TW201038739A - Cancer screening method - Google Patents

Abstract

A method for screening cancer comprises the following steps: (1) providing a test specimen; (2) detecting the methylation state of the CpG sequence in at least one target gene within the genomic DNA of the test specimen, wherein the target genes is consisted of PTPRR, ZNF582, PDE8B and DBC1; and (3) determining whether there is cancer or cancerous pathological change in the specimen based on the presence or absence of the methylation state in the target gene; wherein method for detecting methylation state is methylation-specific PCR (MSP), quantitative methylation-specific PCR (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (DHPLC), and pyrosequencing.

Description

201038739 - VI. Description of the invention: [Technical field to which the invention pertains] The present invention is directed to a method of abbreviating, and _ refers to a method for cancer screening of a biomarker. [Prior Art] Cervical cancer is one of the leading causes of death among women in the world and in Taiwan. According to the World Health Day in 2002 and the uterus cancer of the world, it is the cause of cancer death in women worldwide. Two, second only to breast cancer; Dinglin is the best method for cervical uterine touch. There are two main ways to use cervical cancer screening. One is the most common Pap smear (4) Smear) 'The other is HPV testing, · Pap smear is to remove the secretions from the uterine neck'. Microscopic observation of the epithelial cells in the epithelial cells, whether there is cancerous lesions to produce 'in the early side of the uterus And Qing remuneration is a method of polymerase chain reaction (RT-PCR) or Hybrid Capture. The presence of human papilloma virus (HPV) virus is present in the sample. However, because of the Pap smear, it is necessary to rely on the physician's sampling and the examiner/pathologist to interpret the smear', in addition to prone to the high false negative rate and delay the diagnosis and treatment of precancerous lesions. Moreover, the required human quality and cost are too much. ' k has difficulty in promotion for many developing countries; on the other hand, HPVtesting has high sensitivity, but it is highly sensitive. It is easy to cause high false positive rate, which not only makes patients worry, but also wastes many medical resources 201038739 source in the tracking test of false positive patients; therefore, how to improve the accuracy of cervical cancer test methods and Convenience is one of the important topics to promote cervical cancer testing. Genomic deletions are considered to be important factors in tumor formation. For a long time, we have been accustomed to the idea that the coding in the genome depends on the four bases of ATCG. Knudson proposed the double-invasive theory as early as 1975. Two-hittheory), indicating that mutations or deletions associated with some homologous tumor suppressor genes may cause or contribute to cancer; however, other phenotype-inducing messages may be present in modified bases. In 5-methylcytosine, 5-methylcytosine is found in the palindromic sequence 5'-CpG-3, which is found in mammalian cells, except for some called "CpG islands" in mammalian cells ( Outside the region of CpGislands, CGIs, most CpG dinucleotide pairs are methylated, and CpG islands refer to a large number of GC- and CpG in a region of approximately 1 base pair (1 Kb). -, usually located near the gene, and found near the promoter of a widely expressed gene. The thiolation of cytosine occurs after DNA synthesis, and a sulfhydryl group is transferred from the s-adenosylmethionine (SAM) to the 5th carbon of cytosine. In position, the enzyme reaction is carried out by DNA methyltransferase (DNMTs), which is the major thiol transferase of mammals and is responsible for post-replicative restoration of the hemimethylation site. Thiolation, known as maintenance methylation (negative methylati〇n); conversely, DNMT3A and DNMT3B are thought to be primarily responsible for the new position of methylation, performing a step called de novo methylation .

CpG dinuclear acid loss of methylation, meaning general low methylation, is the first epigenetic abnormality in cancer cells; although 201038739 - and, in the past Studies during the year have shown that site-specific hypermethylation in specific locations (eg, some tumor suppressor genes) is associated with loss of function, which may provide selective advamages during cancer generation; High methylation of CpG islands on the promoter region can be induced by chromatin modification by histone modification _〇ne m〇dificati〇n) followed by gene silencing (chr_tin remodeling) In addition to chromosomal deletions and gene mutations, 'via the hypermethylation of the promoter, the cause of the county, epigenetie silendng is also common in human cancer. Recent epidemiological studies have shown that the concentration of serum folate (a major source of methyl) is associated with HPV infection and clearance; in the metabolism of the methyl cycle, the gene for the enzyme Polymorphism (geneticp) is also reported to be associated with the development of cervical intraepithelial lesions; such as the concept of genetic evolution, DNA methylation and sub (10) Lang association studies are also prevalent, the uterus Dna methylation study days and increases, showing the possibility of using methylation as a screening for cervical cancer; 〇 due to genetic and environmental interactions (4) characteristics, M tumor suppressor gene thiolation degree due to different genes and different Different from ethnic groups, different diseases may have different methylator phenotypes; however, 'the methylation phenotype of cervical cancer and its association with genotype are still unknown' and what is in cervical cancer? The specific genes will be methylated, and how many genes are needed to reach the trampoline (10) needs. These issues are still issues that need to be confirmed in the future.

The inventor of this case has previously been in Taiwan (TW Pat Pub·N〇. 2〇〇8319〇〇), China (CN

Appl. No. 200810094659.2), Malaysia (ui2〇085354) and the United States (us pat pub 201038739 - No.20080311570) filed a related patent application (hereinafter referred to as the previous case), this case is an extension of the previous case, the inventor of the case found novel Cancer screening biomarkers and methods for screening them. It can be seen that there are still many shortcomings in the above-mentioned conventional cervical cancer screening methods, which are not the designers of good products, and need to be improved. In view of the shortcomings derived from the above-mentioned conventional cervical cancer screening methods, the inventors of the present invention have improved and innovated, and after years of painstaking research, they finally succeeded in researching and developing the method for screening cancer. SUMMARY OF THE INVENTION [The present invention] It is an object of the present invention to provide a method for screening for cervical cancer as a cancer screen for first-line cervical cancer. The second objective of the present invention is to provide a method for screening for cervical cancer, which can be used as a screening for second-line cervical cancer in addition to being used as a first-line uterine surface examination to assist human mastoid processes. Virus test (HPVtesting) or uncertain smear results to achieve more accurate cervical cancer screening results. Another object of the present invention is to provide a method for diagnosing cancer. The method can be applied to the detection of cervical cancer, and can also be applied to the detection of other cancers (eg, nest cancer, colorectal cancer) to assist abnormalities. Diagnosis of the specimen. The method for screening for observation of the above-mentioned object is to detect the state of methylation of a target gene in a test subject cell as a screening index for cancer presence, and the method comprises the following steps: Step 1 provides a subject Step 2: detecting (10) sequence 201038739 column methylation status of at least one target gene in the genomic DNA of the test subject, the target gene consisting of DBC, PDE8B, PTPRR and ZNF582; and step 3 according to the The presence or absence of the methylation status of the target gene, whether the sample has cancer or precancerous lesions, or as an indicator of prognosis. The test subject is a Pap smear, ovarian cancer tissue, ascites, blood, urine, feces, sputum, oral mucosal cells, gastric juice, bile, cervical epithelial cells, or cancer tissue after surgery. The method for detecting the methylation status of the CpG sequence of the target gene includes, but is not limited to, methylation-specific polymerase chain reaction (111 over|; 1丫1& line 11 out of 6 (^〇PCR, MSP), quantitative A Chain-specific polymerase chain reaction (qUantitative methylati〇n_speciflc pCR, QMSP), bisulfite sequencing (BS), microarray (micr〇arrays), mass spectrometer (mass spectrometer), denaturing high-performance liquid phase Denaturfng high-performance liquid chromatography (DHPLC) ° wherein the target gene DBC1 has a nucleotide sequence as shown in SEQ ID No: 1. The target gene PDE8B has a nucleus as shown in SEQ N〇: The target acid gene PTPRR has a nuclear sequence as shown in SEQ ID Ng: 3. The target gene ZNF582 has a nucleotide sequence as shown in SEQ ID N:: 4. In addition, the aforementioned screening index and The t-test method can be used for the screening of the uterus and the cancer. The present invention further provides a method for screening for cancer by detecting the state of methylation of the target gene in the test subject cells. Paste nest cancer _ test indicator, the method comprises the following steps: 7 201038739 • Step 1 provides a test subject; Step 2 detects the methylation status of CpG sequence of at least one target gene in the genomic DNA of the test subject, the target gene line It consists of DBC1, FTPRR and ZNF582; and step 3 determines whether the specimen has ovarian cancer lesions or as an indicator of prognosis according to the presence or absence of methylation status of the target gene. The test specimen is ovarian cancer tissue and ascites. , blood, urine, or post-operative cancer tissue, etc. The method for detecting the methylation status of the CpG sequence of the target gene includes, but is not limited to, methylation-specific polymerase chain reaction (MSP), Quantitative methylation-specific polymerase chain reaction (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing HPLC Denaturing high-performance liquid chromatography (DHPLC), pyrosequencing. Wherein the target gene DBC1 has a nucleotide acid sequence as shown in SEQ ID No: 1. Wherein the target gene PPTRR has a nuclear phytic acid sequence as shown in SEQ ID No: 3. Wherein the target gene ZNF582 has a nucleotide sequence as shown in SEQ ID No. 4. The term "tested body" refers to a test sample that is isolated from the body, including the aforementioned Pap smear, ascites, blood, urine, feces, sputum, oral mucosal cells, gastric juice, bile, cervical epithelial cells, or Samples of isolated tissues of cancer tissues after surgery. The cancer screening method of the present invention is for detecting the methylation of a target gene in the isolated samples as a screening index for various types of cancer. The cancer screening method and the screening index provided by the invention can be tested by the testing researcher in the laboratory. 8 201038739 [Embodiment] The present invention is exemplified by the following examples, but the invention is not The following examples are limited. Example 1 Materials and Methods I. Materials The test materials contained a series of complete cervical lesion samples, including: squamous cell carcinoma (SCC, η = 20), adenocarcinoma (adenoCarCinoma, Ac, n=

20) ‘and normal cervical specimens (n=i〇). All cervical samples, ovarian samples, and colorectal cancer samples were taken from the Taipei Military General Hospital, and the genomic DNA of each sample was extracted with the QIAamp DNA kit (QIAGEN) and used for comparative analysis in the whole genome. The case of DNA methylation. The quality of genomic DNA was measured by Bioanalyzer (Agilent) before analysis. In the present example, MeDIP (Methyl DNA IP) was carried out with 1 μμβ fragmented DNA. 2. DNA methylation analysis by MeDIP and CpG is丨-Plus-Promoter arrays (CpG island-Plus-Promoter arrays) First, fragment the genomic DNA to 300~1,000 with BioruptorTM UCS-200 (Diagenode) Bp 〇 again with 30 μl polyclonal Anti-5'-methyl cytosine antibody (Abeam) in final volume 100 μl IP buffer (IP buffer) (0_15% SDS, 1°/. Triton X-100, 150 mM NaCl, ImM EDTA, 0.5 mM The fragmented genomic DNA was immunoprecipitated by EGTA, lOmM Tris and 0.1% BSA) at 4 °C. Will immunize

