0.9M salt; highly stringent conditions include hybridization at a temperature of at least about 65°C in at least about 31% to at least about 50% v / v formamide and at least about 0.01M to at least about 0.15M salt, followed by washing in at least about 0.01M to at least about 0.15M salt; formamide is optional in these hybridization conditions. Other suitable hybridization buffers and conditions are well known to those skilled in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd ed. Cold Spring Harbor Press, Plainview, NY (1989); and Ausubel et al., ShortProtocols in Molecular Biology, 4 th ed., John Wiley & Sons (1999).

[0091] In this application, the term "methylation specificity" refers to certain behaviors or properties that act only on molecules with a specific methylation state or without a specific methylation state. For example, oligonucleotides that hybridize in a methylation-specific manner can only hybridize with nucleic acids with an unmethylated state, or only with nucleic acids with a methylated state, under highly stringent hybridization conditions, but cannot hybridize with both nucleic acids with unmethylated and methylated states.

[0092] In this application, the term "response" refers to an individual's beneficial response to treatment. A beneficial response can manifest clinically in various forms, such as tumor disappearance, reduction in tumor size, tumor growth arrest, relief of one or more tumor-related symptoms, or increased survival time after treatment.

[0093] 2. Screening and monitoring methods

[0094] In one aspect, this application provides a method for diagnosing colorectal cancer in an individual, screening for colorectal cancer formation or a predisposition to form colorectal cancer, or monitoring colorectal cancer progression or prognosis. The method includes detecting the methylation status of a PKNOX2 gene region in a biological sample from the individual and comparing the detected methylation status of the PKNOX2 gene region with the normal methylation status of the PKNOX2 gene region. A change in the methylation status of the PKNOX2 gene region detected in the biological sample from the individual (e.g., a higher methylation status) relative to the normal methylation status of the PKNOX2 gene region indicates that the individual has colorectal cancer, or that the individual has a predisposition to colorectal cancer formation or a predisposition to colorectal cancer development or progression, or that the individual has a poor prognosis or a predisposition to colorectal cancer.

[0095] In one aspect, this application provides a method for monitoring an individual's response to colorectal cancer treatment, the method comprising detecting the methylation status of a PKNOX2 gene region in biological samples from the individual before and after the individual receives colorectal cancer treatment, wherein a change in the methylation status of the PKNOX2 gene region after colorectal cancer treatment (e.g., a lower methylation status after treatment) indicates that the individual has responded to colorectal cancer treatment, relative to the methylation status of the PKNOX2 gene region before receiving colorectal cancer treatment.

[0096] In some embodiments, the aforementioned colorectal tumor is a colorectal tumor. Colorectal tumors are the most common type of colorectal tumor, occurring in the colon, rectum, and cecum. Based on their nature, they can be classified as sessile serrated polyps, colorectal adenomas, and colorectal cancer. Without any theoretical limitations, polyps and adenomas are typically benign tissue proliferations of epithelial origin (derived from epithelial tissue or possessing well-defined epithelial structural features). As they progress, malignant proliferating tumor cells may appear within them, leading to malignant transformation. In some embodiments, the aforementioned colorectal tumor is colorectal cancer.

[0097] In some implementations, the colorectal tumor is precancerous. The term "precancerous" refers to abnormally proliferating cells that are transforming into malignantly proliferating tumor cells. For colorectal tumors, this includes the following categories: Grade 1: Malignant glands infiltrate from the muscular mucosa into the submucosa within the polyp head; Grade 2: The same submucosa invades, but is present at the junction of the head and the styloid process; Grade 3: Invasion of the styloid process; and Grade 4: Invasion of the base of the styloid process at the junction connecting to the colonic wall (this grade corresponds to Dukes A stage).

[0098] In some embodiments, detecting the methylation status of the PKNOX2 gene region includes determining the methylation status of cytosine residues at one or more methylation sites (e.g., CpG sites) in the PKNOX2 gene region from a biological sample from the individual. For example, the methylation status of the entire PKNOX2 gene region, or a portion thereof, such as the methylation status of the target DNA region described above, may be determined.

[0099] 3. Preparation of biological samples

[0100] The methods provided in this application can be used with any suitable biological sample containing DNA obtained from an individual. In some embodiments, the biological sample is selected from histological sections, tissue biopsies, paraffin-embedded tissue, surgically excised samples, isolated cells, body fluids (e.g., whole blood, serum, plasma, urine, saliva, mucus, peritoneal fluid, pleural fluid, pleural effusion, synovial fluid, cerebrospinal fluid, thoracentesis fluid, or peritoneal effusion), colonic effluents (e.g., feces and enema wash samples), and any combination thereof. In cases where the biological sample contains multiple cell populations, it may be necessary to isolate specific cell populations through purification or enrichment operations. In some embodiments, the biological sample is a blood sample (e.g., whole blood, serum, plasma) or a tissue biopsy.

[0101] Biological samples can be used directly in the methods provided in this application, or DNA can be obtained or isolated from the biological samples first and then used in the methods provided in this application. The DNA obtained from the biological samples can be, for example, genomic DNA, intracellular DNA, or extracellular cell-free DNA. Extracellular cell-free DNA, also known as circulating free DNA (cfDNA), is a type of degraded DNA fragment released into bodily fluids (e.g., plasma). The sources of extracellular cell-free DNA are diverse, with common types including, for example, circulating tumor DNA (ctDNA). Elevated levels of circulating free DNA have been observed in cancer patients.

[0102] DNA can be separated from a sample using any standard method in the prior art. The choice of method is influenced by a variety of factors, including time, cost, and the amount of DNA required. When DNA is encapsulated in a cell membrane, the general steps for separating DNA include: breaking up the biological sample and lysing the cells by enzymatic, chemical, or mechanical means; subsequently removing proteins and other contaminants by digestion, for example, with protein kinase K; and then recovering the DNA from the solution by methods including salting out, organic extraction, or binding the DNA to a solid support. When DNA is not encapsulated in a cell membrane (e.g., extracellular free DNA from blood samples), standard methods for separating and / or purifying DNA in the prior art can be used. These include the use of protein-degrading reagents, ethanol precipitation, or propanol precipitation. Devices such as ultrafiltration filters, silica surfaces or membranes, magnetic particles, and positively charged surfaces can also be used.

[0103] In some embodiments, the extracellular cell-free DNA includes circulating tumor DNA (CBDNA). CBDNA is a DNA fragment derived from tumor cells and found in bodily fluids (e.g., blood, urine, saliva, sputum, etc.). The cause of CBDNA formation is not yet clear, but it is presumed to be released by apoptotic or necrotic tumor cells or by tumor cells actively releasing it.

[0104] In some embodiments, the method provided in this application further includes treating the DNA obtained from the biological sample with a reagent capable of distinguishing between methylated and unmethylated CpG sites in the DNA, thereby obtaining the treated DNA.

[0105] It is known in the art that certain reagents can selectively act on unmethylated cytosine bases but not significantly on methylated cytosine residues; or selectively act on methylated cytosine bases but not significantly on unmethylated cytosine residues. For example, some reagents can selectively convert unmethylated cytosine bases into uracil, thymine, or another base that differs from cytosine in hybridization behavior. It is known in the art that methylcytosine has the same base-pairing behavior as guanine; therefore, the treatment can convert methylated DNA into DNA with different hybridization behavior depending on the state of cytosine methylation therein. In this application, DNA treated with a reagent capable of converting unmethylated cytosine bases into uracil is also referred to as transformed DNA or pre-transformed DNA. For example, some reagents can selectively cleave enzyme sites on methylated DNA or selectively cleave enzyme sites on unmethylated DNA.

[0106] In some embodiments, the reagent is a bisulfite. The specific reaction of bisulfites with unmethylated cytosine is known in the art, whereby the unmethylated cytosine is converted to uracil. Methylated cytosine remains unchanged. Commonly used bisulfite reagents include: ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, bisulfite ions, and any combination thereof.

[0107] Bisulfite treatment is typically carried out in the presence of a denaturing solvent, including, for example, n-alkyl glycols, diethylene glycol dimethyl ether (DME), dioxane, or dioxane derivatives. Preferably, the denaturing solvent is used at a concentration of 1% to 35% (v / v). Preferably, the bisulfite treatment is carried out in the presence of a scavenging agent, including, for example, chromane derivatives, trihydroxybenzoic acid, and their derivatives. Even more preferably, the bisulfite treatment is carried out at a reaction temperature of 30°C to 70°C, during which the temperature may be briefly increased to above 85°C.

[0108] In some embodiments, the reagent is a methylation-sensitive restriction enzyme (MSRE). Methylation-sensitive restriction enzymes selectively digest nucleic acids based on the methylation status of their recognition sites, thereby distinguishing (e.g., located at CpG sites) DNA containing methylated and unmethylated cytosine. Some methylation-sensitive restriction enzymes cleave only unmethylated recognition sites, and when their recognition sites are methylated, cleavage does not occur or occurs with significantly reduced efficiency; others cleave only methylated recognition sites, and when the recognition sites are unmethylated, cleavage does not occur or occurs with significantly reduced efficiency. Preferably, a methylation-sensitive restriction enzyme cleaves only the unmethylated cytosine recognition sites. Methylation-sensitive restriction enzymes that can be used in this application include HpaII enzymes, SalI enzymes, etc. Enzymes, including ScrFI, BbeI, NotI, SmaI, XmaI, MboI, BstBI, ClaI, MluI, NaeI, NarI, PvuI, SacII, HhaI, and any combination thereof. In one embodiment, DNA is treated with a methylation-sensitive restriction endonuclease under conditions sufficient to digest nucleic acids to obtain treated DNA, which is then used for the detection of nucleic acid methylation status.

[0109] 4. Detection of nucleic acid methylation status

[0110] DNA treated with the reagents can be used to identify the methylation status or level of methylation sites (such as CpG sites) in DNA obtained from a sample using conventional techniques based on base-specific pairing, such as amplification and hybridization. Any method suitable for detecting DNA methylation can be used in the methods described in this application. Numerous methods are known in the art for detecting methylated DNA at specific sites in the genome in biological samples (such as blood, urine, feces, or saliva) (see Kristensen and Hansen, Clin Chem. 55:1471-83, 2009; Ammerpohl et al., Biochim Biophys Acta. 1790:847-62, 2009; Shames et al., Cancer Lett. 251:187-98, 2007; Clark et al., Nat Protoc. 1:2353-64, 2006). In some embodiments, the detection of the methylation status can use methods including, but not limited to, amplification-based methods, hybridization-based methods, sequencing-based methods, or restriction enzyme digestion-based methods. Exemplary methods include polymerase chain reaction (e.g., real-time polymerase chain reaction, digital polymerase chain reaction), nucleic acid sequencing, quality-based separation (e.g., electrophoresis, mass spectrometry), or target capture (e.g., microarrays).

[0111] A. Amplification-based methods

[0112] In some embodiments, detecting the methylation status of the PKNOX2 gene region includes amplifying the treated DNA using an amplification enzyme and one or more sets of primers to produce at least one amplification product or to prevent amplification of the treated DNA. In some embodiments, the treated DNA (e.g., bisulfite treated) comprises the nucleotide sequences shown in SEQ ID NOs:4-9, 11-12 and any combination thereof, or a nucleotide sequence selected from SEQ ID NOs:4-9, 11-12. In some embodiments, the amplification product comprises the nucleotide sequences shown in SEQ ID NOs:4-9, 11-12 and any combination thereof, or a nucleotide sequence selected from SEQ ID NOs:4-9, 11-12. In some embodiments, the treated DNA (e.g., MSRE treated) comprises the nucleotide sequences shown in SEQ ID NOs:1-3 or 10 and any combination thereof, or a nucleotide sequence selected from SEQ ID NOs:1-3 or 10.