After mixing with 120μ1 Protein G Sepharose (Amersham), react at 2°C for 2 hours, then use 1ml low salinity, high salinity solution, lithium chloride 201038739 » and TE buffer (TE buffer) Wash twice. Then elution buffer (1 % SDS, 0.1Μ)

NaHC〇3) Protein G was treated twice at room temperature for 15 minutes. The methylated DNA is recovered by phenol-chloroform extraction and alcohol precipitation. Enriched methylated DNA and input DNA were amplified (amplify) by the Whole Genome Amplification Kit (Sigma). Enriched DNA and total DNA are labeled with Cy5 and Cy3' at the tail end, respectively. Enriched DNA and total DNA are then hybridized with CpG Island-Plus-Promoter Arrays (co_hybridization) 〇 CpG

Isl^d-Plus-Promoter Arrays were designed and synthesized by NimbleGen Systems, Inc. This array contains 385,000 50-75 bp oligonucleotides (probes) containing approximately 9 HG18 RefSeq promoters per 100 bp (8 bp upstream of the promoter to 200 bp downstream of the promoter) And 28,226 CpG islands. Each eigenvalue in the array has a corresponding binary logarithm (1〇g2) ratio, which represents the normalization of the ratio data to zero concentration (n〇mializati) 〇n) Results. According to a binary log ratio data, a fixed-length window (5 〇〇 bp) can be placed on the D-wire continuous reward® ''Double-area k-monovsov test ^mended Kolmogorovemimov test)' In order to judge the data compared to other binary log ratio data, these probes are obtained in a more meaningful forward distribution. The final ratio of each probe point is based on each probe point. The negative common logarithm (-loglO) of the p__value) obtained by the Kolmonov-Smirnov test of the fixed window is described in detail by Scacheri et al. in Meth〇ds Enzym〇1 2〇〇6 Method (Stati— f0r Chff-chip and DNase experi_s (10)

NimbleGen airays) to select analytical array results. In the present invention, searching for at least two 201038739 * probe points of a p-value minimum cutoff of 2 to detect the peak of the binary log ratio data, and will be between 3 Multiple spikes between 〇〇bp are merged together. Comparing the results of squamous cell carcinoma (see), adenocarcinoma (AC), and normal cervical specimens, if there is a differential methylation region 25 bp upstream and downstream of the transcription initiation region, it is selected to further Evaluation. Finally, the p-value data is checked by SignaMap (NimbleGen). 3. Bisulfite modification, methylation-specific polymerase chain reaction (MSP) and sulfite sequencing (BS) using Chemicon. DNA modification kit (DNA m〇dificati〇n vs Chemicon, Temecula, CA) for sulfite modification: 1 μ§ sample of genomic DNA (genomic DNA), chemical modification of genomic DNA with sodium sulfite, in single-link DNA In this case, all unmethylated cytosines are deaminated and converted to uracil, while methylated cytosines are not modified and remain in the state of 5-methylcytosine; after Q, the reaction will be The resulting sample DNA was dissolved in 70 μl of 55 ° C TE buffer (TE buffer) for methylation specific PCR (MSP). Further, normal DNA of human peripheral blood was subjected to sulfite modification as a control group having a non-methyl group promoter sequence. Take 1 μ§ sample genomic DNA after sulfite modification, and control DNA 'methylation-specific PCR amplification with MSP primer, which is a MSP primer that can specifically recognize methylation gene sequences (M The MSP primer sequence of each target gene is shown in Table 1; the total volume of the methylation-specific PCR reaction is 25 μl, including 1 μ1 of the modified template DNA of 201038739, each primer of 1. 5 pmo b 0.2 mmol /L dNTPs and 1 unit Gold DNA polymerase (Applied Biosystems, Foster City, CA); the mixed reaction was placed at 95t for 5 minutes, followed by dissociation at 95 °C for 30 seconds, appropriate primer bonding temperature The bonding was carried out for 30 seconds at 72 ° C for 30 seconds, and the dissociation, bonding and synthesis steps were repeated for 35 cycles, and then placed at 72 t for 5 minutes. The amplified product was electrophoresed by a 2.5% lipopolyester containing ethidium bromide (EtBr) and exposed to ultraviolet light. Ο Table 1-methylation-specific PCR (MSP) MSP primer sequence gene name primer type _ primer sequence DBC1 Μ PDE8B Μ positive stock (F') 5' gttttcgtcgtttttcgttcggagatc 3, anti-share (R') 5, gctctcgctctcgctattactcgct 3, positive stock (F·) 5, tgtgtatgcggttttttcgttc 3, anti-share (R')5' acctatatatccgccgctccgtc 3' (SEQ ID No: 5) (SEQ ID No: 6) (SEQ ID No: 7) (SEQ ID No: 8) PTPRR Μ Orthonorth (F') 5' cggcgttgggtatgtttagtagtc 3, (SEQ ID No: 9) Strand (R') aattacgaataaaaaaaacaaaaacgctc 3, (SEQ ID No: 10) ZNF582 normal stock (F) 5, tgacggttttttgtttattcggttattcS, (SEQ ID No: ll)

The primer type M represents a Msp primer that can specifically recognize the methylation gene sequence. All samples are subjected to at least two independent sulfite modification and methylation-specific PCR. In the use of specifically recognizing methylation gene sequences In the PCR reaction performed by MSj> primer (Μ), if the same sample cannot synthesize the pCR product twice or more, it is considered that the sample is not methylated; an MSP primer that can specifically recognize the methylation gene sequence will be used (M The amplified PCR product was cloned into the pCR4_T0P0 vector (Invitr〇gen, carisbad, ca), and at least 5 independent strains (d〇nes) were selected for sulfite sequencing (BS), sulfite The primers used in the order (BS) are shown in Table 2, using the 377 automatic sequencer (Applied 12 201038739)

Biosystems, Foster City, CA) for sulfite sequencing, the results of which are sequenced

As shown, the sequence numbers for sulfite sequencing are: DBC1_BS (SEQ ID No: 21), PDE8B_BS (SEQ ID No: 22), PPRRR_BS (SEQ ID No: 23), and ZNF582_BS (SEQ ID)

No: 24). Table 2 Sequence name of the primer used for sulfite sequencing (BS) Primer type Primer sequence DBC1 Positive strand (F) Anti-strand (R') 5f ggttaagtttttttttyggygtagtt 3, 5*tactccctctacctcccractctctc 3' (SEQ ID No: 13) (SEQ ID No: 14) PDE8B positive (F) anti-strand (R,) 5f ttgtggygtagaggattattagtttggt 3, 5rctaaaaacrcaacccatccctc 3r (SEQ ID No: 15) (SEQ ID No: 16) PTPRR positive (F) anti-strand (R. 5rggaattttattttgaaatttttttgtt 3, 5rccccacttcaaataaaatactattaaaaaaaac 3f (SEQ ID No: 17) (SEQ ID No: 18) ZNF582 positive strand (F) anti-strand (R') 5' tagtgayggttttttgtttattyggttatt 3, 57 taaacrtaaaaacaacaacccracct 3f (SEQ ID No: 19) (SEQ ID No: 20) o Example 2 Screening of target genes for cervical cancer methylation Screening by CpG island-Plus-Promoter array (CpG island-Plus-Promoter 〇arrays), screening for four target genes in cervical cancer Cells may have a high degree of methylation, DBC1 (SEQ ID No: 1), PDE8B (SEQ ID No: 2), PTPRR (SEQ ID No: 3), and ZNF582 (SEQ ID No: 4), details As shown in Table 3; Three found 'in addition to these four genes known to be associated with bladder cancer DBC1, the study shows that there is little correlation between these genes and the cervical cancer. Table 3: CpGisland_Phis-Promoter array for screening for details of methylated genes in cervical cancer cells 13 201038739 Gene Name UniGene Number Chromosome Localization Gene Full Name SEQ ID No DBC1 NM_014618 9q32-q33 deleted in bladder cancer 1 SEQ ID No: 1 PDE8B NM_003719 5ql4.1 phosphodiesterase 8B SEQ ID No: 2 PTPRR NM_002849 12ql5 protein tyrosine phosphatase, receptor type, R SEQ ID No: 3 ZNF582 NM_144690 19ql3.43 zinc finger protein 582 SEQ ID No: 4 Example Trisulfite sequencing ( BS) Analysis of methylation status of target genes in cervical lesion samples

Target factor: PPRRR test sample group: 1. HeLa cervical cancer cell line (HeLa_0); 2. HeLa cervical cancer cell line treated with 10 μΜ DNA methyltransferase inhibitor (AZC) (HeLa_10D>; 3 Simultaneous treatment of 10 μΜ DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC) and 〇·33 μΜ of TSA (Sigma)

Chemical Co., St. Louis, MO) HeLa cervical cancer cell line (HeLa_DT); 4. Cervical adenocarcinoma sample (AC); 5. Cervical squamous cell carcinoma sample (SCC); 6. Control group ( Normal): normal cervical blood DNA as a non-thiolated control 201038739 ' After each sulfite modification of each test sample, the sulfite sequencing (BS) analysis target gene (PTPRR) was used in each test sample. Whether there is a high degree of hysteration (hype_hylati〇n) phenomenon, as shown in Fig. 1, black indicates a methylated region and white indicates an unmethylated region. The target gene PPTRR was not methylated in the control group and adenocarcinoma. In the HeLa cervical cancer cell line (HeLa-0) and cervical squamous cell carcinoma (scc), the target gene PTPRR was highly methylated. . Therefore, the degree of methylation of pTpRR can be used to screen for cervical cancer. 〇 In addition, in order to confirm whether the degree of methylation of the target gene in the cervical cancer sample is regulated by DNA methylation, the 1〇μΜ DNA methyltransferase inhibitor 5'_aza-2'-de〇XyCytidine ( AZC) (Sigma Chemical Co.) treats the HeLa cervical cancer cell line, and then the DNA sample extracted from the cell is subjected to sulfite modification followed by sulfite sequencing (BS). The results are shown in Figure A, compared to the cervical squamous cell carcinoma sample (scc) and the HeLa cervical cancer cell line (HeLa_0) 'ALa-treated HeLa cervical cancer cell line (HeLa-10D)' The target gene PTprr showed partial methylation removal. © hchostath A (TSA) is a histone deacetylase (HDAC) inhibitor that can also be used to reduce and weaken the degree of methylation. The HeLa cervical cancer cell line was simultaneously treated with AZC and TSA (HeLa-DT). The results are shown in Figure 1. Compared with cervical squamous cell carcinoma (SCC) and HeLa cervical cancer cell line (HeLa_0), The HeLa cervical cancer cell line (fjeLa_DT) treated with AZC and TSA showed that the target gene PTPRR showed high degree of demethylation. According to the above results, in the cervical cancer sample, the target gene pTpRR was indeed methylated by DNA methylation. 15 201038739 _Target gene: Z1VF you 2 test sample group: 1 · HeLa cervical cancer cell line (HeLaj)); 2. Treatment of 1〇μΜ DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC) HeLa cervical cancer cell line (HeLa_10D); 3. Simultaneous treatment of 1〇μΜ DNA thiotransferase inhibitor 5 '-aza-2, deoxycytidine (AZC) and 0.33 μΜ of TSA HeLa cervical cancer Cell line (HeLa_DT); 4. Cervical squamous cell carcinoma sample 1 (SCC 1); 5. Cervical squamous cell carcinoma sample 2 (SCC2); 6. Control group (normal): normal cervical blood DNA No methylation control group. Among them, cervical squamous cell carcinoma samples 1 and 2 were samples of different patients. The above test samples were analyzed by sulfite sequencing (BS) to analyze whether the target gene (ZNF582) had hypermethylation in each test sample. The results are shown in Figure 1b, compared with the control group. The gene ZNF582 exhibits southern methylation in cervical squamous cell carcinoma sample 1 (SCC), cervical squamous cell carcinoma sample 2 (SCC), and HeLa cervical cancer cell line (HeLa_0). Therefore, the target gene 2NF582 is highly thiolated in cervical cancer samples.

Target: PDE8B test sample group: 201038739 • 1. SiHa cervical cancer cell line (siHa_0); 2. Treatment of 10 μΜ DNA methyltransferase inhibitor 5,-aza_2,-deoxycytidine (AZC) SiHa Cervical cancer cell line (siHa_l〇D); 3. Simultaneous treatment of 10 μΜ of DNA thiotransferase inhibitor 5'-aza-2'-deoxycytidine (AZC) and 0.33 μΜ of TSA of SiHa cervical cancer cell line ( SiHa_DT); 4. Cervical squamous cell carcinoma sample 1 (SCC 1); 〇 5. Cervical squamous cell carcinoma sample 2 (SCC2); 6. Control group (normal) ·• Normal cervical blood DNA as no Methylated control group. Among them, cervical squamous cell carcinoma samples 1 and 2 were samples of different patients. The above test samples were analyzed by sulfite sequencing (BS) to analyze whether the target gene (pDE8B) had hypermethylation in each test sample. The results are shown in Figure 1c, compared to the control group. The gene PDE8B is highly methylated in cervical squamous cell carcinoma sample S1 (SCC), cervical squamous cell carcinoma sample 2 (SCC), and SiHa cervical cancer cell line (SiHa_0). Therefore, the target gene PDE8B is highly methylated in cervical cancer samples. Target gene: DBC1 test sample group: 1. SiHa cervical cancer cell line (SiHa__0); 2. Treatment of 10 μΜ DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine 17 201038739 - (AZC) SiHa cervical cancer cell line (SiHa_l〇D); 3. Simultaneous treatment of 10 μΜ DNA methyltransferase inhibitor 5'-aza-2'-de〇xycytidine (AZC) and 0.33 μΜ TSA of SiHa cervical cancer Cell line (SiHa_DT); 4. Cervical squamous cell carcinoma sample 1 (SCC1;); 5. Cervical squamous cell carcinoma sample 2 (SCC2); 6. Control group (normal): normal cervical blood DNA Non-methylated control sputum group. Among them, cervical squamous cell carcinoma samples 1 and 2 were samples of different patients. The above test samples were analyzed by sulfite sequencing (BS) for the presence of hypermethylati〇n in each test sample. The results are shown in Figure D, 'Compared with the control group, the target gene DBC1 exhibits methylation in the cervical squamous cell carcinoma sample "see", Zilu neck squamous cell carcinoma sample 2 (SCC), and SiHa cervical cancer cell line (SiHa-〇). Therefore, the target gene DBC1 It is highly methylated in cervical cancer samples. Example 4 Methylation analysis of target genes in cervical cancer samples The squamous-specific PCR (MSP) analysis of the four target genes in cervical scales The methylation status of the cell carcinoma (see) sample, the methylation status analysis results are shown in Table 4, the results show that the frequency of methylation of DBC1, PDE8B, PTPRR (10) in the normal child Weigan towel 11%, 0%, 9%, and 6%, respectively; the frequency of thiolation of 'DBC1, PDE8B, PTPRR, and ZNF582 in uterine sputum cell carcinoma samples was 201038739 brain, 47%, face, and 97%, respectively. This shows that in the cervical squamous cancer sample, the four genes It is greatly methylated. Therefore, the methyl silkness of DBC1, pDE8B, pTpRR and ZNF582 can be used as a screening index for uterine occupation. Table 4: F-basedization of target genes in squamous cell carcinoma of the uterus (IV) Normal cervical specimens (4) 4) Cervical squamous cell carcinoma-like (n=3〇) DBC1 PDE8B PTPRR n〇/〇100% 〇% 47% 9% ιηη〇Λ

Example 5 Methylation analysis of target genes in ovarian tumor samples The methylation status of target genes in the nested tumor samples was analyzed by methylation-specific PCR (MSP). The methylation status analysis results are shown in Table 5. In the (4) malignant tumor samples and the methylation status of the three genes of DBa, pTpRR and Tiaojiao of the ovarian I., the results showed that DBa, pTpRR and Hall 582 in the nested malignant tumor samples. The base frequency was 50.3%, 5〇.0%, and 56_3%, respectively. The bases of the benign tumor samples DBC1, PPRRR, and leg 82 were 25%, Ο.Ο/d, and 12.5% 〇. The degree of methylation difference was 53 8%, 5 〇 and 43 respectively. Therefore, compared with the benign tumor samples of the sputum, the methylation of the three genes in the nest of malignant tumors was significantly higher. Therefore, the degree of methylation of DBC1, pTpRR and indeed can be used as an indicator for the detection of sputum cancer. Methylation status analysis of target genes in the five-in-situ tumor samples 19 201038739 Ovarian malignant tumors (11=28) Benign ovarian tumors (n=28) Difference in methylation degree DBC1 56.3% 2.5% 53.8% PTPRR 50.0% 0.0% 50.0% ZNF582 56.3% 12.5% 43.8% Methylation analysis of the target gene in the sample colorectal cancer sample by methylation-specific PCR (MSP) analysis of the methyl hydrazine state of the target gene in colorectal cancer samples The results of methylation status analysis are shown in Table 6. The methylation status of DBC, PDE8B, PTPRR and ZNF582 in colorectal cancer samples was analyzed. The results showed that DBC1, PDE8B, PTPRR and ZNF582 were found in colorectal cancer samples. The methylation frequencies were 100_0%, 100.0, 1〇〇·〇%, and 100%, respectively. The methylation frequencies of DBC1, PDE8B, PTPRR, and ZNF582 in normal large intestine tissue samples were 25,0°/〇, respectively. 25.0%, 25.0%, and 25.0%. Therefore, compared with normal colorectal tissue samples, the four genes were significantly more methylated in colorectal cancer samples. Therefore, the degree of methylation of dbci, PDE8B, PTPRR and ZNF582 can indeed be used as a screening index for screening colorectal cancer. Methylation status of target gene in rabbit g colorectal cancer samples Methylation frequency of target gene (%) Normal colorectal tissue sample (n=24) Large cancer sample fn=20) DBC1 25% 100% PDE8B 25% 100% PTPRR 25% 100% ZNF582 25%_ _ 100% 201038739 The method for cancer diagnosis provided by the present invention has the following advantages when compared with the conventional techniques described above: 1. The cancer ladder of the present invention The method is based on the degree of methylation of a specific gene in an ex vivo sample as a diagnostic indicator for the presence or absence of cancer, and the sensitivity of the cancer diagnostic method of the present invention compared with the conventional Pap smear and HPV testing method. And specificity is higher than the previous two. 0 2_ The method for screening cancer provided by the present invention can be combined with or assisted by HPV testing as a second-line cervical cancer in addition to screening for first-line cervical cancer. Screening to achieve a more accurate screening effect of uterine head cancer. 3. The prescription of the 24th remedy of the present invention can also be applied to the detection of other cancers (such as: nested cancer, colorectal cancer) to assist in the diagnosis of abnormal samples. DETAILED DESCRIPTION OF THE INVENTION The detailed description above is a specific description of one of the possible embodiments of the present invention, but the implementation of the invention is equivalent to or equivalent to the invention of the present invention. The equivalent embodiment of the change in the degree of methylation of each target gene in the subject's sample, etc., should be included in the patent scope of the present application. In summary, the method of cancer diagnosis provided in this case is indeed innovative, and it can be used in the case of cervical cancer, and the above-mentioned rural items, Wei and Hong, fully meet the statutory invention patent requirements of the Qing and progressive. Please, the _ t bureau approves the invention patent application, in order to invent the invention, to the sense of virtue. [Simplified illustration] 21 201038739 Figure 1A is the target gene PPRRR used in the cancer screening method of the present invention, in various types In the cervical sample, the results of sulfite sequencing (BS) analysis were performed; Figure 1B is the target gene ZNF582 used in the cancer screening method of the present invention, and sulfite sequencing is performed in various cervical samples (BS). The results of the analysis; Figure 1C is the result of the sulfite sequencing (BS) analysis of the target gene pDE8B used in the cancer screening method of the present invention; and Figure 1E) The target gene DBC1 used in the cancer screening method of the present invention is subjected to sulfite sequencing (BS) analysis in various sub- & 颂 samples.