[0113] A variety of nucleic acid amplification methods known in the art can be used, including but not limited to polymerase chain reaction (PCR), allele-specific PCR (ASPCR), single base extension (SBE), allele-specific primer extension (ASPE), strand displacement amplification (SDA), transcription-mediated amplification (TMA), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), primer extension, rolling circle amplification (RCA), autonomous sequence replication (3SR), using Qβ replicase, nick translation or loop-mediated isothermal amplification (LAMP), or any combination thereof.

[0114] PCR is a well-known method in the field (see CR Primer: A Laboratory Manual, Cold Spring Harbour Laboratories, NY, 1995). Generally, in a PCR reaction, the double-stranded template DNA (the DNA to be amplified) denatures and breaks down into single strands at high temperature; at hybridization / annealing temperatures, two oligonucleotide primers bind to the single strands of template DNA according to the principle of complementary base pairing; at a suitable temperature, DNA polymerase extends the primers along the 5'-3' direction, synthesizing the complementary strand of the template DNA single strand. Through repeated temperature cycles, the template DNA is amplified exponentially, yielding the amplified product.

[0115] In this application, the term "DNA polymerase" refers to an enzyme that catalyzes the synthesis of polynucleotides from monodeoxyribonucleoside triphosphates (dNTPs). DNA replication and repair both require the use of DNA polymerases. In addition to its activity of catalyzing DNA synthesis, DNA polymerase may also possess exonuclease activity (e.g., 5'-3' exonuclease activity or 3'-5' exonuclease activity), which means removing mononucleotides from the 5' or 3' end of a polynucleotide chain.

[0116] In some implementations, the DNA polymerase is a thermostable DNA polymerase or a polymerase lacking 5'-3' exonuclease activity. Thermostable DNA polymerases can withstand high temperatures that denature and unwind DNA double strands without inactivation; examples of thermostable DNA polymerases include Taq DNA polymerase. High-fidelity DNA polymerase or DNA polymerases lacking 5'-3' exonuclease activity are unable to remove single nucleotides from the 5' end to the 3' end of a polynucleotide chain. Examples of DNA polymerases lacking 5'-3' exonuclease activity include... High-fidelity DNA polymerase, polymerase, DNA polymerase, (exo-)DNA polymerase, Deep DNA polymerase, T4 DNA polymerase, and T7 DNA polymerase.

[0117] In some embodiments, the primers comprise a sequence substantially complementary to or substantially identical to at least nine consecutive nucleotides of the PKNOX2 gene region sequence in the treated DNA, wherein said consecutive nucleotides comprise at least one CpG site, or wherein said amplification product comprises at least one CpG site. In some embodiments, the treated DNA comprises the nucleotide sequences shown in SEQ ID NOs:4-9, 11-12, and any combination thereof. In some embodiments, the treated DNA is selected from the nucleotide sequences composed of SEQ ID NOs:4-9, 11-12. In some embodiments, the treated DNA comprises MSRE digestion products of the nucleotide sequences shown in SEQ ID NOs:1-3 or 10.

[0118] In this application, the term "complementarity" or "complementarity" refers to the base pairing between nucleotides or nucleic acids, such as between the two strands of a double-stranded DNA molecule or between the binding sites of an oligonucleotide primer and a primer on a single-stranded nucleic acid to be sequenced or amplified. Complementary or paired bases are typically: A and T (or A and U), or C and G. The percentage of complementarity (%) represents the percentage of nucleotide residues on one nucleic acid that can pair with residues on another nucleic acid, and can be obtained by dividing the number of paired residues by the total number of residues on the shorter of the two nucleic acid chains. Two nucleic acid chains are considered substantially complementary when they have at least 80% complementarity (typically at least about 90% to 95%, more preferably about 98% to 100% complementarity). In some embodiments, substantially complementary nucleotide chains can be at least 80% complementary, at least 85% complementary, at least 90% complementary, at least 95% complementary, or 100% complementary.

[0119] In the context of describing two or more nucleic acid sequences, the term "identical" or "percentage of identity (%)" refers to the degree of similarity of nucleotide residues after sequence alignment of two or more nucleic acid molecules. Gaps may be introduced during alignment to achieve the maximum degree of similarity or pairing of nucleotide residues. Two nucleic acid chains are considered substantially identical when they have at least 80% identity (typically at least about 90% to 95%, more preferably about 98% to 100% identity). In some embodiments, substantially identical nucleotide chains can be at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical.

[0120] The percentage of identity (%) or the percentage of complementarity (%) can be obtained by dividing the number of identical nucleotide residues obtained in the alignment by the total number of nucleotide residues in the shorter nucleic acid sequence in the alignment. Those skilled in the art can use publicly available tools, such as BLASTN, which is available on the website of the National Center for Biotechnology Information (NCBI), to perform nucleic acid sequence alignment to obtain the percentage of identity (see, for example, Altschul SF et al., J. Mol. Biol., 215: 403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25: 3389-3402 (1997)).

[0121] In some embodiments, the primer comprises a sequence that is substantially complementary or substantially identical to at least nine consecutive nucleotides selected from the sequences of SEQ ID NOs:1-12. In some embodiments, the primer comprises a sequence that is at least 85% complementary, at least 90% complementary, at least 95% complementary, or 100% complementary to at least nine consecutive nucleotides selected from the sequences of SEQ ID NOs:1-12, or at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical.

[0122] In some embodiments, the primers are methylation-specific. As used herein, a methylation-specific primer is one that amplifies a specific target on the treated DNA in a methylation-specific manner. The primer can specifically amplify DNA molecules with a specific methylation state but not DNA molecules without a specific methylation state. For example, the primer may specifically hybridize with a specific methylated CpG site under moderately, strictly, or highly strictly conditions, but not specifically hybridize with that specific unmethylated CpG site, thus the primer will specifically amplify the target methylated at that specific CpG site. As another example, the primer may specifically hybridize with a specific unmethylated CpG site under moderately, strictly, or highly strictly conditions, but not specifically hybridize with that specific methylated CpG site, thus specifically amplifying the target unmethylated at that specific CpG site.

[0123] In this application, methylation-specific primers can be used to amplify treated DNA, thereby distinguishing between methylated and unmethylated DNA. The methylation-specific primers contain at least one primer that hybridizes to a bisulfite-treated CpG dinucleotide, i.e., containing at least one CpG dinucleotide for amplifying bisulfite-treated methylated DNA, or containing at least one TpG or CpA dinucleotide for amplifying bisulfite-treated unmethylated DNA.

[0124] In some embodiments, the primer comprises, or consists of, sequences selected from, the following group:

[0125] GTTTTAGGAGTTATTTGGGTTTGC(SEQ ID NO:13) PKNOX2 upstream primer sequence ACTATAACACCTCGCTACTAACGCT(SEQ ID NO:14) PKNOX2 downstream primer sequence

[0126] Methods for designing primers, for example, used in PCR, are known in the art (see PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995). Several software packages for designing optimal primers for various assays are also publicly available, such as Primer3 from the Center for Genome Research, Cambridge, Mass., USA. Of course, the possible uses of the primers should also be considered during primer design. For example, primer sequences designed for the purposes of this application may contain at least one CpG site, or the amplification products obtained from said primers may contain at least one CpG site. Primer design tools for detecting DNA methylation status are known in the art, such as MethPrimer (see Li LC and Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002 Nov; 18(11):1427-31).

[0127] Methods for preparing oligonucleotide primers are known in the art (see Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, 1984). For example, primers can be obtained by biological synthesis (e.g., by digesting nucleic acids with restriction endonucleases) or by chemical synthesis. For short sequences (up to about 100 nucleotides), chemical synthesis is preferred. For longer sequences, standard replication methods used in molecular biology are preferred, for example, obtaining single-stranded DNA using M13 (see Messing, Methods Enzymol., 101, 20-78, 1983).

[0128] In some implementations, amplification is performed in the presence of a blocking agent. Methylation-specific amplification can also be achieved by using a blocking agent. Examples of such use are known in the art (see Yu et al., BioTechniques 23:714-720, 1997).

[0129] In some embodiments, the blocking reagent comprises a blocking oligonucleotide that cannot be extended and / or degraded by DNA polymerase. For PCR methods using blocking oligonucleotides, to effectively disrupt the amplification of nucleic acids specifically bound to the blocking oligonucleotide, the blocking oligonucleotide needs to be prevented from being extended by polymerase. In some embodiments, the blocking oligonucleotide does not contain a free hydroxyl group at its 3' end, for example, it is 3' deoxygenated or substituted with a group other than a hydroxyl group (e.g., 3'-O-acetyl), thus preventing polymerase extension. Additionally, it is necessary to prevent DNA polymerase-mediated degradation of the blocking oligonucleotide. In some embodiments, the blocking oligonucleotide has a thioester bridge at its 5' end, thereby preventing degradation by polymerases with 5'-3' exonuclease activity. In some embodiments, the binding sequence of the primer is designed to overlap with the binding sequence of the blocking oligonucleotide, thereby preventing degradation by polymerases with 5'-3' exonuclease activity. In other embodiments, a polymerase lacking 5'-3' exonuclease activity is used. Peptide nucleic acid (PNA) oligomers can also be used as blocking oligonucleotides in this application. PNA is neither broken down nor extended by DNA polymerase, making it an ideal blocking oligonucleotide.

[0130] In some implementations, the blocking oligonucleotide is capable of hybridizing with CpG sites on the treated DNA in a methylation-specific manner. For example, to detect methylated nucleic acids against a background of unmethylated nucleic acids, for bisulfite-treated DNA, blocking oligonucleotides containing "CpA" or "TpG" at the corresponding sites can be used to inhibit the amplification of unmethylated nucleic acids at those sites; correspondingly, if it is necessary to inhibit the amplification of methylated nucleic acids, blocking oligonucleotides containing "CpG" at the corresponding sites can be used.

[0131] In some implementations, amplification is performed in the presence of detection reagents.

[0132] In some embodiments, the detection reagent comprises primers labeled with a detectable marker, oligonucleotide detection probes labeled with a detectable marker, or DNA double-stranded insertion dyes. Examples of detectable markers include, for example, fluorescent molecules, chromophores, biotin, radioisotopes, etc. In some embodiments, the detection reagent is capable of reporting information about the amplification products in real time.

[0133] In some embodiments, the oligonucleotide probe comprises a sequence capable of hybridizing with the amplification product under stringent, moderately stringent, or highly stringent conditions. In some embodiments, the oligonucleotide probe comprises, or consists of, sequences selected from, the group consisting of, [a specific sequence group].

[0134] CGGTGGTTCGTAGGGGTCGCG (SEQ ID NO:15) PKNOX2 probe sequence

[0135] In some embodiments, the oligonucleotide probe contains a fluorescent dye (e.g., FAM, HEX / VIC, TAMRA, Texas Red, or Cy5) at the 5' end and a quencher (e.g., BHQ1, BHQ2, BHQ3, DABCYL, or TAMRA) at the 3' end.

[0136] Probes can be prepared using methods similar to or the same as primers. Labeling the probe with a detectable marker can be done directly or indirectly. Direct labeling involves directly (covalently or non-covalently) coupling the marker to the oligonucleotide probe. Indirect labeling involves coupling the marker to the oligonucleotide probe via an intermediate reagent that can specifically bind (covalently or non-covalently) the marker. There can be multiple intermediate reagents (e.g., two or three binding sequentially in a specific manner), and multiple intermediate reagents are often used to amplify the detectable signal. Detectable markers suitable for indirect coupling include antibodies and the streptavidin-biotin system.