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201038739 Sequence Listing <110> Lai Hongzheng <120> - Method for Cancer Screening <160> 24

≪ 210 > 1 < 211 > 5000 < 212 > DNA < 213 > person who is wise species (Homo sapiens) < 400 > 1 ggtcttcacc tgaagttgcc tgactacgtt gtctcagatt gatttcagaa ctgcattatc agcagggcag ctgttgccta ctaacagtca tggaagtaga tctcagctgg gggatgatgg ggaaacaaag agaaagatag ggacgaataa aggaaggaat ggggcacaga cagaaagaga gagagagaaa agaaagaaag gaggagaggc aagagatagt gacctggggt ttcataccgc ggtgggtggg agaaggagtg tgtgtggtgg gcgtatttct tcgtggtcag aatcaaagtc agaactgacc tggggaacct gtgttcaaga cctgaacctg gaccagaagc caagggaaag agaaagttgc ccaagaaaaa agggagaaat ttccaccagg cgaaaataga aatcgctgac tgggtttgtg gctggagagc tgtccgtggt gctgactcct cctcattggg attccagtga aggctgagaa gctctgcact ggctgctcct cctctccctt tcctctcccc tcagtcctga ccttctgaga cccaggatca ttctggctag ctcgatgtct tctcctctct ttctcccttt ctctttgtcg attaaataat ttttcccgag ttcccagttc atcactcaaa catttctctt ccattttgtt tcaagactcc Tcttcctcca attagttcac tggctcctag attgttctag gcctccctgg tggtcacatc tctctcttat catcctttga attaaaaaac aaacaaaaaa catacaatgg caatcataaa agcagacaac atgc actgag cactcactag gtgtcaggga gcagtctaag tgcctttgca tacattaact catgagcagt ctaagtgcct ttgcatatat taactcatta caattccaca tcaaccctat tagatgggta gatatattat tattctatta aacagacaag gaattgaggc ttaaggggta agtgatttca ccaagaccag acagctacag gactccaacc tctctggagc ctgcacttct actgaatctg gctcttttac atattccaac accttttcct atgacttcct gcccctccat ctggtagcct taatgctttc tattcttgcc taatcttcag gggactaggg atctgcattg ctgatttggt ttaaatcaga agggaagcaa agtaaacaaa cataccaaat gtgcgctgtg tgggaattat catacgaggg ctttattttc tgcttcagga agaggcccta tgttagcagc cccagcctgc attcaggctg attgcagagt attttgcttt ttattttcat gtcttagtcc ctgtaccctc gccccttccc cgcctctggt ggtctccaga gaacttcgtg tcccctcagc ttctccctcc tacatcctgc ctacgtagag aagctcttgc ttcattctgg gaggttacgt gggctctcgc ctacacaccg agagaaacaa acagtgtcaa acactcacag agagacgcgc agacacaaac ggacccacac gggcaactcc cgagacaaaa cccacactcg atggatccac gcggccgtgg aaacacctgc cgccccagaa acactcaggt actcgcgaca cacacagtac agtcacgctt aagggcacca ggattccggg tttgcgcgta tgcgcggtcc ctttggatgc tcgtgcgcat aga Cacaaca ccctacacgc cccagaccca cgaaactccc tacggctcag ccccagccca cccgggccgc ccttccctcg 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800

201038739 aggcggcctc ccgtctctcc tcctctcgct tctcctcctc ctccgcctaa agatgtacaa aacactcctc ggaagcaacc ccggcgttca gctcctccct ccccgccccc cggccgccgc tcccccattc attttcggcc gtcgccggct aagtccctcc cccggcgtag cccggcctcc gccgctcccc gcccggagac cgcggcgcac ttggacttcc ctctccattc gccagccgcc tcgctcccgg accccacggc tgcaaactga tctggcgcgc ggggaggagg agagcgcagg cgagcgaacc cgcgagagag ggagagagcg agcgagcaac agcgagagcg agagcgagag agccgggagg cagagggagt agtgaccgcc ttccggagcc gggattcatg cctgtcctcg ggaccagcga aggggacttt acggctgagt atgagccagg ctgctaggag ccaggtaccc ccacgcctgc agtccccgcg ccgtgcccgg aatgcgagct gcacgcaggg ctctcccaag ttcccaccga gccgaataaa aagcgtcctc ccgcagctct ccgccaaaga cggacattga ctccaggtaa ggcggcgccg ggtgcagcgc cccgcagccc cgctgccctt ggacccggcc ccgggccgca ttcggggcgt ccccgcgctc ctctgcccct ccccctaccg gcacccttgt ccgctcttca cctggccgcc ccgccgcctc caagtcttct ccagttctag ggagggggtt cctgtgcctg gggctcaaag ggctaattgc gggtttgagt gagtggcgtg tgtgtccccg cgcgctcccg acgtgtgcac catggtggga acttgatgtg gtgctagtgt gtttgcgtgt gcggcgtcgt ttgcgtttga tgcgtgtgtt cgtggtgtgt gtgtgtgtcc gtgtgtgtaa gggaggggtg aagagagaga ggtcctataa cctacttacg gcgcgatgtg tgtgtgcatg tttgtacgta tgtgtttgta tgtgtgccct cgtgtgtctt tttaattagg tctctccagc ttacacggaa tgggaccctt actataggat cacgtagtca ccgggaaacc cgctgtggac ttcctcttgg ggctctgggc ttggggtttg gggaggatta tggggctgta gatggcacct tatttagccc aatgttggta cgcttgaagg aaaattcctc caaacggtgg aatcctgcta cactgggacc cacagcttaa tatgaaagag acatggccaa cccccgaggc aaatgagacg ctgtcacttt aaattctaca ctgggcagac tccaagattc tgatgggaat ttggagacac tggatagt £ g gtgacagaga aagggggaag tcagcggtgg gctccttatc tggggcttgg aaagtctggg atagggattt accctgcatc cccgtttgca atcaacagag cccctccggc ttctgttggt tttggggagg atctggcaag tttgcatgga tccccctcag gggaaaggag aaagcgtcct cggggacttg ctcatccatc acagttgcaa agggtctcag aggaaatttc atctggggcg gctgtggatg atatggaagg agatggatgg ggcactttcc taagacagat tcgtctttct ttccccattt caggcgggga agcccccaga gagtctctcc ctaaatatgc ctctccatgg ctcccctgga gttaggggga tattgagaga aggcagagag gtggagaggg agagagaga g agcaagagcg agagagaggg agacagagag agagagtgtt tcagtactga gggagatcta caatttgaaa aggggctgtg agtgtggaac ccatgacaga atgtggcagt aattgactta aactgctgtc ggtttgcacc tcgcgtctct cacttggctc caaaatactg ccaagggcag gggggcggtg gaggaatctc agccaggaag gtagtttggg tcaaggccct ctttcttctt tcctccatct cctgggaggc tctttaggta ggtcccttgg ccaccctggc ttggtactgt tgggcttggg ctctggggcc agccattgat ttgattccca gctgcctctt aaaggcttgc cctactcagc aaaaatgctg agttttactg ctgttagcat ggcttccaga ctcggcagct gttactttct caaggtaagc ggcagccgtt ctgctctcag gcagggaggc taggagaaga caagggctgt gacgctggga cacagcctct tgtctagctc agcacggagg ggccgctgaa aagccctctt aggggaaaaa agtagaaata tatttggcct aaaaaatgta cacatatatt ctaggaagat atatatatat atatatatat caatcacaca cacacacaca cacacacatg tgttcatatt ctggggagag ataaaagcaa acatgtattt gtaggaacct gccctagtgg gtgggttcta cctgcaggca cttgcagaca taccattctc tgtccagagg ctgatacctc aggctgaagc cttcacatag ccacagagaa tctccttcag gaaactcatg 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 2

201038739 tcagggcaga gctccctgga ttatcttggg tagggggtac tagggccaag gaggagaccc atttttggga actgcctcat tttctctctt tcctaattca cagacgatcg aaactggaag gaatattaaa gagcagaaag gcaggactct gtggcctaaa gagggaatga gaattcccca acattcagta gtgggttagt ggcatgccca ggactagaat ctaggcttat caatcagcta gatcaatacc ctttccactc ctcccagcaa tatacactct aggactgtat attgctccca cccctatctt gccaacctgc caccatgacc cctaccaaga caatcgcttg tcttccctca tcaaacttca cattttcaga gatgcatttc agggtttttc tatctggaaa catggccctg aagagaaaag atccaatgcc tcaatcacta ttcccgaaag acacacacac acacacacac acacacacac Acacacacaca acacacacac acacacacgg tggtgggggg aggactgaga gagagacaga gagagaacat

<2l〇> 2 <211> 5000 <212> DNA <213>Homo sapiens <400> 2 agaacaactc tgaagcatca tcccaccttc agaatcctcc ctgcagggat gcctgaggct tggtcaattt ctccctctgc tcaagcttgc ctccctccat tccctccctt tatttcctcc cacaaatgtt gactacaaga gcacttecta aaaagcctcc ctctgcctgc taaacaccat ctcagaatct gettetagga ggacccaacc tgtgcagaaa ttttatgagc aatttgcctg tccattaaaa aatacagcat agactttttc caggttagct ggagatggag tttttttgta tcaaaaaaag agagctaaac tgagaagaga agcaaaggta gaagaataag gttagactct ccctgtataa ctgaatttgg gattgggaat gatatettge ttctctatga aatttagaaa ctgtcttttg aagtcttcat ccagtcacat ccaagagtgg tccaggaaga aaaaggaaaa taagaatgaa aaaaaaacaa aaaacaaacc ttaaaattgg ataettettt gtgeettgea ttaagcctct atgttactgc ccttgtccgt ataagcaact ctagataaaa teetttetta gaccatctaa tttatctccc cgtgccattg tgaaaatcgg gcaggaatta ctgcgggaac Cagtgactga atgggcagtg gtgtctcgca gctgtaactc agagtttaca agggaaggat gaacagcagc catccttcta aaggeattet tcctctcctc cagcacagct cagtctgtgc atgcctcacc aaggagaatg agggaaggee ttgt tgactg cccagcccag agtgtgagga acagcagttg acatgggcgg ctggacggga tagaaaccag catgaacatt tagtgacacc taaggaagag gtgaaaatgg gggagaaagc caagaaccat cagacagctt ctttctgttc ttgccttgga tcttagggga acgtatgtgt tcaagggtgc cttcctccgc agggcctccc atcccttggc acaaacttcc aaaegatgag tagacatgga atgggcttct tctgaagact gggtcaccat ccaactcagc etteggagta ggcggtgggg ctgggggaag aggacggaac catgaggtct ctccatcact tcccgggtgt gttgagagge aagcaaatgc ggtgggtggg cctgggctgt agggcccctg ccatttcggg aggggcctcc tggtgtttag ctctgggatg tgtgaaaatg tgttggtgaa aagegagagg cttacacagc ccttcagggg aagaggggct ggggcgctgg gggcggcgtc gggatgagtg cagaagagac gaggcctctg gacagcggag gaggagggga gggegeegag gcgcggtgcc agctgccgcg cacaggggcc ccgcggcgga gccgagccgc gggcacgctc tgccctgtcg ggagagctcc gggagcggcg ggaggggcgg aggggcgcag tggggcccgg geggetgegg ccgcggagcc ggggcacctg aggaggaagg 4500 4560 4620 4680 4740 4800 4860 4920 4980 5000 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 3 Ο 〇2010387 39 agggtgggag cgagggaggg aggggacggg cgcagaccga aagtggggaa agaaggtgca ggcaggcggg caggcgggcg ggcgccctgg cccagggccg cgggtgcggg agcccggcga ggtcgagctg ggcggcggcg ggggccgcgc cgagggagga ggggaaggcg gaggcgcggg gagcgtgttt ggggcgccgc ggcggggagg gtggcggccg ctggtgcgcg cggggcgctg tgtatgcgcg ctcccccgct cggggaggaa gatggcccaa aagggaaagt tggggtgacg cgcgcggtcc ccggaggctc ggcggggggc accgcggcca gcccgacgga gcggcggaca cacaggccgg gg ££ cgcgca gtccgggcgc cgccgcggcc gccccctcac tgcaggtggc agcgggtgcg ctgggtcccg gcggccgcgg gcgcgggcgg gcgcgcgggg gagcccggcc gagggatggg ctgcgccccc agcatccatg tctcgcagag cggcgtgatc tactgccggg actcggacga gtccagctcg ccccgccaga ccaccagcgt gtcgcagggc ccggcggcac ccctgcccgg cctcttcgtc cagaccgacg ccgccgacgc catccccccg agccgcgcgt cgggaccccc cagcgtagcc cgcgtccgca gggcccgcac cgagctgggc agcggtagca gcgcgggttc cgcagccccc gccgcgacca ccagcagggg ccggaggcgc cactgctgca gcagcgccga ggccgagact cagacctgct acaccagcgt gaaggtaaat gccccgcgct ggcacacgcc gtgggggccg tccgccccgt cggcggggct cgcacgggta gggggctccg gcgga gttgg gtgaccgtga ggcggttggt ttggagaggt tgtcactaag gaggagttta cttttcattt gtggagatga tgggagccca ggaaatgtgg tcagaaaaag gcccctggag gggtcctgga agcgtcctta gctggtcctg ggggactggg cggggaaggg agcgcagaag gaagcaggtg ggctggcctg ttcctccttg agggcaggaa ggctgtggct tggtttatgc aggaagaggg gtggggacca ttgagagcat tcggtggcca gtcctgttga atgaaatctg agcactgagc tggatttgcg tgccttgtag gtgactggtg cagttgcagc accaggatag atagtgcccc atattccgat ttttacctgg gattaccagc caggctggag tctcagcaca ggaaccgagc gtagggattt gtgaatgaat gagtgttcgt gttttaagag atgtgggaac ggagcagagt ggaacctgtt gtttgtcact gtaacgtttc tctgggttgg ctgcatccta gacagaattg agagaaccgg gccatgagtt cggagtgtca gcagagccac cgtgagggga cgtggtttcc agtgcagtac agctgtctga ggatgattct gcacatacaa ctgatcttcc cagagagtgg gattttgagg aagtggaaaa aattgtttag atggttaaag cagtcgctaa atatttattt cttaaagaca gaagaaaaat aattatttaa atagtgtcct cccgtatgtt ctcaaagtac cgttaaatcc aagaggcttt tcattgtgta aatctgggca ctgggtcttt tttcctttca gcaaacaaga taacaatggc atatcctatt gtacagagag aaaaaaatac tcctaatgtc agat agaatc acagccttta cctggtcaat aggttaccaa gaaccattcc atggattatt tgtcaaagac acatttgtat ttttaatagt taaaaacttg gtcttcattg gatgaaggca gcccacagtg gaggagtgag gagaaggata acatgttttg gcttcaatct ggaagtccaa aagtttttta atgaacctat ttttttttta acagcatctg attgtttaac atgaagcttg actgatgttt gcttgtggtt gcagtgtttc tctggcagta atcataatca ccattatagt aaaaacatcg tttattgagc acctactatg tgccaggagt aagctttctc tcctaactat tgaatgttaa cagtgtgatc ttagcatagc atgtctgaag actagagtct aggatttatg acagtacaaa cctaccagtt gcccattcgt tacagaggca tgtgctgaac gctatacttt gcttttgtca agctctctct ctctccccct ccctgttcac acaaagttag ctttaggagc aattttcagg gatgaaaatg ttaaatttgg tgaaaatatt aagttgggat attatcagta aagaccatcc ttttgcctga cggttgaaga cctgaagcag tagttcaaaa ttgtgacagc tcatgaactt tgtgatactt tttttggttg ctccagagaa attgtgaatt tttgttttct agaatatcta taatctcagt ttagtacttg gcaactctag ctactccatg tttgttcagt agaagaaaga atactgaatg aacccattct gtacattttt aatacttccc 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2 700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200