[0137] Nucleic acid amplification-based methods can also be used to analyze DNA digested by methylation-sensitive restriction enzymes (MSREs). For example, primers can be designed with binding sites located flanking or adjacent to a specific methylation-sensitive restriction enzyme recognition site within a target DNA region. This results in amplification products being generated only when the restriction recognition site is not cleaved; that is, the presence or amount of amplification products can indicate the methylation status of cytosine within the specific methylation-sensitive restriction enzyme recognition site in the target DNA region. In some embodiments, the presence of amplification products when MSRE cleaves unmethylated DNA indicates that the MSRE recognition site is methylated; in other embodiments, the presence of amplification products when MSRE cleaves methylated DNA indicates that the MSRE recognition site is unmethylated. Real-time quantitative PCR can be used to analyze the amplification products, with the amount indicating the level of methylation.

[0138] In some embodiments, the methylation status of the PKNOX2 gene region is determined based on the presence and properties of the amplified product. The amplified nucleic acid product can be analyzed using methods commonly used in the art, such as fluorescence detection, non-denaturing agarose gel electrophoresis, non-denaturing polyacrylamide gel electrophoresis, mass spectrometry, liquid chromatography (e.g., HPLC), or capillary electrophoresis. High-throughput detection methods can also be used in this application, such as matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF), electrospray ionization (ESI), tandem mass spectrometry (e.g., LC MS / MS), biosensor technologies, etc.

[0139] In some implementations, methylation-specific PCR (MSP) is used to detect methylation status by amplifying treated DNA (see Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1992). For example, this amplification system contains one or more primers that hybridize specifically to unmutated sequences under moderate and / or highly stringent conditions, producing amplification products only from a template containing methylated cytosine. Similarly, methods for selectively amplifying and detecting methylated or unmethylated components from mixtures of bisulfite-treated DNA can be found in Cottrell et al., Nucl. Acids Res. 32:e10, 2003 (Heavy MethylPCR), Rand et al., Nucl. Acids Res. 33:e127, 2005 (Headloop PCR), Rand et al., Epigenetics 1:94-100, 2006 (Bisulfite Differential Denaturation PCR) and PCT / AU07 / 000389 (End-specific PCR).

[0140] In some implementations, real-time quantitative PCR is used to detect DNA methylation status by amplifying treated DNA (see Holland et al., Proc. Natl. Acad. Sci. USA, 88:7276-7280, 1991; Lee et al., Nucleic Acid Res. 21:3761-3766, 1993). For example, the MethyLight method uses a modified TaqMan assay to detect cytosine methylation of CpG dinucleotides (see Eads et al., Nucl. Acids Res. 28:E32, 2000). In short, this method involves treating a sample containing nucleic acids with bisulfite followed by nucleic acid amplification. The amplification reaction comprises a DNA polymerase (e.g., Taq DNA polymerase) with 5' to 3' exonuclease activity and three oligonucleotides. Three oligonucleotides are used: a forward primer and a reverse primer that anneal to the target region, and a probe that hybridizes to one or more methylated CpG dinucleotide sites between these two primers. The 5' and 3' segments of the probe oligonucleotide are labeled with a fluorescent reporter molecule and a quencher, respectively. When the probe is intact, the quencher approaches the fluorescent reporter molecule, quenching the fluorescence. After the probe oligonucleotide anneals to the amplification product, the 5' to 3' exonuclease activity of DNA polymerase cleaves the probe, causing the quencher to release the fluorescent reporter molecule, resulting in enhanced fluorescence. The methylation level of the initial nucleic acid template can be estimated by the enhancement of the fluorescence signal. In the above method, the detectable labeled probe can use a molecular beacon, a system independent of the 5' to 3' exonuclease activity of DNA polymerase (see Mhlanga and Malmberg, Methods 25:463-471, 2001).

[0141] Another method for detecting DNA methylation status using real-time quantitative PCR amplification of bisulfite-treated DNA is the HeavyMethyl assay (see Cottrell et al., Nucl. Acids Res. 32:e10, 2003). This method's nucleic acid amplification system also includes one or more non-extension blocking reagents (such as blocking oligonucleotides) that specifically bind to unmethylated bisulfite-treated DNA in a methylation-specific manner under moderate to high stringency conditions. Amplification is performed using one or more pairs of primers that flank or partially or completely cover the binding sequence of one or more blocking reagents. When unmethylated nucleic acids are present, the blocking reagent binds, and the nucleic acids are not amplified; when methylated nucleic acids are present, the blocking reagent does not bind, and the nucleic acids are amplified. In practical applications, assays using TaqMan or molecular beacons, essentially as described above, can be used to determine the methylation level of nucleic acids in a sample. In other embodiments, the blocking reagent binds specifically to methylated DNA treated with bisulfite in a methylation-specific manner under moderate to highly stringent conditions; that is, when methylated nucleic acids are present, the blocking reagent binds and the nucleic acids are not amplified.

[0142] B. Based on hybridization method

[0143] In some implementations, after treating nucleic acids with a reagent (e.g., bisulfite) to convert unmethylated cytosine into another base that behaves differently from cytosine, the presence or level of pre-conversion methylated cytosine is determined by hybridization. This method can be used on amplified, treated DNA or directly on the treated DNA.

[0144] Any nucleic acid hybridization technique known in the art can be used for detection, such as DNA blotting, dot blotting, slit blotting, or others (Kawai et al., Mol. Cell. Biol. 14:7421-7427, 1994; Gonzalgo et al., Cancer Res. 57:594-599, 1997). In some embodiments, the probe nucleic acid used for the hybridization assay is detectably labeled. In some embodiments, the probe of the probe nucleic acid used for the hybridization assay is unlabeled, the unlabeled probe is immobilized on a solid support such as a microarray, and can hybridize with a detectably labeled target nucleic acid molecule.

[0145] Hybridization conditions can be determined based on the melting temperature (Tm) of the nucleic acid duplex containing the probe. Generally, low to moderately stringent hybridization and / or washing conditions are used for shorter oligonucleotide probes, while highly stringent hybridization and / or washing conditions are used for GC-rich or longer probes. Those skilled in the art will understand that, in addition to general rules, optimal hybridization reaction conditions should also be determined empirically for each probe.

[0146] In one implementation, methylation-specific microarrays (MSOs) are used to distinguish between mutated and non-mutated sequences (see Adorjan et al., Nucl. Acids Res., 30:e21, 2002). Nucleic acids are treated with a reagent (e.g., bisulfite) to selectively transform unmethylated cytosine. Using the treated nucleic acid as a template, nucleic acid amplification is performed with detectably labeled primers (e.g., fluorophores), amplifying both transformed and untransformed nucleic acids. The labeled amplification products are then hybridized with oligonucleotides on the microarray under conditions capable of detecting single nucleotide differences. After washing away unbound amplification products, hybridization is detected using, for example, a microarray scanner. This method allows for semi-quantitative determination of the methylation status of a large number of methylable sites.

[0147] Hybridization-based analysis can also be used for nucleic acids treated with methylation-sensitive restriction enzymes, for example, by designing probes that selectively hybridize with undigested nucleic acids. In other embodiments, digested and undigested nucleic acids are distinguished by electrophoresis, and then detected using probes that can hybridize with both digested and undigested nucleic acids. Similarly, this method can be used to amplify treated DNA or directly on treated DNA.

[0148] C. Nucleic acid sequencing-based methods

[0149] In one implementation, after treating nucleic acids with a reagent (e.g., bisulfite) to mutate unmethylated cytosine to uracil (and thus thymine after amplification), DNA sequencing is used to determine the presence or number of methylcytosines (see Frommer et al. Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992; Clark et al., Nucl. Acids Res. 22:2990-2997, 1994). For example, methylcytosines in the DNA sequence can be identified by comparing the sequencing results or known nucleotide sequences of the target region obtained from untreated samples with the sequencing results from bisulfite-treated samples: any thymine detected in the bisulfite-treated sample at a cytosine site corresponding to that in the control sample can be considered a mutation caused by bisulfite treatment, i.e., the presence of methylcytosine at that site. This method can be used on amplified, treated DNA or directly on the treated DNA.

[0150] Various DNA sequencing methods are known in this field, such as dideoxy chain termination or Maxam-Gilbert sequencing (see Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd edition), CSHP, New York 1989), pyrosequencing (e.g., Uhlmann et al., Electrophoresis, 23:4072-4079, 2002), solid-phase pyrosequencing (see Landegren et al., Genome Res., 8(8):769-776, 1998), solid-phase microsequencing (see e.g., Southern et al., Genomics, 13:1008-1017, 199), microsequencing using FRET (see e.g., Chen and Kwok, Nucleic Acids Res. 25:347-353, 1997), ligation sequencing, or ultra-deep sequencing (see Marguiles et al.). al.,Nature437(7057):376-80(2005)).

[0151] One method for determining the sequence of bisulfite-treated DNA is methylation-sensitive single nucleotide primer extension (Me-SnuPE) or SNaPmeth (see Gonzalgo and Jones, Nucl. Acids Res., 25:2529-2531). An oligonucleotide is hybridized to a nucleic acid region adjacent to a site of methylated cytosine, followed by primer extension using a polymerase and a detectable label (e.g., a fluorophore-labeled) free nucleotide diphosphate or dideoxynucleotide triphosphate, wherein the free nucleotide diphosphate or dideoxynucleotide triphosphate corresponds to one of the two bases that may appear at this site after bisulfite treatment (i.e., thymine or cytosine). After primer extension, the unbound free nucleotide diphosphate or dideoxynucleotide triphosphate is detected, and the incorporated labeled nucleotide is identified, thus revealing the base at that site.

[0152] D. Restriction endonuclease-based methods

[0153] In some implementations, the presence of methylated cytosine in a DNA sequence is detected by combined bisulfite restriction analysis (COBRA) (see (Xiong and Laird, Nucl. Acids Res., 25:2532-2534, 2001). This method utilizes the selective modification of unmethylated cytosine with reagents, followed by differentiation between unmethylated and methylated cytosine at restriction enzyme recognition sites.

[0154] In short, after bisulfite treatment introduces methylation-dependent sequence differences into genomic DNA, the target DNA region containing methylation sites is amplified using specific primers. The amplified product is then digested with a restriction endonuclease, which can be selected to indicate the presence or absence of methylation. For example, the restriction endonuclease Taq1 cleaves the sequence TCGA. If the cytosine in this recognition sequence is unmethylated, after bisulfite treatment, the sequence will be TTGA and will therefore not be cleaved. The products after restriction endonuclease digestion can be detected using detection methods known in the art, such as electrophoresis and / or mass spectrometry.

[0155] In other embodiments, based on the differences in nucleotide sequence and / or secondary structure after treatment with reagents that selectively mutate unmethylated cytosine residues, different techniques are used to detect nucleic acid differences in the amplified products, such as methylation single-strand conformation analysis (MS-SSCA) (Bianco et al., Hum. Mutat., 14:289-293, 1999), methylation-specific denaturing gradient gel electrophoresis (MS-DGGE) (Abrams and Stanton, Methods Enzymol., 212:71-74, 1992) and methylation-specific denaturing high-performance liquid chromatography (MS-DHPLC) (Deng et al., Chin. J. Cancer Res., 12:171-191, 2000).