201038739 tcaagaggct gcttagagac caaatgaagt aaaaactgaa gggacaatga gccccaatta ttagcagctt gtataaactt gtattaatct cattgctact tggcttaagt attaaaaggc ttgacctggg taggagttta actcattttg ctttatcaag aagattccca aataagagct taattcactc taacccatta ctgggaatgt tggaaatgaa acccctggtt cattgactta acactacttt gcattgtgtg gatctcagaa agtatatatt tgaagtgaaa tttgagacca gagttattta tgcaaatctt acagaattat tatcacagaa ctgccctaat atatttaata gtatcctttt aggcctgtga ctcggtgttg tattgtcttt tctcaatcgt aaagttcaca tgttctgtgc ccttttgttg ctgagataat tttataaaaa ctttacattc atttttttgt agactaagca ttcttttttt gccgtaagag tttgagatag ttgctaggca cgtggtggtg ccccacccct ttccaaagca cacaggtgga aaaataaaat ctttaccatt tggtcctgtt tacagtggtg gaaggttgta atagtttcca ccccctcccc ctggccattt tacatcaatt atgaagtgta tcctcattat ctacttggcc tctggtattc cccagagtgc aaaaatgtgt gcttgatctc tctctctctc ttttctagca ataattcatc tcattaagct tttagaatga tgaggcatta tgatgtcaaa square < 210 > 3.

≪ 211 > 5000 < 212 > DNA < 213> person who is wisdom species (Homo sapiens} < 400 > 3 aagaaaaata gacctattgt aagtgggata caattatttt tccctaacct ccagccctgc agcatttcta gtaacagatg ttagatgaga agagttattc ttctaagctt tacagaatgc taaataaatt atctcaaagg ttgactcagt aagaatttca aagagttaat tgtcagtatt gaactttgtc aactatcatt ttcatttatt catttttgag tatctatttt gcatgaggca ttgctctagg gagttgggac atatcaataa agtcatcaaa gttcttcaac attggagctc aaattctgtt ttgcgggggg gagtagggaa atagatacta aacaatagat tttataagta attattttgg tttgttagaa agtgatacgg gaaaagcaaa ctggaggaca gtgaatcagg aataggagaa gaggttgcag cttagagtgg tttgggatga aatgatagtg ccacagaaat ggagtgtgcc acacgcatgg gggagtaggg ggtggagcac gttgctagag aggggctaga catggagata tgtctctatt ttcagcagag aaggcctgtc aagatattaa aagtacatct cattgtcccc ttctcccact tgttggagtg cttcctggcc ttgtctctca gagctgaaga tgacataaat cataaaagaa ataaatggta ccgcctgggc tctctgatct tataaggtcc cacctaattt Aagctctggt ggatagccta tttcccatta gttgtctcaa ttcacagagg atggagaaag aaagcactga atgagatgtg cagctcaact caatgggca c ttgttagccc caattatttg gctaggacaa ctttattcaa gttaatggaa tacaaaactc agcagttctt atggagactt caatgtgctg tagaacaaac cacagataaa tgaactgtct gaaagaataa tatgaggcac ggctaaacaa gtgttgtgga agagaaagat cactgtaatc tagagttagt tagagttagg gctttctgga aaagtgagac caacattaca ccttcgaaaa ctagtgtggt ggtggtgcaa gtggaggaaa atgggaaaga ggcaacagtg tccataaaga cacagaggca gaaagggcag aggccctggg atagttaaaa gagaagtcta gctgagggac ttcttgactt gactagcagc aattagccat gactaataag gctttccaca ctccaaagac ttagatggag ggataaaaaa ccatctaatg gcagactgtg gtagcctccc tagagacaca gagctgggcc 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5000 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 5

201038739 ggatgagtcc aggcactgac gtgatccatt atctttcacc ttaaagagta aaagggaaac taaagttaat tacctccacg aaacaaaaag gtgccttctt gtgcttcaat tacatggata tattctacta gtctaaaagt atcttctcac ttctttctgt cactgtgagg acttgagtca gaagaaagtt taaatacagt cattgagctg gaaagagtgg aaagagaagc aaagaggggg aagctgtagg aaggacgaag tcacccccaa gatacatggt tactgcttac accaagcaag ctgccttggg aacgcttccc ccgagcagcc agaatgctca gcagtggaag acacctctat tcctgtaggc gagtcctggg aagctggtca atctgcaaat gccaattccc agcagtgagc tcggtccacg tgtaaatcaa gatttgggga aagagtaggg tgggtggcat ggttgacaat gtcatcagct ccctcctctg actcctgtgg tcgtgccccc atctactctc actcagctac accccacctt cggatttgtg atggacgctg ggtccctagt aaccacagca agtgtctccc ccgcacttcc cccttcccca cccccacccc cacccccaac caccacccca gcgatggagc ctactctgct ccaagccgcc gctaagaccc ggagaagcgg aatttcactt tgaaattccc ttgcctcgtg agggccggcg ctgggcatgc tcagtagccg cggcgctgct gctgggctgc tgggctggcg cggagtccac cctgccgtct ccgccttggc ttctgggcgt ccagaaggcc aggcatttgc cgcctctgag cgcttctgtt ccccttaccc gcaacctcct actgctcttc ctctctccct ctcttaggga ggttgaagct ggtgctggtt tctgtcggcg ccacagactg actgctctgc aaaccccagc cgaggacctg aatcccggag actagaagac ccttggcggt ggctctttct aatagcactt tacctgaagt ggggtcgtgg tggagtttct cctccacctc tcaatgcaaa cactatgcgg agagcagtct gcttccctgc gctgtgcctg ctccttaatc ttcacgctgc aggtaagggg ttgccaggct tagagccgga gctgtgcatg agatgggaaa ctgcacatgc ttaaggactc taggaaaagc ttgccttgcg aagaaagcct ttctaaaaag gtaaaacagg acagtacctg acagggaagg ggtgagggag tcgtgcacct gttggaaaac tgaggccgaa aacttaacct aaaattagct cttgattttt ctttacttta tacgaaaata gtgaacattt tcaatatgaa tacaggtttg ctcccccctt cgcctcccct ccgtctagat tccctgctct tgtttctagg ctatgcactt aactgtcaat ttttcaggag agggataaga catcctgcta gatgtaacct tttctactgc agcggctact acattcataa ggtctcttgg tctagcgagc gctcatagga aggcattggc tgtaacctga ctccgcccaa aagatcgaaa tacatggttc acattagtga tgctgcacca ggctcctttg gcccctgctc ttgtcactcg aattttctaa gccaataaga ggaagaaaag gtttcaaata gaatcctctt gtctctttca gcaccgtggc tagcgtccgc tggcatatta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa tggaccacat acaattacag ttccaaagct gacaaccctg ggttaggcac tgtccctcgg acagatttga taaggtgtaa ccaagagtaa cagtgagact gctcccaaat ataaatagat ttgaggcacc agaatcaata tcaagtctcg tgaggaagca aaccttggga agtacctgag agatattata tgtggtgtcc ctgctttttg catacatagc acctctattt aatgcagaca gacttgctag gctgaattcc ttatcattgt cacacacaca tgaaccaaaa taaaatatgg cagggagatt ttagaaaccc tactgtgaca gcagttgtcc atgcagtagt cctgtaacaa ccagagacga ggcattttcc ctgagtgtgc ttctcatcca atcacacata tccattcatt ccttcaagca acatttgctg agtgtatact atgttctagg ggtaatgagt aaaaataatt gttctgaaat tacaatcctt gcagtatctt ttttaaaaga gtgggtgaat tttattctct gtgtacaata gtaggagtga aatatggtac tttcttattc cagctaacgc tattatttaa atgtatcatg aatcttttga gcaaataatg cataggatta ttaaagctat tattaaatac acatgttaat tgttataatt atgatgttat attgtggagt tttcaatagt tctttggcac attttgagaa gtagaaaata gctatgactt gattattaaa ttatttgcaa acagctgtag tgaattattt cgttaccaca tacaattctt caatgctgaa atgtgggatg aaaacataca 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2 400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 6 201038739 cattgggttg agttttattt atagtattgc ttaataatag actgacacaa acattcatta cagaaaaatc tcaattctta ctaaggtagc aatgatttag ctggtctgcc aacaatatca gtgtagtcat tttttcaaaa ctaacatata aaaaagttta aagatcacat ttgatacatc ttgagtaatt atatcttaaa agttataaat aaactgaagc atgtgaaaaa catgtcttga tttactgtag atgaatattc gggatgagaa aaatgaggac ttttgctcat cagatgagaa acctagaagc ctgtagccct agattttacc tggagttctg ccaaagggta tattaccaaa gccaatccaa tccatctcta cttcaacttt ttaatttgca aaacaaagat aaaaagtaag attaatcaac caattttaat ttaataagaa aatgcattta aggacatttt tatatttcaa ttgcaagata ctattcattg tattcatatt taatatagta aaatacattg gtgttttatc acagcagatt gtaagtttaa tattatagtt ctcttgccaa tttcatttta aatgcatttt aattttaaac agcaatttgt gggtcacaag ctgtcatcag attaaaattt atcacagtta tctaataaca ttgttagggc ttttcatcag aactaaaaca aacacagctc Cttggtctaa gcttctatac atttggagat taagaaaata agtggatggg atgacaaaga tcaagaa atc aagatattaa tctctacttg ttgaaggtgt gagtttgtta cttttatgga agacttttct ctcagaacag aggctgactt cataaagaag ggaaatgaat tctatacatt caggcctctt taactgtaaa gctaattgat ccattttggt tacgcccagg gtatcagcaa ctgctttatg ctggtggaac tgatgacacc ttataatgct ttagagatgg gaggaatgtg ctaacgggat gtaagtgttc tttttagcta

<210> 4 <211> 5000 <212> DNA <213> Human Genus (Homo sapiens) 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5000

≪ 400 > 4 gacgaccctg tagactcaca tcagagtcag gtgagaaagt gtttggtctt cactcaacac agaacatagg gtcttgaacc ctcacaagtg aataaccaac aagctgcaag aaccgagata tctaattcta taccctctcc gcagaaagaa ttttacatat actttcaaat agtcttagag aatgatccct gctgattttg aagtggccat aaaaatggta tttccttcct agtgggatgt aaagatgatg acaaataagg tgaaattcaa ctctgagctt cccagcatta ggcacgaaga aaagtgtgct tgaagaatgt agcattagct cagttactcc gtggagaccg atggttaagg gcctcctagt tgaccaagag ttcatcatcc tagaagaaag ttcctccata taaagggaaa actttttgca tcaagtagga ccatccttgc tgcttatatt cattcttcct aactaataaa ttccaccaca gtaaagaggc tcagtcatat tccccaacaa aggcacatct gttcacactc acttatactc tataatggtg tctaaatttc ttagcctgag agcaaatact ctagagccac tctaccctct taaatctaca tgaagttgtc attaaatcat tttatggaca tggctgaaat tcaccgcacc aatatggtgt tagcatcctt ctcaatgaat ctaagactgg ttaaccattt ttgtgtagcc tatgacttca tctttggcag agatggctgg gacgcatgta tcgccccttc cctcttgaac actgtctcct ttttaatatc tttatttgac aaaaaaaaaa gctatttgaa tacaaggaaa gttttggaaa atccagcagc tagatgccac agttcaggca attacac Cat tccacctact gaggcttggt attaatgaat ctttgtacct catatagaca ccaagatcag tcgcaacact caactgtccg agtaatcagc cctcaggaca aacaaaacaa tccacatctg atgcagcctc aagcatcttt agtagcatta aatcatcctc ttctggctcc agagctcacc 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080

201038739 aggtttgagg gaaacaggat caaatcaaaa gagtcactca ggactccagt cctcactcaa ggacaaactg ttccacctcg gacagggaga gtttccgcat tctgagaccc agcataacag gtcctgaccg gcatctggca ctcggactcc caatcatact ggatcacact ggctcgggat gtgtaaagtc cagggcttct cacatttgat gacaccaaag ccgcctaaaa acaagagaga attaacaact acctacggcg gtctgatatt tgcccaagag atgccgcccc ataaaactcc tttacatctt tataacgttt ttattttgcg ttctccttca taacccacat ttaactcacc atagatgtaa tgtttaaaat tagttaccag ataaactctt acgcttccaa actttaaggt tccttcgaaa ccttctggta aaactgttgt tccacggaaa tgggaacgta acggatgagg caatcttcca cagccgcaca cagttgtgta tccaccgcta aacggtccca gtcatacatt caacgaccca cgcggagtca gaagctacca ccacacactg tcaaaatcac gcacacacag tgacggcccc ttgcccactc ggtcactcgc ccacaatctc tcgctagaga atcacacgca gatagcacac ccagcaccac agaccccagg aagcaaccca gggactcgaa cacacgaaca gcactcctcc gcgcactgcg caggcacgcc tgcgtccggc tcaccctgaa acatcgcgag atccggcttc aaggccgggc tgctgccttt acgcctaaag actatgtttc ccggaagaca ctgcggcgcc ggccctatca tggcgcagca tcggtgtgct ttgtgcgtct gcgccatctt ccggctgcgc acggcgaatc caccggtacc gtggtggaag cgcgccctgg gctgccgggg gcgcggccgc ggtggcactt ggacccgagg aggcggcagg tgagaggttc cggagctttc caggcgctct ggggtccagc aggagctggt gcccgggccg gttgggtctc aggcctgaga agaacgcaga cgtctcgcct catcgtcgct ctgtggcttt accggcgtga gactacattt cccgccggcc ctcgcggcgt gcgctttctg gcgccccctt tctgcttcca gcctcatagc ccaggtgcat ggacccctta ggtgggtgcg caggggtctc cgaccccctg aaattgcgga tgttttgcat tcactgttat gcgtgccttt tttttttttt aatctgagag gaaatcttgt ttcatgaggt tctcagagag ttacaagacc ccagaagact tagaaccgct agtttagaaa aacttatact tgggaacatt ttatatgttc aaattctatg tccagcaatg gggaaacact tgagtggatc acaggccatc ctttgtaaat aggatatcgt gcagttaata caaataatgc ttataaaagt ggtcataata aggaaaagct gcctattaca tcatattaag caacattcat aattatttca cttacacgtt acttagaacc aaatgtttgt aatgcagtag gcaccttata tactccttta cagagtactt cttgtttaac agtgagctca agaactggtt aaatgacaac actgcgaata cttgggtgtt ttttcttcca atttttttgt tttgttttgt ttttagcctg tgtgttagtg agtttgactt ctttttgctg aatgttctca aaggattcag tagttaaiat ctttgcttgt cagtttttga tagtcatttt tgatagtcac ttggcatccc attatatggc tgtgccaaaa atgattcaac ctcttggcta cttgtggact tgcctactta cgtactaatt ttcagttctc ctatttagca aatattattg gacatgcagc tttgtgcact gctgtgtctc tatggaagga cacatttatg tgctgataat ggagcactgg gcgtgctgtg tgtaatttgt ctcttactgt tccttccaac tctatcccat ttgttgatgc aaggcatgct ccatgaagtg gcatgcacaa agcctcagtt tgggctgtag agctttttgt atatttggcg gttgggaaaa gagccaggta gactttcaaa ggtgttatat ttgctgcagg agagagttaa atggcctagt cctggaaagg ctttattcta ggctggggct atgcaaatga tgcaggcctt ctatgctccc tggcttgagg gaagccctcc ttgtccttga ggtaacatgt gggtctcagt cactagaaca gccctcagca tccctgtctt agacttgcat ttgcagcaaa acagaaccac tggatgttta ggggaaatgg gagggaaagg accactggtt cccctttgcc ttgcagcagc taaagctggt ttcctggggt tgggtgagga tccctttgcc tccccctcct tctcattttc ttggtgggca gagggcaagc aggtggggac ttgtaacttg gcattctagg aactgtgagg caagcctagc ctctcatgtt tgctctttcc tcccccagct ctaccgtcgc aggactctgc ccttccccaa 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2 160 2220 2280 2340 2400 2460 2760 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 201038739 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5000

gagaggaacc agaaggagga cctatcagcc cttgaaattc taaaagtcat agtttttaaa gtcccttgtg atgtaattga ggtttggctg ggaatccatc tcccataaat atttgctttc cttatcttga aactttccac ctgggttcca tccagaaatt tggttctcag actttcccat tttagggaat aaaaggacca ggcaggagtc tcctctccca ttgtcctctc ccctctgaca aatgcccaca gatttattct ctatagcatt ctgcatctcc tgatggtttt cgtctgctag gaggagaaac tcatttccct gtatgaaatg agagcatcct ttgtaagaac tgaagtttga agagatggaa tgaatgctta ggagcaatca catatagaaa agcaggggag gtggtgttgg ttggtttcag tttcagcgca tggtacagtg tcctgctttg ggaatgatta atgattggat aaaaatttga tgccagaatt aaagtctagt ttgacgttgg acagagcatt tgaatctggg acatcctcca ttagtttctt tagcttaaga ggaagaaaag tacagagctg aggatggtga gcgatgtagc aagtgagatg atcatctagg tgggtgagtg gtcagggctt ggaaaagcct tagagatctg atccaacctc gggatccaaa ataagaggat gtttgagtca gttgtttcac ttgatagaaa attgtgcact caataaaagt tatctccatg gtttttccca tgaagaaaaa tgcttgtgtt atgttccaac ataatgatga actttcatct ccaagaaatt cttgacttac atgaaaatcc tgtatctaag atagaaaaca tgtttccata attttaaaaa ataaaatatt tggggaaaat ccatcagaaa tattttttgt tgttattgtt attccaaaag atagaagtga taggtctcac cttgagaatt tattgtatgc caagtataga gtgggcagtg tgctttatta cactgtttct taacaataag gaaaactcca tttagcttga ttttaatttt catcccagac ctgtacacag agcattgatt tgcagttaaa aggaatgttt gagaacaatt tgatcattct gttttactca tccccatttc ttctgtcacc tttcacattc agtcccaccc ttcttgttca acaaaacaac cccccctccc gcaaagacct gccccatctc ctttcatccc actgtgaacc attgaaatac atatatatca < 210 > 5 < 211> 27 < 212 & gt DNA <213>Artificial sequence<220><223> Forward introduction<400> 5 gttttcgtcg tttttcgttc ggagatc 27 <210> 6 <211> 25 <212> DNA <213> Artificial sequence < 220> < 223 > reverse introduction <400> 6 gctctcgctc tcgctattac tcgct 25 201038739 <210> 7 <211> 23 <212> DNA <213> artificial sequence <220><223> To the primer <400〉 7 tgtgtatgcg cgttttttcg ttc