[0156] In one aspect, this application provides a method for diagnosing colorectal cancer in an individual, screening for colorectal cancer formation or a predisposition to formation, or monitoring colorectal cancer progression or prognosis, comprising:

[0157] (a) Obtaining a biological sample containing DNA from the individual;

[0158] (b) The DNA in the biological sample obtained in step (a) is treated with a reagent that can distinguish between methylated CpG sites and unmethylated CpG sites in the DNA, thereby obtaining the treated DNA;

[0159] (c) Contact the DNA treated in step (b) with an amplification enzyme and one or more sets of primers suitable for amplifying a target DNA region, the target DNA region comprising at least nine consecutive nucleotides of a PKNOX2 gene region or its complementary sequence, wherein the consecutive nucleotides comprise at least one CpG site.

[0160] (d) Determine the methylation status of the target DNA region based on the presence and properties of the amplification products;

[0161] (e) Compare the methylation state of the target DNA region determined in step (d) with the normal methylation state of the target DNA region.

[0162] The change in the methylation state of the target DNA region determined in step (d), relative to the normal methylation state of the target DNA region, indicates that the individual has colorectal cancer, or the individual has a tendency to form or develop colorectal cancer, or the individual has a tendency to develop or progress colorectal cancer, or the individual has a poor prognosis or a tendency to have a poor prognosis for colorectal cancer.

[0163] In some embodiments, the reagent (such as bisulfite) converts the unmethylated 5'-cytosine base into uracil, thymine, or another base that differs from cytosine in hybridization behavior. In other embodiments, the reagent is a methylation-sensitive restriction enzyme.

[0164] In some implementations, the method for amplifying the target DNA region and determining the methylation status of the target DNA region based on the presence and nature of the amplification product can be selected from any suitable method known in the art.

[0165] In some implementations, a higher methylation state of the target DNA region determined in step (d) relative to the normal methylation state of the target DNA region indicates that the individual has colorectal cancer, or the individual has a tendency to form or develop colorectal cancer, or the individual has a tendency to develop or progress colorectal cancer, or the individual has a poor prognosis or a tendency to have a poor prognosis for colorectal cancer.

[0166] In some implementations, the normal methylation state of the PKNOX2 gene region or target region represents the methylation state of the PKNOX2 gene region or target region in individuals who do not have colorectal cancer, or individuals who do not have colorectal cancer formation or a tendency to form colorectal cancer, or individuals who do not have colorectal cancer development or a tendency to develop colorectal cancer, or individuals who have a good prognosis or a tendency to have a good prognosis for colorectal cancer.

[0167] Comparison with normal methylation states can be performed manually or with computer assistance. With computer assistance, a computer program can compare the detected methylation state of a biological sample with normal methylation states stored in a database. This program can further evaluate the comparison results and automatically provide the desired assessment in a suitable output format.

[0168] In some implementations, the method provided in this application has a sensitivity of at least 60% and a specificity of at least 85% in identifying individuals with colorectal cancer, a tendency to form or develop colorectal cancer, a tendency to develop or progress colorectal cancer, or a tendency to have a poor prognosis or poor prognosis of colorectal cancer.

[0169] Sensitivity is calculated as: Sensitivity = TP / (TP+FN); specificity is calculated as: Specificity = TN / (FP+TN), where a true positive (TP) result is a positive result indicating the presence of the disease state, a false positive (FP) result is a positive result indicating the absence of the disease state, a true negative (TN) result is a negative result indicating the absence of the disease state, and a false negative (FN) result is a negative result indicating the presence of the disease state. Sensitivity measures the ability of a test to correctly detect a target disease or state in the tested individuals. The method provided in this application has high sensitivity, which helps to reduce the occurrence of delayed disease treatment due to missed diagnoses.

[0170] 5. Detection tools and their applications

[0171] In one aspect, this application provides an oligonucleotide for use as a detection tool, comprising at least nine consecutive nucleotides of a PKNOX2 gene region or its complementary sequence, or consisting of at least nine consecutive nucleotides of a PKNOX2 gene region or its complementary sequence.

[0172] In one aspect, this application provides an oligonucleotide for use as a detection tool, comprising at least nine consecutive nucleotides of a treated DNA sequence of the PKNOX2 gene region or its complementary sequence, or consisting of at least nine consecutive nucleotides of a treated DNA sequence of the PKNOX2 gene region or its complementary sequence. In some embodiments, the treatment is adapted to convert at least one unmethylated cytosine residue in the PKNOX2 gene region into a uracil residue, a thymine residue, or other residues detectably different from cytosine in hybridization. In some embodiments, the treated DNA comprises the nucleotide sequences shown in SEQ ID NOs:4-9, 11-12, and any combination thereof. In some embodiments, the treated DNA is selected from the nucleotide sequences composed of SEQ ID NOs:4-9, 11-12. In some embodiments, the treated DNA comprises the MSRE digestion product of the nucleotide sequences shown in SEQ ID NOs:1-3 or 10.

[0173] In some embodiments, the oligonucleotide comprises one or more CpG, TpG, or CpA dinucleotides.

[0174] In some embodiments, the oligonucleotide may be modified by chemically linking it to one or more conjugates to improve its activity, stability, or detectability. Suitable conjugates include chromophores, fluorophores, cholesterol, bile acids, thioethers, aliphatic chains, phospholipids, polyamines, polyethylene glycol, palmityl groups, peptides, hybridization-triggered crosslinking agents, transporters, hybridization-triggered cleavage agents, etc. The sugar and / or bases of the oligonucleotide may contain at least one known modification, and the backbone of the oligonucleotide may also be modified, for example, by containing non-natural nucleoside internucleotide bonds. In some embodiments, the oligonucleotide may be in the form of peptide nucleic acid (PNA) or locked nucleic acid (LNA) molecules for better pairing.

[0175] In some embodiments, the oligonucleotide can be used as a primer (such as a PCR primer), a detection probe, or a blocking oligonucleotide. Preferably, the hybridization of the oligonucleotide with DNA in the sample is performed under highly stringent conditions.

[0176] In some embodiments, multiple oligonucleotides may be bound to a solid surface in the form of an array (such as a rectangular or hexagonal lattice). The material of the solid surface may be silicon, glass, polystyrene, metal, nitrocellulose, nylon, etc. For the preparation of oligomeric arrays, see Nature Genetics Supplement, Volume 21, January 1999.

[0177] In some embodiments, the oligonucleotide comprises a sequence that is substantially complementary or substantially identical to at least nine consecutive nucleotides selected from the sequences of SEQ ID NOs:1-12. In some embodiments, the oligonucleotide comprises a sequence that is at least 80% complementary, at least 85% complementary, at least 90% complementary, at least 95% complementary, or 100% complementary to at least nine consecutive nucleotides selected from the sequences of SEQ ID NOs:1-12, or at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical. In some embodiments, the oligonucleotide contains, or consists of, sequences selected from the group consisting of SEQ ID NOs:13, 14, and 15.

[0178] In some embodiments, the oligonucleotide can be used in methods for diagnosing colorectal cancer in an individual, screening for colorectal cancer formation or a predisposition to formation, monitoring colorectal cancer progression or prognosis, or monitoring response to colorectal cancer treatment. In other embodiments, the oligonucleotide can be used to prepare kits for methods for diagnosing colorectal cancer in an individual, screening for colorectal cancer formation or a predisposition to formation, monitoring colorectal cancer progression or prognosis, or monitoring response to colorectal cancer treatment.

[0179] In another aspect, this application provides a reagent (e.g., a bisulfite reagent or a methylation-sensitive restriction enzyme (MSRE)) for distinguishing methylated and unmethylated CpG sites in a target DNA region. This reagent can be used to prepare kits for methods of diagnosing colorectal cancer in an individual, screening for colorectal cancer formation or predisposition, monitoring colorectal cancer progression or prognosis, or monitoring response to colorectal cancer treatment. The method includes contacting DNA isolated from a biological sample of the individual with the at least one reagent and one or more oligonucleotides that hybridize with the target DNA region under stringent, moderately stringent, or highly stringent conditions. In some embodiments, the reagent is a bisulfite reagent or a methylation-sensitive restriction enzyme (MSRE).

[0180] In another aspect, this application provides a kit for diagnosing colorectal cancer, screening for colorectal cancer formation or a tendency to form, or monitoring colorectal cancer progression or prognosis, comprising a first reagent containing one or more oligonucleotides as provided in this application for use as detection tools.

[0181] In some specific embodiments, the kit further includes a second reagent comprising one or more reagents as provided in this application for distinguishing methylated and unmethylated CpG sites in a target DNA region.

[0182] In some embodiments, the first reagent and the second reagent are packaged in a single container or in separate containers. In some embodiments, the kit further includes a container suitable for holding biological samples from the individual. In some embodiments, the kit further includes instructions for use and / or an interpretation of the kit's test results.

[0183] In some embodiments, the kit may further comprise a computer program product stored on a computer-readable medium. When executed by a computer, the computer program product identifies an individual as having a predisposition to colorectal cancer development or progression, or a poor prognosis for colorectal cancer, based on a higher methylation state of the PKNOX2 gene region detected in a biological sample from the individual, relative to the normal methylation state of the PKNOX2 gene region. This application contemplates any medium capable of storing and transmitting such computer-executable instructions to an end user. Such media include, but are not limited to, electronic storage media (e.g., disks, magnetic tapes, cartridges, chips), optical media (e.g., CD-ROMs), etc. Such media may include the URL of an Internet website providing such instructional material.

[0184] Computer programs can also be encoded and transmitted using carrier signals suitable for transmission over wired, optical, and / or wireless networks conforming to various protocols, including the Internet. Therefore, such program-encoded data signals can be used to create computer-readable media according to embodiments of this application. Computer-readable media with encoded program code can be packaged with compatible devices or provided separately from other devices (e.g., for download via the Internet). Any such computer-readable medium can reside on or within a single computer product (e.g., a hard disk drive, CD, or an entire computer system) and can exist on or within different computer products within a system or network.

[0185] In some embodiments, the kit may contain a natural or synthetic control DNA sequence representing a methylated or unmethylated form of the target DNA region. In some embodiments, the kit may also contain a detectable marker optionally linked to the first reagent. In some embodiments, the kit includes other materials useful for carrying out the methods provided in this application, such as test tubes, pipettes, etc.

[0186] In some implementations, the kit includes,

[0187] (a) Bisulfite reagent or methylation-sensitive restriction enzyme reagent;

[0188] (b) A container suitable for containing the reagents and biological samples from the individual;

[0189] (c) One or more sets of primers comprising a sequence that is complementary to or identical to at least nine consecutive nucleotides of the PKNOX2 gene region sequence in DNA treated with bisulfite or methylation-sensitive restriction enzymes; and optionally,

[0190] (d) Instructions for use and / or interpretation of the test results from the kit. Detailed Implementation

[0191] Example 1. Validation of methylation-specific primers and probes

[0192] Reference DNA treated with bisulfite was selected to verify primer specificity. First, primers and probes were designed for the target DNA region within the PKNOX2 gene region and the internal reference gene ACTB, respectively. Then, gradients were prepared from 100%, 50%, 25%, and 10% methylated DNA (Qiagen 59655) to unmethylated DNA (Qiagen 59665), with a total DNA volume of 4 ng. Finally, the designed primers were used to amplify the DNA in each gradient DNA mixture, repeated twice. The sequences of the primers and probes used to detect PKNOX2, and the unmethylated specific primers and probes used to detect the internal reference gene ACTB, are shown in Table 1. In the PCR reaction system, the final primer concentration was 500 nM, and the final probe concentration was 100 nM.

[0193] Table 1. Primer and probe sequences for the target DNA region within the PKNOX2 gene region and the internal reference gene ACTB.

[0194]

[0195]

[0196] The PCR reaction system is as follows: 10 μL of 4 ng DNA mixture with different methylation ratios; 2.5 μL of primer and probe premix shown in Table 1; 12.5 μL of PCR reagent (EpiTect MethyLight PCR kit, Qiagen).