<210> 8 <211> 23 <212> DNA <213> artificial sequence <220><223> reverse introduction <400> 8 acctatatat ccgccgctcc gtc <210> 9 <211><212> DNA <213>Artificial sequence <220><223> Forward introduction <400> 9 cggcgttggg tatgtttagt agtc <210> 10 <211> 29 <212> DNA <213> Sequence <220><223> Reverse Initiative <400> 10 aattacgaat aaaaaaaaca aaaacgctc 201038739 <210> 11 <211> 28 <212> DNA <213>Artificial Sequence <220><223> Forward introduction <400> 11 tgacggtttt ttgtttattc ggttattc

<210> 12 <211> 21 <212> DNA <213> artificial sequence <220><223> reverse introduction <400> 12 cgaacgcaaa cgtacctacg c <210> 13 <211><212> DNA <213>Artificial sequence<220><223> Forward introduction <400> 13 ggttaagttt ttttttyggy gtagtt <210> 14 <211> 26 <212> DNA <213> Sequence <220><223> Reverse Initiative <400> 14 tactccctct acctcccrac tctctc 201038739 <210> 15 <211> 28 <212> DNA <213> Artificial Sequence <220><223> Forward introduction <400> 15 ttgtggygta gaggattatt agtttggt 28

<210> 16 <211> 22 <212> DNA <213> artificial sequence <220><223> reverse introduction <400> 16 ctaaaaacrc aacccatccc tc 22 <210> 17 <211> 27 <212> DNA <213>Artificial sequence <220><223> Forward introduction <400> 17 ggaattttat tttgaaattt ttttgtt 27 <210> 18 <211> 33 <212> DNA <213> Artificial sequence <220><223> Reverse introduction <400> 18 ccccacttca aataaaatac tattaaaaaa aac 33 12 201038739 <210> 19 <211> 30 <212> DNA <213>Artificial sequence <220>;<223> forward introduction <400> 19 tagtgayggt tttttgttta ttyggttatt 30

O

<210> 20 <211> 26 <212> DNA <213> artificial sequence <220><223> reverse introduction <400> 20 taaacrtaaa aacaacaacc cracct 26 <210> 21 <211> 5000 <212> DNA <213>unknown<220><223> DBC1 sequence by sulfite sequencing <400> 21 60 120 180 240 300 360 420 480 540 600 660 720 ggtttttatt tgaagttgtt tgattaygtt gttttagatt gattttagaa ttgtattatt agtagggtag ttgttgttta ttaatagtta tggaagtaga ttttagttgg gggatgatgg ggaaataaag agaaagatag ggaygaataa aggaaggaat ggggtataga tagaaagaga gagagagaaa agaaagaaag gaggagaggt aagagatagt gatttggggt tttatatygy ggtgggtggg agaaggagtg tgtgtggtgg gygtattttt tygtggttag aattaaagtt agaattgatt tggggaattt gtgtttaaga tttgaatttg gattagaagt taagggaaag agaaagttgt ttaagaaaaa agggagaaat ttttattagg ygaaaataga aatygttgat tgggtttgtg gttggagagt tgttygtggt gttgattttt ttttattggg attttagtga aggttgagaa gttttgtatt ggttgttttt tttttttttt tttttttttt ttagttttga ttttttgaga Tttaggatta ttttggttag ttygatgttt tttttttttt tttttttttt ttt Tttgtyg attaaataat ttttttygag tttttagttt attatttaaa tatttttttt ttattttgtt ttaagatttt ttttttttta attagtttat tggtttttag attgttttag 13

201038739 gtttttttgg tggttatatt ttttttttat tattttttga attaaaaaat aaataaaaaa tatataatgg taattataaa agtagataat atgtattgag tatttattag gtgttaggga gtagtttaag tgtttttgta tatattaatt tatgagtagt ttaagtgttt ttgtatatat taatttatta taattttata ttaattttat tagatgggta gatatattat tattttatta aatagataag gaattgaggt ttaaggggta agtgatttta ttaagattag atagttatag gattttaatt tttttggagt ttgtattttt attgaatttg gtttttttat atattttaat attttttttt atgatttttt gtttttttat ttggtagttt taatgttttt tatttttgtt taatttttag gggattaggg atttgtattg ttgatttggt ttaaattaga agggaagtaa agtaaataaa tatattaaat gtgygttgtg tgggaattat tataygaggg ttttattttt tgttttagga agaggtttta tgttagtagt tttagtttgt atttaggttg attgtagagt attttgtttt ttatttttat gttttagttt ttgtatttty gttttttttt ygtttttggt ggtttttaga gaatttygtg tttttttagt tttttttttt tatattttgt ttaygtagag aagtttttgt tttattttgg gaggttaygt gggttttygt ttatatatyg agagaaataa atagtgttaa atatttatag agagaygygt agatataaay ggatttatay gggtaatttt ygagataaaa tttatattyg atggatttay gyggtygtgg aaatatttgt ygttttagaa atatttaggt attygygata tatatagtat agttaygttt aagggtatta ggatttyggg tttgygygta tgygyggttt ttttggatgt tygtgygtat agatataata ttttataygt tttagattta ygaaattttt tayggtttag ttttagttta ttygggtygt ttttttttyg aggyggtttt tygttttttt ttttttygtt tttttttttt tttygtttaa agatgtataa aatattttty ggaagtaatt tyggygttta gttttttttt tttygttttt yggtygtygt ttttttattt attttyggty gtygtyggtt aagttttttt ttyggygtag ttyggtttty gtygttttty gttyggagat ygyggygtat ttggattttt ttttttatty gttagtygtt tygttttygg attttayggt tgtaaattga tttggygygy ggggaggagg agagygtagg ygagygaatt ygygagagag ggagagagyg agygagtaat agygagagyg agagygagag agtygggagg tagagggagt agtgatygtt tttyggagty gggatttatg tttgttttyg ggattagyga aggggatttt ayggttgagt atgagttagg ttgttaggag ttaggtattt ttaygtttgt agttttygyg tygtgttygg aatgygagtt gtaygtaggg tttttttaag tttttatyga gtygaataaa aagygttttt tygtagtttt tygttaaaga yggatattga ttttaggtaa ggyggygtyg ggtgtagygt ttygtagttt ygttgttttt ggattyggtt tygggtygta ttyggggygt tttygygttt ttttgttttt ttttttatyg gtatttttgt tygtttttta tttggtygtt tygtygtttt taagtttttt ttagttttag ggagggggtt tttgtgtttg gggtttaaag ggttaattgy gggtttgagt gagtggygtg tgtgttttyg ygygttttyg aygtgtgtat tatggtggga atttgatgtg gtgttagtgt gtttgygtgt gyggygtygt ttgygtttga tgygtgtgtt ygtggtgtgt gtgtgtgtty gtgtgtgtaa gggaggggtg aagagagaga ggttttataa tttatttayg gygygatgtg tgtgtgtatg tttgtaygta tgtgtttgta tgtgtgtttt ygtgtgtttt tttaattagg tttttttagt ttatayggaa tgggattttt attataggat taygtagtta tygggaaatt ygttgtggat ttttttttgg ggttttgggt ttggggtttg gggaggatta tggggttgta gatggtattt tatttagttt aatgttggta ygtttgaagg aaaatttttt taaayggtgg aattttgtta tattgggatt tatagtttaa tatgaaagag atatggttaa ttttygaggt aaatgagayg ttgttatttt aaattttata ttgggtagat tttaagattt tgatgggaat ttggagatat tggatagtgg gtgatagaga aagggggaag ttagyggtgg gttttttatt tggggtttgg aaagtttggg atagggattt attttgtatt ttygtttgta attaatagag ttttttyggt ttttgttggt tttggggagg atttggtaag tttgtatgga ttttttttag gggaaaggag 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 14 Ο 201038739 aaagygtttt yggggatttg tttatttatt atagttgtaa agggttttag aggaaatttt atttggggyg gttgtggatg atatggaagg agatggatgg ggtatttttt taagatagat tygttttttt ttttttattt taggygggga agtttttaga gagttttttt ttaaatatgt ttttttatgg tttttttgga gttaggggga tattgagaga aggtagagag gtggagaggg agagagagag agtaagagyg agagagaggg agatagagag agagagtgtt ttagtattga gggagattta taatttgaaa aggggttgtg agtgtggaat ttatgataga atgtggtagt aattgattta aattgttgty ggtttgtatt tygygttttt tatttggttt taaaatattg ttaagggtag gggggyggtg gaggaatttt agttaggaag gtagtttggg ttaaggtttt tttttttttt ttttttattt tttgggaggt tttttaggta ggttttttgg ttattttggt ttggtattgt tgggtttggg ttttggggtt agttattgat ttgattttta gttgtttttt aaaggtttgt tttatttagt aaaaatgttg agttttattg ttgttagtat ggtttttaga ttyggtagtt gttatttttt taaggtaagy ggtagtygtt ttgtttttag Gtagggaggt taggagaaga taagggttgt gaygttggga tatagttttt tgtttagttt agtaygg agg ggtygttgaa aagttttttt aggggaaaaa agtagaaata tatttggttt aaaaaatgta tatatatatt ttaggaagat atatatatat atatatatat taattatata tatatatata tatatatatg tgtttatatt ttggggagag ataaaagtaa atatgtattt gtaggaattt gttttagtgg gtgggtttta tttgtaggta tttgtagata tattattttt tgtttagagg ttgatatttt aggttgaagt ttttatatag ttatagagaa ttttttttag gaaatttatg ttagggtaga gttttttgga ttattttggg tagggggtat tagggttaag gaggagattt atttttggga attgttttat tttttttttt ttttaattta tagaygatyg aaattggaag gaatattaaa gagtagaaag gtaggatttt gtggtttaaa gagggaatga gaatttttta atatttagta gtgggttagt ggtatgttta ggattagaat ttaggtttat taattagtta gattaatatt ttttttattt tttttagtaa tatatatttt aggattgtat attgttttta tttttatttt gttaatttgt tattatgatt tttattaaga taatygtttg ttttttttta ttaaatttta tatttttaga gatgtatttt agggtttttt tatttggaaa tatggttttg aagagaaaag atttaatgtt ttaattatta ttttygaaag atatatatat atatatatat atatatatat atatatatat atatatatat atatataygg tggtgggggg aggattgaga gagagataga gagagaatat