[0197] The PCR reaction conditions were as follows: 95℃ for 5 minutes; 95℃ for 15 seconds, 56℃ for 40 seconds (fluorescence acquisition), for 50 cycles. For different gene probe modifications, the corresponding fluorescence detection channel was selected, and detection was performed using an ABI 7500 Real-Time PCR instrument.

[0198] result

[0199] The Ct value for each PCR reaction was calculated, and the Ct values ​​obtained from the PCR reactions of each gene in each DNA mixture were analyzed. The results showed that for PKNOX2, the Ct value decreased with increasing percentage of methylated DNA transformed in the DNA mixture (e.g., ...). Figure 1 As shown in Figure A), this indicates that the primers used to amplify the target region of the PKNOX2 gene are methylation-specific primers. For the internal reference gene ACTB, the curves for each DNA mixture overlap (as shown in Figure A). Figure 1 (As shown in B), this indicates that although the percentage of methylated DNA converted in the DNA mixture increases, the Ct value remains unchanged, suggesting that the primers used to amplify the internal reference gene ACTB are non-methylation specific primers.

[0200] Example 2. Comparison of methylation abundance of PKNOX2 in different tissues

[0201] We used blood cells from individuals with negative colonoscopy results, adjacent and colorectal cancer tissues from individuals with colorectal cancer, and high-grade adenoma tissues from individuals with adenomas to detect the methylation abundance of the target PKNOX2 gene region, exploring the potential of the target in blood screening for colorectal cancer.

[0202] The detection includes the following steps:

[0203] 1. Obtain 10 DNA samples each from hematopoietic cells, adjacent normal tissue, high-grade adenoma tissue, and colorectal cancer tissue (i.e., a total of 40 samples). Hematopoietic cell DNA was extracted using the commercially available QIAamp DNA Mini Kit according to the manufacturer's instructions. DNA from adjacent normal tissue, high-grade adenoma tissue, and colorectal cancer tissue was extracted using the commercially available QIAamp DNA FFPE Tissue Kit according to the manufacturer's instructions.

[0204] 2. Using the commercially available bisulfite conversion reagent MethylCode TM The Bisulfite Conversion Kit is used to convert the DNA sample obtained in step 1 to sulfite to obtain the converted DNA.

[0205] 3. The transformed DNA was subjected to fluorescent PCR detection using the primer and probe sequences shown in Table 1, and the internal reference gene ACTB was also detected as a control.

[0206] The final primer concentration was 500 nM, and the final probe concentration was 100 nM. The probe was labeled with the fluorescent group VIC and the quencher group BHQ1.

[0207] The PCR reaction system is as follows: 10 μL of transformed DNA; 2.5 μL of primer and probe premix shown in Table 1; PCR reagent (PCR mix) Universal Probe qPCR Master Mix(NEB))12.5ul.

[0208] The PCR reaction conditions were as follows: 95℃ for 3 minutes; 95℃ for 30 seconds, 56℃ for 60 seconds (fluorescence acquisition), for 15 cycles. For different gene probe modifications, the corresponding fluorescence detection channel was selected, and detection was performed using an ABI 7500 Real-Time PCR instrument.

[0209] 4. Calculate and summarize the Ct values ​​of each test, and compare the distribution of Ct values ​​in blood cell, adjacent normal tissue, high-grade adenoma tissue, and colorectal cancer tissue samples.

[0210] result

[0211] The results showed that the methylation abundance of the PKNOX2 gene region in blood cells was significantly lower than that in tissue samples, and even lower in adjacent normal tissues than in high-grade adenoma tissue and colorectal cancer tissue (see [link to study]). Figure 2A It has the potential to perform blood screening. Figure 2B The internal reference gene ACTB was used as a control.

[0212] Example 3. Detection of PKNOX2 methylation using extracellular cell-free DNA

[0213] Plasma samples from 99 individuals with colorectal cancer and 116 individuals with negative colonoscopy results were analyzed using fluorescent PCR. Among the 99 plasma samples from the colorectal cancer patients, there were 20 stage I samples, 26 stage II samples, 28 stage III samples, 19 stage IV samples, and 6 samples with unclear staging information.

[0214] The detection includes the following steps:

[0215] 1. Cell-free extracellular DNA was extracted from the plasma samples described above using the commercial Qiagen QIAamp Circulating Nucleic Acid Kit.

[0216] 2. Using the commercially available bisulfite conversion reagent MethylCode TM The Bisulfite Conversion Kit converts extracted extracellular free DNA to sulfite to obtain converted DNA.

[0217] 3. The transformed DNA was diluted 10-fold and used for fluorescent PCR detection. Primers and detection probe sequences were used as shown in Table 1, and the internal reference gene ACTB was also detected simultaneously as a control. The final primer concentration was 500 nM, and the final probe concentration was 200 nM. A commercially available PCR mix reagent from NEB was selected. Universal Probe qPCR Master Mix (NEB) is used as a PCR reagent.

[0218] 4. The PCR reaction system was the same as in Example 2. The PCR reaction conditions were as follows: 95℃ for 5 minutes; 95℃ for 15 seconds, 56℃ for 40 seconds (fluorescence acquisition), 50 cycles. The corresponding fluorescence detection channel was selected based on the fluorescence modification of different gene probes.

[0219] 5. Calculate and summarize the Ct values ​​of each colorectal cancer plasma sample and colonoscopy-negative plasma sample. The results are as follows: Figure 3 As shown in the figure. The Ct value for samples without any amplified signal was set to 50.

[0220] 6. Interpretation criteria: A PKNOX2 Ct value less than or equal to 26 and an ACTB Ct value less than or equal to 25 are considered positive; a PKNOX2 Ct value greater than 26 and an ACTB Ct value less than or equal to 25 are considered negative; an ACTB Ct value greater than 25 indicates a failed test, and retesting is required.

[0221] result

[0222] Depend on Figure 3 It can be seen that the methylation level of the PKNOX2 gene region in the plasma samples of individuals with colorectal cancer is significantly higher than that in the plasma samples of individuals with negative colonoscopy. The SPSS independent samples equal variance t-test yielded a p-value of 0.018, which is less than the commonly used statistical significance threshold of 0.05.

[0223] Table 2 shows the number of positive and negative colorectal cancer samples detected using the method described in this application, and compares them with the results of colonoscopy. The results show that, out of 99 colorectal cancer samples, the method described in this application detected 62 cases (sensitivity as high as 62.6%); for 116 colonoscopy-negative samples, the method described in this application detected 100 cases (specificity as high as 86.2%). Therefore, the method described in this application has high sensitivity and specificity for colorectal cancer screening. In comparison, Septin9, currently recognized for plasma testing, has a colorectal cancer detection rate of 48.2% in clinical trial samples (see TRChurch et al., Gut.; 63:317–325 (2014)).

[0224] Table 2. Comparison of detection results and colonoscopy results using the method described in this application.

[0225] sequence list <110> Shanghai Kunyuan Health Technology Co., Ltd. <120> Methods and kits for screening colorectal cancer by detecting the methylation status of the PKNOX2 gene region. <130> 071918-8010CN02 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 4899 <212> DNA <213> Homo sapiens <400> 1 agtaggactg tgatctctgc tgagtgaatg gaggcgcttg tgggtctaca catgcctgct 60 tttgtcttcc actgtagagg gcatgtaagg agcccatttc ctccctgatg tagatgaagc 120 caccccagca ggcccaggct gaaacagccc agctggtctt tccctttata tgactgcacc 180 ccagccccca gaactctggg aactcactga gtttcttgag gactttttat tagaatgaaa 240 atagcagtgg cttagaacag gcttgcctga catttgcaaa gaagtgcttt actgacaaag 300 cctatccact ctttcctctc tccctctgcc ccaccttgaa ccagacaccc cgggaagccc 360 acagcagaaa aagctcttga gaccaggaat aactaaccag agtgtcatct gaacaccttt 420 tctggggcca gggtaatgag aattattcaa atgcaatcat aaaaatgagg ccagccaggc 480 aggaaaaggg ggagggggag tgctggtgga aggaaggatt tcttgcccgt tttaagtttc 540 atcatttctg gggagttctc ttggggtcca cagaaaatct cacctcatct cggtcccctc 600 agcaccggtg atatcttgga actcggcccc ggttgttcgc taaggaggac ttggcttggg 660 ggcgggaggg tgctccagag ttcctggggg tccaccgcac tccccatccg aggctcctcg 720 cgagcggagc cgcttctcca ggccgcccgc ctccctgccg caggagcagg cccgcgcccg 780 ccgcccgaga gcctccggta gagggagaca ctccagcggc tcctccgact tggcagcggc 840 cgggccggga gtgggtgggg gtgatggggg gtggggagaa aagggggagt ggggagaggg 900 gaggtgtgtg aagggggggt ccggggggcg ggtccctgtg ccgctgacgt cccgagcagt 960 gctgggaagt ataggctgtg ttgtcacgcc ggtgtcagtc tgatgaagat tggcatcagg 1020 taagctgtca ttcatttcca tgtcagagac gcttttgcag gcggcggcgg cggcgcggcg 1080 gcggctgctg ctgcgggcgg ctgcctcaga gcgcgtgtgt tttattccag tccccaagcc 1140 agagtattat tcattgcgac agggcaagga ggagagaggg agagagggag gcagcaggga 1200 ggagagagag ggaggcagca gggaggaggg aggcagggag cagcgaggga cggcgggagc 1260 gtgcagagag aagctgggga agcgccggga gagcgcggag cggagcagcg cgaggggcgg 1320 cgaggccggg cacggaggct gcgagagccc cgcgggccgc ccgctcccct gcccggccag 1380 cgctcagccc cgccgccgcc gccgccgccg cctcgccgcg cttgggcccg tggccggccg 1440 cgcattgtcc tcgggtgcaa ggagccgggc tgcggactcg aatcgccgcg ggcccaaccc 1500 cgtagcgggc gggcggggag ctgtgcgcca ggagcgccag gggacccgag aataggaaca 1560 ggcacgccgg cccgagcccg ggtgcagaag gctcccggcc gggcgctccg cggggagagg 1620 ctgggaaccg cgggcaggct ccaggttctc tttctcccgg cttcgggcgt ccttggggcc 1680 ggcgcttact ccgcgccacc agcctggaga tgctttccag cggctggagg cgggagcggt 1740 cccaggctgg ggccaggtga ccggaggagt cgaggagccg cggccgcggc ctggggaggc 1800 agggcgaatg agggtttgca gacggatcag tggagacagg ggaacaccgg cggggcccgg 1860 gaagccagga tccgaggggc tacacgcacg gaccctcacc cagggaggag cgagaatgtg 1920 tagggtcacc ggctttccat gattgctggg gatctttggt gaggcgggcg taggggcccg 1980 cgcgaggctt gggaatcggg agcccttctg gctcgagaac taggggatga gttcgtaaaa 2040 gagggaactg aaagcgatcg agagcggaga gctgaggggg atggcgcagg atcccgaatc 2100 tgccgctcaa agtttgcatt tctttcgggt taggagacgg gctttcctgg ctcccgatcc 2160 ccaggaagaa acgagcgaaa tgggccgtcc tttcccgggg ctcttcacgg gggagccggg 2220 ggtttccgcg cggtggggag actcgggttg ggaattgagg ggtaggggct tgtggatcgg 2280 cctgaattag ggctgggttt taggaccagt ctagagttcg gtttatagga tccagactgt 2340 ttacggaatc gggatcgagg ggccgataag tagtttacac gccggccaga gcagaggggct 2400 ggaggtcgga gttggggggct ggaggaacgg gtggcgtttt taggattcag taacagggatc 2460 acagcttttt cttgtggtgg aagctattgg aatttgggga gggtagcacg aggggtcctg 2520 cagctccgcg tgtgaaaaag cgtttaggta ggcgatgaaa gtagttgatc tgagccatgg 2580 caggcgagcc ccgaattttt gctgcttccc cctgaaagtg tttctttagg aggagaggac 2640 ttgggccaca caggacccgg tcctaagaga gcgattccgg gaagcggaca gatcgaagag 2700 accttctggg cgaagcggca gggcagcctc gcggggctgg gagtggatct gaggtcccga 2760 cccaggcggc tcggagtgct ccaggagcca cctgggtctg cgggcgcagc gcggcggggc 2820 gggagcggtg gcccgcaggg gccgcggcct gcgatgaagg ccggggggca gcgctagcag 2880 cgaggtgcca cagtgggccg aggagtctgg gctgtggccc agggtaggac cggctcaaac 2940 tccagtgccc tgattggagc cgcttcctgt gcttacccgc gccggactga gaagcccaca 3000 aacccggcct ttggtgcgcc cgggggagga ggaagcttgg agtgccctac tgtcatctct 3060 cctgtccgga gacttcagga tattagcttt tcgggttca aatgctctat aaccggtggc 3120 agccaaaagc ttcgcatttt aggcagttta gacgatcctc cccgccccaa atctgagaat 3180 gatggtgttc aaacataaca cggtgtatta cccaaagccc ctgcccttgc cctcctgctc 3240 tctctgccgt tctggccttc ggaggcccca ggaccttggt ggaagcgggg agggagggtc 3300 ccgtttctgg tctcataatc tcccactcgg gctgcatccc catagcttgg gcctagctgt 3360 ggggtgtggg gtgggaaggg gtcccgacca gggaggtagg ttattctctg ggcatctaga 3420 gaaagagtgt gtgtgggggg tgggagcggg ggggaggagg gcattgaggg gaggaagatt 3480 tgagatttcc tcctcttaat attaaaccca ccatcccagc cccctgcacc cctggcaggg 3540 agagccagac agcccttaca ggacacggaa gacgcacgac ccacgaccca cgagtggcct 3600 cggcggcccg gggcgcggcg actgccgccg cggagctcca aggctgttgt tttgtaggct 3660 tggaattcgt gagagctctc tcccgctgag agcccgaggg gggagggagc cgtgacccca 3720 cggagactcc ctggtcaccg cctcccctttt gtgagcggga aaggcttgaa agccaagaga 3780 aagagaggct gctcagaata aggggcaggg ggttaccgga gggagatggg cccgaccacc 3840 gctcacagta aaatgcctcg tgcgaattgc actgaagtat acccaacttg agactggctg 3900 tttatgatc ctctctggga gtttactgct ctctgctttt aagtctatag attgctttaa 3960 gttaatgaaa gtgctgcttt caaaaggggc ttttattgtg tggcagtggc aaatccagta 4020 cctcccctt actcttccaa aatgggcctg tttaaaaaga gggagcttgc tggatcccac 4080 atggcagagc ttgggggagg gtgccatgtg acaatgagaa ttaataaaag ttaatgtcca 4140 agtgattctg aaagacgagt ttgcttcttt tccagtccgg tttcatctgt tcattctcgt 4200 gttgccttg ggctttcact gagtctgcct ttaaatgttg cttggtgcct cccagcagc 4260 ctctcccagc attgctttag gtaggcctga cccctgttca gttaaaccca aactggattt 4320 caggatttca gcccagctgc caattcaaac tgcaactaaa atatttactt gggactgggg 4380 gaggggcttt gcctcgcgtt tagtaagata aatgtgttta aatgcctggg aaccttgaga 4440 aggaacaagg caggctgttt ttcagtgctt tttgcttttt caatctgttt ggcacaaaag 4500 gtgagatgac aaggtttttg actaggtcct aacacagaaa ctgcagtggg ctcaggctgt 4560​​​​​​​​​​​​​​​​​​​​​​​cagcacagag ggccagagcc agtggtgtgg tgcttctaga ctcagtcggg tgccagccct 60 agctctagta tgccctggtt gtgtgactgg ggaccaattc ctatttttcc ttgggcctca 120 gtttccccag aaatgcaatg gggaggacat tgccctaccc ttcaaggaca tgcctcggtt 180 cataaaggag acctcctctc tctagagcca agctccccca ccccaccccc atcctgccag 240 ggcaggctca gccgctccct tcccgtccct agcagtgggg gaagccgcat gaggaggagc 300 ggcgtgggag caaatggagg ggatctgctg gcgggcctag tttaggcaaa cagcagggac 360 tgctggcgac ggctccagcc cccactcccg cggtaacagc tgcagtccag ctgctggcat 420 ccctgctggg ggagcgtgct aaacctctcc ggggtcagca ctgtggtatg tcggtttcca 480 tagcaaccag ccaacgggcc actttctcag aacaattccc ctactcagcc gggttctgga 540 cttagctcag cccaagggca tgtggctgag gagcagcgcg ggagggggag agctgccgag 600 ggccagactg ggccccacag aacgtggcta tctcagtccg gtcttcgcac ggagggcgtg 660 gagccgagga tgagccatgc agactcttcc ggcgctggct ctccctggcc tctctctgtg 720 ccacgccgcc cccaccaccc gtccacagac tggccagctt ggcccagttg gaagaagtgt 780 caggtctttg tctgaatctg gagaacagct gccttggctg ggttcagagc tgggaggaca 840 aagtcctgga atggcgtata agggggtgcg gggagggggc ttttcctggc ttccttctcc 900 gcacccttcc atccagctct tgcaggacca tgggaatccc agtgacctca ggggatgggg 960 ggctaatcag tcctccccca catttagagg gtgggagagg atcacctttc tcaagcccct 1020 acaagagtta actcc 1035 <210> 3 <211> 419 <212> DNA <213> Homo sapiens <400> 3 acataccgta tacattcatt tgctaatctt ttgatacagg actaaggtct ttttctaatt 60 gttagtcatc ttccttgcat aaacctctcc tatactgtaa catctataaa ttccacctca 120 atagaaagtg gaattacagg ccgggtgcgg tggctcacgc ctgtaatccc agcactttgg 180 gaggccgagg cgggcggatc acgaggtcag gagatcgaga ccatcccggc taaaacggtg 240 aaaccccgtc tctactaaaa atacaaaaaa ttagccgggc gtggtggcgg gcgcctgtag 300 tcccagctac ttgggaggct gaggcaggag aatggcgtga acccgggagg cggagcttgc 360 agtgagccga gatcccgcca ctgcactcca gcctgggcga cagagcgaga cgccgtctc 419 <210> 4 <211> 4899 <212> DNA <213> Synthetic sequence <220> <223> Synthetic <400> 4 agtaggattg tgatttttgt tgagtgaatg gaggcgtttg tgggtttata tatgtttgtt 60 tttgtttttt attgtagagg gtatgtaagg agtttatttt ttttttgatg tagatgaagt 120 tattttagta ggtttaggtt gaaatagttt agttggtttt tttttttata tgattgtatt 180 ttagttttta gaattttggg aatttattga gttttttgag gattttttat tagaatgaaa 240 atagtagtgg tttagaatag gtttgtttga tatttgtaaa gaagtgtttt attgataaag 300 tttatttatt tttttttttt tttttttgtt ttattttgaa ttagatattt cgggaagttt 360 atagtagaaa aagtttttga gattaggaat aattaattag agtgttattt gaatattttt 420 tttggggtta gggtaatgag aattatttaa atgtaattat aaaaatgagg ttagttaggt 480 aggaaaaggg ggagggggag tgttggtgga aggaaggatt ttttgttcgt tttaagtttt 540 attatttttg gggagttttt ttggggttta tagaaaattt tattttattt cggttttttt 600 agtatcggtg atattttgga attcggtttc ggttgttcgt taaggaggat ttggtttggg 660 ggcgggaggg tgttttagag tttttggggg tttatcgtat tttttattcg aggtttttcg 720 cgagcggagt cgttttttta ggtcgttcgt ttttttgtcg taggagtagg ttcgcgttcg 780 tcgttcgaga gttttcggta gagggagata ttttagcggt tttttcgatt tggtagcggt 840 cgggtcggga gtgggtgggg gtgatggggg gtggggagaa aagggggagt ggggagaggg 900 gaggtgtgtg aagggggggt tcggggggcg ggtttttgtg tcgttgacgt ttcgagtagt 960 gttgggaagt ataggttgtg ttgttacgtc ggtgttagtt tgatgaagat tggtattagg 1020 taagttgtta tttattttta tgttagagac gtttttgtag gcggcggcgg cggcgcggcg 1080 gcggttgttg ttgcgggcgg ttgttttaga gcgcgtgtgt tttattttag tttttaagtt 1140 agagtattat ttattgcgat agggtaagga ggagagaggg agagagggag gtagtaggga 1200 ggagagagag ggaggtagta gggaggaggg aggtagggag tagcgaggga cggcgggagc 1260 gtgtagagag aagttgggga agcgtcggga gagcgcggag cggagtagcg cgaggggcgg 1320 cgaggtcggg tacggaggtt gcgagagttt cgcgggtcgt tcgttttttt gttcggttag 1380 cgtttagttt cgtcgtcgtc gtcgtcgtcg tttcgtcgcg tttgggttcg tggtcggtcg 1440 cgtattgttt tcgggtgtaa ggagtcgggt tgcggattcg aatcgtcgcg ggtttaattt 1500 cgtagcgggc gggcggggag ttgtgcgtta ggagcgttag gggattcgag aataggaata 1560 ggtacgtcgg ttcgagttcg ggtgtagaag gttttcggtc gggcgtttcg cggggagagg 1620 ttgggaatcg cgggtaggtt ttaggttttt tttttttcgg tttcgggcgt ttttggggtc 1680 ggcgtttatt tcgcgttatt agtttggaga tgttttttag cggttggagg cgggagcggt 1740 tttaggttgg ggttaggtga tcggaggagt cgaggagtcg cggtcgcggt ttggggaggt 1800 agggcgaatg agggtttgta gacggattag tggagatagg ggaatatcgg cggggttcgg 1860 gaagttagga ttcgaggggt tatacgtacg gatttttatt tagggaggag cgagaatgtg 1920 tagggttatc ggtttttat gattgttggg gatttttggt gaggcgggcg taggggttcg 1980 cgcgaggttt gggaatcggg agtttttttg gttcgagaat taggggatga gttcgtaaaa 2040 gaggggaattg aaagcgatcg agagcggaga gttgaggggg atggcgtagg atttcgaatt 2100 tgtcgtttaa agtttgtatt tttttcgggt taggagacgg gtttttttgg ttttcgattt 2160 ttaggaagaa acgagcgaaa tgggtcgttt tttttcgggg ttttttacgg gggagtcggg 2220 ggttttcgcg cggtggggag attcgggttg ggaattgagg ggtaggggtt tgtggatcgg 2280 tttgaattag ggttgggttt taggatagt ttagagttcg gtttatagga tttagattgt 2340 ttacggaatc gggatcgagg ggtcgataag tagtttatac gtcgggtaga gtagagggtt 2400 ggaggtcgga gttggggtt ggaggaacgg gtggcgtttt taggatttag taataggatt 2460 atagttttt tttgtggtgg aagttattgg aatttgggga gggtagtacg aggggttttg 2520 tagtttcgcg tgtgaaaaag cgtttaggta ggcgatgaaa gtagttgatt tgagttatgg 2580 taggcgagtt tcgaattttt gttgtttttt tttgaaagtg tttttttagg aggagaggat 2640 ttgggttata taggattcgg ttttaagaga gcgatttcgg gaagcggata gatcgaagag 2700 attttttgg cgaagcggta gggtagtttc gcggggttgg gagtggattt gaggtttcga 2760 tttaggcggt tcggagtgtt ttaggagtta tttgggtttg cgggcgtagc gcggcggggc 2820 gggagcggtg gttcgtaggg gtcgcggttt gcgatgaagg tcgggggta gcgttagtag 2880 cgaggtgtta tagtgggtcg aggagtttgg gttgtggttt aggtaggat cggtttaaat 2940 tttagtgttt tgattggagt cgttttttgt gtttattcgc gtcggattga gaagtttata 3000 aattcggttt ttggtgcgtt cggggagga ggaagtttgg agtgttttat tgttatttt 3060 tttgttcgga gattttaggga tattagttt tcgggtttta aatgttttat aatcggtggt 3120 agttaaaagt ttcgtatttt aggtagttta gacgattttt ttcgttttaa atttgagaat 3180 gatggtgttt aaatataata cggtgtatta tttaaagttt ttgttttgt ttttttgttt 3240 tttttgtcgt tttggttttc ggaggtttta ggattttggt ggaagcgggg agggagggtt 3300 tcgtttttgg ttttataatt ttttattcgg gttgttttt tatagtttgg gtttagttgt 3360 ggggtgtggg gtgggaaggg gtttcgatta gggaggtagg ttattttttg ggtatttaga 3420 gaaagagtgt gtgtgggggg tgggagcggg ggggaggagg gtattgaggg gaggaagatt 3480 tgagattttt tttttttaat attaaattta ttattttagt tttttgtatt tttggtaggg 3540 agagttagat agtttttata ggatacggaa gacgtacgat ttacgattta cgagtggttt 3600 cggcggttcg gggcgcggcg attgtcgtcg cggagtttta aggttgttgt tttgtaggtt 3660 tggaattcgt gagagttttt tttcgttgag agttcgaggg gggagggagt cgtgatttta 3720 cggagatttt ttggttatcg tttttttttt gtgagcggga aaggtttgaa agttaagaga 3780 aagagaggtt gtttagaata aggggtaggg ggttatcgga gggagatggg ttcgattatc 3840 gtttatagta aaatgtttcg tgcgaattgt attgaagtat atttaatttg agattggttg 3900 ttttatgatt ttttttggga gtttattgtt ttttgttttt aagtttatag attgttttaa 3960 gttaatgaaa gtgttgtttt taaaaggggt ttttattgtg tggtagtggt aaatttagta 4020 tttttttttt atttttttaa aatgggtttg tttaaaaaga gggagtttgt tggattttat 4080 atggtagagt ttgggggagg gtgttatgtg ataatgagaa ttaataaaag ttaatgttta 4140 agtgattttg aaagacgagt ttgttttttt tttagttcgg ttttatttgt ttattttcgt 4200 gttgtttttg ggtttttatt gagtttgttt ttaaatgttg tttggtgttt ttttagtagt 4260 tttttttagt attgttttag gtaggtttga tttttgttta gttaaattta aattggattt 4320 taggatttta gtttagttgt taatttaaat tgtaattaaa atatttattt gggattgggg 4380 gaggggttt gtttcgcgtt tagtaagata aatgtgttta aatgtttggg aattttgaga 4440 aggaataagg taggttgttt tttagtgttt tttgttttt taatttgttt ggtataaaag 4500 gtgagatgat aaggtttttg attaggtttt aatatagagaaa ttgtagtggg tttaggttgt ggttttgatc gtatgttata attatttaaa ttgtggttag ttgtttaaag tttaaagttt 4620 ttttaagttg ggttttatta attttatatt agttgtttgg aagtttagta gtaataaatt 4680 gtataaatat atttgaagta agttagtgat gaaataagta gaattattgt tttgtgagtg 4740 gagaattttg aatttatgga gttttagttt aattggaaat atattttttt ttttttggtt 4800 ttgaagttta tgggtgtatt agagtgtgtg ttaaattttt ttttgttatg tttattttta 4860 atttgggttg taatttatta gagattaagg tatgtttag 4899 <210> 5 <211> 1035 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 5 tagtatagag ggttagagtt agtggtgtgg tgtttttaga tttagtcggg tgttagtttt 60 agttttagta tgttttggtt gtgtgattgg ggattaattt ttattttttt ttgggtttta 120 gtttttttag aaatgtaatg gggaggatat tgttttattt tttaaggata tgtttcggtt 180 tataaaggag attttttttt tttagagtta agttttttta ttttattttt attttgttag 240 ggtaggttta gtcgtttttt tttcgttttt agtagtgggg gaagtcgtat gaggaggagc 300 ggcgtgggag taaatggagg ggatttgttg gcgggtttag tttaggtaaa tagtagggat 360 tgttggcgac ggttttagtt tttattttcg cggtaatagt tgtagtttag ttgttggtat 420 ttttgttggg ggagcgtgtt aaattttttc ggggttagta ttgtggtatg tcggttttta 480 tagtaattag ttaacgggtt atttttttag aataattttt ttatttagtc gggttttgga 540 tttagtttag tttaagggta tgtggttgag gagtagcgcg ggagggggag agttgtcgag 600 ggttagattg ggttttatag aacgtggtta ttttagttcg gttttcgtac ggagggcgtg 660 gagtcgagga tgagttatgt agattttttc ggcgttggtt ttttttggtt tttttttgtg 720 ttacgtcgtt tttattattc gtttatagat tggttagttt ggtttagttg gaagaagtgt 780 taggtttttg tttgaatttg gagaatagtt gttttggttg ggtttagagt tgggaggata 840 aagttttgga atggcgtata agggggtgcg gggagggggt tttttttggt tttttttttc 900 gtattttttt atttagtttt tgtaggatta tgggaatttt agtgatttta ggggatgggg 960 ggttaattag ttttttttta tatttagagg gtgggagagg attatttttt ttaagttttt 1020 ataagagtta atttt 1035 <210> 6 <211> 419 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 6 atatatcgta tatatttatt tgttaatttt ttgatatagg attaaggttt ttttttaatt 60 gttagttatt ttttttgtat aaattttttt tatattgtaa tatttataaa ttttatttta 120 atagaaagtg gaattatagg tcgggtgcgg tggtttacgt ttgtaatttt agtattttgg 180 gaggtcgagg cgggcggatt acgaggttag gagatcgaga ttatttcggt taaaacggtg 240 aaatttcgtt tttattaaaa atataaaaaa ttagtcgggc gtggtggcgg gcgtttgtag 300 ttttagttat ttgggaggtt gaggtaggag aatggcgtga attcgggagg cggagtttgt 360 agtgagtcga gatttcgtta ttgtatttta gtttgggcga tagagcgaga cgtcgtttt 419 <210> 7 <211> 4899 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 7 aataaaacta taatctctac taaataaata aaaacgctta taaatctaca catacctact 60 tttatcttcc actataaaaa acatataaaa aacccatttc ctccctaata taaataaaac 120 caccccaaca aacccaaact aaaacaaccc aactaatctt tccctttata taactacacc 180 ccaaccccca aaactctaaa aactcactaa atttcttaaa aactttttat taaaataaaa 240 atacataa cttaaaaaaaacttacctaa cattacaaa aaatacttt actaaaaaaa 300 cctatccact ctttcctctc tcctacctacc ccaccttaaa ccaacaccc cgaaaaaccc 360 aaaaaaa aaacttta aaccaaaaat aactaccaa atatcatct aaacacctttt 420 tctaaaacca aaataaaa aattattcaa atacaatcat aaaaaaaaaccaaac 480 aaaaaaaaaaaaaaaaaaaaaaaaaatt tcttaccgt tttaaatttc 540 atcatttcta aaaaattctc ttaaaatcca caaaaatct cacctcatct cgatccccctc 600 aacaccgata atatcttaa actcgacccc gattattcgc taaaaaaac ttaacttaaa 660 aacgaaaaaa tactccaaaa ttcctaaaaa tccacccac tcccatccg aaactccctcg 720 cgaacgaaac cgctctcca aaccgcccgc cccctaccg caaaaaaaa cccgcgcccg 780 ccgcccgaaa acctccgata aaaaaaaaaaca ctccacgac tcctccgact taacaacgac 840 cgaaccgaaa attaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaa 900 aaaatatata aaaaaaaaat ccgaaaaacg aatccctata ccgctaacgt cccgaacaat 960 actaaaaaat ataaactata ttatcacgcc gatatcaatc taataaaaat taacatcaaa 1020 taaactatca ttcatttcca tatcaaaaac gcttttacaa acgacgacga cgacgcgacg 1080 acgactacta ctacgaacga ctacctcaaa acgcgtatat tttattccaa tccccaaacc 1140 aaaatattat tcattacgac aaaacaaaaa aaaaaaaaaa aaaaaaaaaa acaacaaaaa 1200 aaaaaaaaaa aaaaacaaca aaaaaaaaaa aaacaaaaaa caacgaaaaa cgacgaaaac 1260 gtacaaaaaa aaactaaaaa aacgccgaaa aaacgcgaaa cgaaacaacg cgaaaaacga 1320 cgaaaccgaa cacgaaaact acgaaaaccc cgcgaaccgc ccgctcccct acccgaccaa 1380 cgctcaaccc cgccgccgcc gccgccgccg cctcgccgcg cttaaacccg taaccgaccg 1440 cgcattatcc tcgaatacaa aaaaccgaac tacgaactcg aatcgccgcg aacccaaccc 1500 cgtaacgaac gaacgaaaaa ctatacgcca aaaacgccaa aaaacccgaa aataaaaaca 1560 aacacgccga cccgaacccg aatacaaaaa actcccgacc gaacgctccg cgaaaaaaaa 1620 ctaaaaaccg cgaacaaact ccaaattctc tttctcccga cttcgaacgt ccttaaaacc 1680 gacgcttact ccgcgccacc aacctaaaaa tactttccaa cgactaaaaa cgaaaacgat 1740 ccccaaactaa aaccaaataa ccgaaaaaat cgaaaaaccg cgaccgcgac ctaaaaaaac 1800 aaaacgaata aaaatttaca aacgaatcaa taaaaacaaa aaaacaccga cgaaacccga 1860 aaaaccaaaa tccgaaaaac tacacgcacg aaccctcacc caaaaaaaaa cgaaaatata 1920 taaaatcacc gactttccat aattactaaa aatctttaat aaaacgaacg taaaaacccg 1980 cgcgaaactt aaaaatcgaa aacccttcta actcgaaaac taaaaaataa attcgtaaaa 2040 aaaaaaacta aaaacgatcg aaaacgaaaa actaaaaaaa ataacgcaaa atcccgaatc 2100 taccgctcaa aatttacatt tctttcgaat taaaaaacga actttcctaa ctcccgatcc 2160 ccaaaaaaaa acgaacgaaa taaaccgtcc tttcccgaaa ctcttcacga aaaaaccgaa 2220 aatttccgcg cgataaaaaa actcgaatta aaaattaaaa aataaaaact tataaatcga 2280 cctaaattaa aactaaattt taaaaccaat ctaaaattcg atttataaaa tccaaactat 2340 ttacgaaatc gaaatcgaaa aaccgataaa taattacac gccgaccaaa acaaaaaact 2400 aaaaatcga ataaaaact aaaaaacga ataacgtttt taaattcaa taacaaaatc acaacttttt cttatataa aaactattaa aatttaaaaa aatacacg aaaaatccta caactccgcg tataaaaaaa cgtttaaata aacgataaaa ataattaatc taaccataa 2640. caaacgaacc ccgaattttt actacttccc cctaaaaata tttctttaa aaaaaaaac ttaaccaca caaaacccga tcctaaaaaa aacgattccga aaaacgaaca aatcgaaaaa accttctaaa cgaaacgaca aaacaacctc gcgaactaa aataatct aaaatcccga cccaaacgac tcgaaatact ccaaaaacca cctaaatcta cgaaacgcaac gcgacgaac gaaaacgata acccgcaaaa accgcgacct acgataaaaa ccgaaaaca acgctaacaa cgaaatacca caataaccg aaaaatctaa actataacc aaaataaac cgactcaaac tccaataccc tattaaaac cgcttcctat acttacccgc gccgaactaa aaaacccaca aacccgacct ttaatacgcc cgaaaaaaaa aaaaacttaa aataccctac tatcatctct cctatccgaa aacttcaaaa tattaacttt tcgaatttca aatactctat aaccgataac aaccaaaaac ttcgcatttt aaacaattta aacgatccctc cccgccccaa atctaaaaat 3180 aaatattc aaacaataca cgatatatta cccaaaaccc ctacccttac cctcctactc 3240 tctctaccgt tctaccttc gaaaacccca aaaaacgaaa aaaaaaatc 3300 ccgtttctaa tctcataatc tcccactcga actacatccc cataactaa acctaacta 3360 aaaatataaaaaaaaaa atcccgacca aaaaaaa ttattctcta aacatctaaa 3420 aaaaaaaaaaaaaaaaaaaacgaaaaaaaaaaaaaaaaaaaaaaatt 3480 taaaatttcc tcctcttaat attackaaccca cccctacacc cctacaaaa 3540 aaaaccaaac aacccttaca aaacacgaaa acgcacgac ccacgaccca cgaataacct 3600 cgacgacccg aaacgcgacg actaccgccg cgaaactcca aaactattat tttataaact 3660 taaaattcgt aaaaactctc tcccgctaaa acccgaaa aaaaaaaaac cgtaacccca 3720 cgaaaactcc ctaatcaccg cctcccctttt aaaacttaa aaaaaaaaaa 3780 aaaaaaaaact attaccgaa aaaaaaaa cccgaccacc 3840 gctcacaata aaataccctcg tacgaattac actaaaatat acccaactta aaactaacta 3900 ttttaatc ctctctaaaa atttactact cttactttt aaatctaa attactttaa 3960 attaataaaa attacktacttt haaaaaaaac ttttatta taacaatac aaatccaata 4020 cctccccttt actctccaa aaaaccta tttaaaaaaa aaaaacttac taaatcccac 4080 ataaaaac ttaaaaaaaa ataccatata acataaaa ttaaaaaa ttaatatcca 4140 aaattcta aaaaacgaat tactcttt tccaatccga ttcatctat tcattctcgt 4200 attaccttta aactttcact aaatctacct ttaatatta cttaatacct cccacaac 4260 ctctcccaac attactttaa aaaccta cccctattca attackaccca aactaaattt 4320 caaaatttca acccactac cattcaac tacaactaa attackttt aaactaaaa 4380 aaaaaacttt acctcgcgtt taaaaata attattatta atacctaaaaccttaaaa 4440 aaaaaaaaa caactattt ttcatactt ttcactttt caatcttt aacacaaaaa 4500 aaaatac aaaattttta actaaatcct aacaaaaa ctacataaa ctcaactat 4560 aactttaacc gcatatcata accatttaaa ctataattaa ttattcaaaa ctcaaaactt 4620 ctccaaacta aatttcatta actctacacc aattacctaa aaattcaata acaataaatt 4680 atataaacac atttaaaata aattaataat aaaataaata aaattactat cttataaata 4740 aaaaatccta aattcataaa attctaactt aactaaaaac atacctcctc tcccttaacc 4800 ttaaaattta taaatacacc aaaatatata ttaaatttct ttctaccata tccatcttca 4860 acctaaacta taactcatta aaaaccaaaa catacctaa 4899 <210> 8 <211> 1035 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 8 caacacaaaa aaccaaaacc aataatataa tacttctaaa ctcaatcgaa taccaaccct 60 aactctaata taccctaatt atataactaa aaaccaattc ctatttttcc ttaaacctca 120 atttccccaa aaatacaata aaaaaaacat taccctaccc ttcaaaaaca tacctcgatt 180 cataaaaaaa acctcctctc tctaaaacca aactccccca ccccaccccc atcctaccaa 240 aaaaactca accgctccct tcccgtccct aacataaaa aaaaccgcat aaaaaaaaac 300 gacgtaaaaaaaaaa aaatctaacta acgaaccta ttaaaaaaaaaaaac 360 tactaacgac gactccaacc cccactcccg cgatacaac tacaatccaa ctactaacat 420 ccctactaaa aaacgtact aaacctctcc gaatcaca ctatatata tcgatttcca 480 taacaaccaaccgaacc actttctcaaacaatcccc ctactcaacc gattctaaa 540 cttaactcaa cccaaaaa tataactaa aaaaaaaaaaaaaaaaaaaaaactaccgaa 600 aaccaaacta aaccccaca aacgtaacta tctcaatccg atctcgcac gaaaaacgta 660 aaaccgaaaa taaaccatac aactcttcc gacgctact ctcctacc tctctctata 720 ccacgccgcc cccacccc gtccacaac ccaccactt ccaccatta aaaaaaatat 780 aaatcttta tctaaatcta aaaaaacact accttaacta attcaaac taaaaaaaca 840 aaatcctaaa ataacgtata aaaaaatacg aaaaaaaac ttttcctaac ttccttctcc 900 gcacccttcc atccaactct tacaaaacca taaaaatccc ataacctca aaaaaayaa 960 aactaatcaa tcctccccca catttaaaaa ataaaaaaaa atcacctttc tcaaacccct 1020 acaaaaatta actcc 1035 <210> 9 <211> 419 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 9 acataccgta tacattcatt tactaatctt ttaatacaaa actaaaatct ttttctaatt 60 attaatcatc ttccttacat aaacctctcc tatactataa catctataaa ttccacctca 120 ataaaaaata aaattacaaa ccgaatacga taactcacgc ctataatccc aacactttaa 180 aaaaccgaaa cgaacgaatc acgaaatcaa aaaatcgaaa ccatcccgac taaaacgata 240 aaaccccgtc tctactaaaa atacaaaaaa ttaaccgaac gtaataacga acgcctataa 300 tcccaactac ttaaaaaact aaaacaaaaa aataacgtaa acccgaaaaa cgaaacttac 360 aataaaccga aatcccgcca ctacactcca acctaaacga caaaacgaaa cgccgtctc 419 <210> 10<00009​​​​​​​​tggcaggcga gccccgaatt tttgctgctt ccccctgaaa gtgtttcttt aggaggagag 60 gacttgggcc acacaggacc cggtcctaag agagcgattc cgggaagcgg acagatcgaa 120 gagaccttct gggcgaagcg gcagggcagc ctcgcggggc tgggagtgga tctgaggtcc 180 cgacccaggc ggctcggagt gctccaggag ccacctgggt ctgcgggcgc agcgcggcgg 240 ggcgggagcg gtggcccgca ggggccgcgg cctgcgatga aggccggggg gcagcgctag 300 cagcgaggtg ccacagtggg ccgaggagtc tgggctgtgg cccagggtag gaccggctca 360 aactccagtg ccctgattgg agccgcttcc tgtgcttacc cgcgccggac tgagaagccc 420 acaaacccgg cctttggtgc gcccggggga ggaggaagct tggagtgccc tactgtcatc 480 tctcctgtcc ggagacttca ggatattagc ttttcgg 517 <210> 11 <211> 517 <212> DNA <213> Synthetic sequence <220> <223> Synthetic <400> 11 tggtaggcga gtttcgaatt tttgttgttt ttttttgaaa gtgttttttt aggaggagag 60 gatttgggtt atataggatt cggttttaag agagcgattt cgggaagcgg atagatcgaa 120 gagatttttt gggcgaagcg gtagggtagt ttcgcggggt tgggagtgga tttgaggttt 180 cgatttaggc ggttcggagt gttttaggag ttatttgggt ttgcgggcgt agcgcggcgg 240 ggcgggagcg gtggttcgta ggggtcgcgg tttgcgatga aggtcggggg gtagcgttag 300 tagcgaggtg ttatagtggg tcgaggagtt tgggttgtgg tttagggtag gatcggttta 360 aattttagtg ttttgattgg agtcgttttt tgtgtttatt cgcgtcggat tgagaagttt 420 ataaattcgg tttttggtgc gttcggggga ggaggaagtt tggagtgttt tattgttatt 480 ttttttgttc ggagatttta ggatattagt ttttcgg 517 <210> 12 <211> 517 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 12 taacaaacga accccgaatt tttactactt ccccctaaaa atatttcttt aaaaaaaaaa 60 aacttaaacc acacaaaacc cgatcctaaa aaaacgattc cgaaaaacga acaaatcgaa 120 aaaaccttct aaacgaaacg acaaaacaac ctcgcgaaac taaaaataaa tctaaaatcc 180 cgacccaaac gactcgaaat actccaaaaa ccacctaaat ctacgaacgc aacgcgacga 240 aacgaaaacg ataacccgca aaaaccgcga cctacgataa aaaccgaaaa acaacgctaa 300 caacgaaata ccacaataaa ccgaaaaatc taaactataa cccaaaataa aaccgactca 360 aactccaata ccctaattaa aaccgcttcc tatacttacc cgcgccgaac taaaaaaccc 420 acaaacccga cctttaatac gcccgaaaaa aaaaaaaact taaaataccc tactatcatc 480 tctcctatcc gaaaacttca aaatattaac ttttcga 517 <210> 13 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 13 gttttaggag ttatttgggt ttgc 24 <210> 14 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 14 actataacac ctcgctacta acgct 25 <210> 15 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 15 cggtggttcg taggggtcgc g 21 <210> 16 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 16 gtgatggagg aggtttagta agtt 24 <210> 17 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 17 ccaataaaac ctactcctcc cttaa 25 <210> 18 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 18 accacccaccc aacacacaat aacaaacaca 30

Claims

1. Use of reagents for detecting the methylation status of the PKNOX2 gene region in the preparation of kits for methods of diagnosing colorectal tumors in individuals. The method includes detecting the methylation status of the PKNOX2 gene region in a biological sample from the individual, and comparing the detected methylation status of the PKNOX2 gene region with the normal methylation status of the PKNOX2 gene region. The higher methylation state of the PKNOX2 gene region detected in the biological sample from the individual, relative to the normal methylation state, indicates that the individual has colorectal cancer. The PKNOX2 gene region is selected as follows: a) The sequence of the PKNOX2 gene region is Hg19 coordinate chr11:125036431-125036547 and its upstream and downstream 200bp sequences; Or b) The corresponding regions listed in a) above after bisulfite conversion.

2. The use according to claim 1, characterized in that, The detection of methylation status of the PKNOX2 gene region described herein includes determining the methylation status of cytosine residues at one or more CpG sites in the PKNOX2 gene region in a biological sample from the individual.

3. The use as described in any one of claims 1-2, wherein the reagent for detecting the methylation status of the PKNOX2 gene region is a group of oligonucleotides, wherein the oligonucleotides are: SEQ ID NOs: 13, 14 and 15.

Images