Q &lt;210> 22 &lt;211>5000 &lt;212> DNA &lt;213>unknown <220> &lt;223>PDE8B sequence of sulfite sequence &lt;400> 22 agaataattt tgaagtatta ttttattttt agaatttttt ttgtagggat gtttgaggtt tggttaattt ttttttttgt ttaagtttgt ttttttttat tttttttttt tatttttttt tataaatgtt gattataaga gtatttttta aaaagttttt ttttgtttgt taaatattat tttagaattt gtttttagga ggatttaatt tgtgtagaaa ttttatgagt aatttgtttg tttattaaaa aatatagtat agattttttt taggttagtt ggagatggag tttttttgta ttaaaaaaag agagttaaat tgagaagaga agtaaaggta gaagaataag gttagatttt 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5000 60 120 180 240 300 360 15

201038739 ttttgtataa ttgaatttgg gattgggaat gatattttgt ttttttatga aatttagaaa ttgttttttg aagtttttat ttagttatat ttaagagtgg tttaggaaga aaaaggaaaa taagaatgaa aaaaaaataa aaaataaatt ttaaaattgg atattttttt gtgttttgta ttaagttttt atgttattgt ttttgttygt ataagtaatt ttagataaaa ttttttttta gattatttaa tttatttttt ygtgttattg tgaaaatygg gtaggaatta ttgygggaat tagtgattga atgggtagtg gtgtttygta gttgtaattt agagtttata agggaaggat gaatagtagt tattttttta aaggtatttt tttttttttt tagtatagtt tagtttgtgt atgttttatt aaggagaatg agggaaggtt ttgttgattg tttagtttag agtgtgagga atagtagttg atatgggygg ttggayggga tagaaattag tatgaatatt tagtgatatt taaggaagag gtgaaaatgg gggagaaagt taagaattat tagatagttt ttttttgttt ttgttttgga ttttagggga aygtatgtgt ttaagggtgt agggtttttt attttttggt ataaattttt aaaygatgag tagatatgga atgggttttt tttgaagatt gggttattat ttaatttagt tttyggagta ggyggtgggg ttgggggaag aggayggaat tatgaggttt ttttattatt tttygggtgt gttgagaggt aagtaaatgy ggtgggtggg tttgggttgt agggtttttg ttatttyggg aggggttttt tggtgtttag ttttgggatg tttttttygt tgtgaaaatg tgttggtgaa aagygagagg tttatatagt tttttagggg aagaggggtt ggggygttgg gggyggygty gggatgagtg tagaagagay gaggtttttg gatagyggag gaggagggga gggygtygag gygyggtgtt agttgtygyg tataggggtt tygyggygga gtygagtygy gggtaygttt tgttttgtyg ggagagttty gggagyggyg gga £ gggygg aggggygtag tggggttygg gyggttgygg tygyggagty ggggtatttg aggaggaagg agggtgggag ygagggaggg aggggayggg ygtagatyga aagtggggaa agaaggtgta ggtaggyggg taggygggyg ggygttttgg tttagggtyg ygggtgyggg agttyggyga ggtygagttg ggyggyggyg ggggtygygt ygagggagga ggggaaggyg gaggygyggg gagygtgttt ggggygtygy ggyggggagg gtggyggtyg ttggtgygyg yggggygttg tgtatgygyg ttttttygtt yggggaggaa gatggtttaa aagggaaagt tggggtgayg ygygyggttt tyggaggtty ggyggggggt atygyggtta gttygaygga gyggyggata tataggtygg ggggygygta gttygggygt ygtygyggty gtttttttat tgtaggtggt agygggtgyg ttgggtttyg gyggtygygg gygygggygg gygygygggg gagttyggty gagggatggg ttgygttttt agtatttatg tttygtagag yggygtgatt tattgtyggg attyggayga gtttagttyg tttygttaga ttattagygt gtygtagggt tyggyggtat ttttgttyg g ttttttygtt tagatygayg tygtygaygt tattttttyg agtygygygt ygggattttt tagygtagtt ygygttygta gggttygtat ygagttgggt agyggtagta gygygggttt ygtagtttty gtygygatta ttagtagggg tyggaggygt tattgttgta gtagygtyga ggtygagatt tagatttgtt atattagygt gaaggtaaat gtttygygtt ggtataygty gtgggggtyg ttygtttygt yggyggggtt ygtaygggta gggggtttyg gyggagttgg gtgatygtga ggyggttggt ttggagaggt tgttattaag gaggagttta ttttttattt gtggagatga tgggagttta ggaaatgtgg ttagaaaaag gtttttggag gggttttgga agygttttta gttggttttg ggggattggg yggggaaggg agygtagaag gaagtaggtg ggttggtttg tttttttttg agggtaggaa ggttgtggtt tggtttatgt aggaagaggg gtggggatta ttgagagtat tyggtggtta gttttgttga atgaaatttg agtattgagt tggatttgyg tgttttgtag gtgattggtg tagttgtagt attaggatag atagtgtttt atatttygat ttttatttgg gattattagt taggttggag ttttagtata ggaatygagy gtagggattt gtgaatgaat gagtgttygt gttttaagag atgtgggaay ggagtagagt ggaatttgtt gtttgttatt gtaaygtttt tttgggttgg ttgtatttta 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 16

201038739 gatagaattg agagaatygg gttatgagtt yggagtgtta gtagagttat ygtgagggga ygtggttttt agtgtagtat agttgtttga ggatgatttt gtatatataa ttgatttttt tagagagtgg gattttgagg aagtggaaaa aattgtttag atggttaaag tagtygttaa atatttattt tttaaagata gaagaaaaat aattatttaa atagtgtttt ttygtatgtt tttaaagtat ygttaaattt aagaggtttt ttattgtgta aatttgggta ttgggttttt ttttttttta gtaaataaga taataatggt atattttatt gtatagagag aaaaaaatat ttttaatgtt agatagaatt atagttttta tttggttaat aggttattaa gaattatttt atggattatt tgttaaagat atatttgtat ttttaatagt taaaaatttg gtttttattg gatgaaggta gtttatagtg gaggagtgag gagaaggata atatgttttg gttttaattt ggaagtttaa aagtttttta atgaatttat ttttttttta atagtatttg attgtttaat atgaagtttg attgatgttt gtttgtggtt gtagtgtttt tttggtagta attataatta ttattatagt aaaaatatyg tttattgagt atttattatg tgttaggagt aagttttttt ttttaattat tgaatgttaa tagtgtgatt ttagtatagt atgtttgaag attagagttt aggatttatg atagtataaa tttattagtt gtttattygt tatagaggta tgtgttgaay gttatatttt gtttttgtta agtttttttt tttttttttt ttttgtttat ataaagttag ttttaggagt aatttttagg gatgaaaatg ttaaatttgg tgaaaatatt aagttgggat attattagta aagattattt ttttgtttga yggttgaaga tttgaagtag tagtttaaaa ttgtgatagt ttatgaattt tgtgatattt tttttggttg ttttagagaa attgtgaatt tttgtttttt agaatattta taattttagt ttagtatttg gtaattttag ttattttatg tttgtttagt agaagaaaga atattgaatg aatttatttt gtatattttt aatatttttt ttaagaggtt gtttagagat taaatgaagt aaaaattgaa gggataatga gttttaatta ttagtagttt gtataaattt gtattaattt tattgttatt tggtttaagt attaaaaggt ttgatttggg taggagttta atttattttg ttttattaag aagattttta aataagagtt taatttattt taatttatta ttgggaatgt tggaaatgaa atttttggtt tattgattta atattatttt gtattgtgtg gattttagaa agtatatatt tgaagtgaaa tttgagatta gagttattta tgtaaatttt atagaattat tattatagaa ttgttttaat atatttaata gtattttttt aggtttgtga ttyggtgttg tattgttttt ttttaatygt aaagtttata tgttttgtgt ttttttgttg ttgagataat tttataaaaa ttttatattt atttttttgt agattaagta tttttttttt gtygtaagag tttgagatag ttgttaggta ygtggtggtg ttttattttt ttttaaagta tataggtgga aaaataaaat ttttattatt tggttttgtt tatagtggtg Gaaggttgta atagttttta tttttttttt ttggttattt tatattaatt atgaagtgta tttttattat ttatttggtt tttggtattt tttagagtgt aaaaatgtgt gtttgatttt tttttttttt ttttttagta ataatttatt ttattaagtt tttagaatga tgaggtatta tgatgttaaa &lt;210> 23 <211>5000 &lt;212&gt; DNA &lt;213>unknown&lt;220> &lt;223>PTPRR via sulfite Sequence of salt sequencing &lt;400> 23 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5000 17

201038739 aagaaaaata gatttattgt aagtgggata taattatttt tttttaattt ttagttttgt agtattttta gtaatagatg ttagatgaga agagttattt ttttaagttt tatagaatgt taaataaatt attttaaagg ttgatttagt aagaatttta aagagttaat tgttagtatt gaattttgtt aattattatt tttatttatt tatttttgag tatttatttt gtatgaggta ttgttttagg gagttgggat atattaataa agttattaaa gttttttaat attggagttt aaattttgtt ttgygggggg gagtagggaa atagatatta aataatagat tttataagta attattttgg tttgttagaa agtgataygg gaaaagtaaa ttggaggata gtgaattagg aataggagaa gaggttgtag tttagagtgg tttgggatga aatgatagtg ttatagaaat ggagtgtgtt ataygtatgg gggagtaggg ggtggagtay gttgttagag aggggttaga tatggagata tgtttttatt tttagtagag aaggtttgtt aagatattaa aagtatattt tattgttttt tttttttatt tgttggagtg ttttttggtt ttgtttttta gagttgaaga tgatataaat tataaaagaa ataaatggta tygtttgggt tttttgattt tataaggttt tatttaattt aagttttggt ggatagttta ttttttatta gttgttttaa tttatagagg atggagaaag aaagtattga atgagatgtg tagtttaatt taatgggtat ttgttagttt taattatttg gttaggataa ttttatttaa gttaatggaa tataaaattt agtagttttt atggagattt taatgtgttg tagaataaat tatagataaa tgaattgttt gaaagaataa tatgaggtay ggttaaataa gtgttgtgga agagaaagat tattgtaatt tagagttagt tagagttagg gttttttgga aaagtgagat taatattata ttttygaaaa ttagtgtggt ggtggtgtaa gtggaggaaa atgggaaaga ggtaatagtg tttataaaga tatagaggta gaaagggtag aggttttggg atagttaaaa gagaagttta gttgagggat tttttgattt gattagtagt aattagttat gattaataag gttttttata ttttaaagat ttagatggag ggataaaaaa ttatttaatg gtagattgtg gtagtttttt tagagatata gagttgggty ggatgagttt aggtattgay gtgatttatt attttttatt ttaaagagta aaagggaaat taaagttaat tatttttayg aaataaaaag gtgttttttt gtgttttaat tatatggata tattttatta gtttaaaagt atttttttat ttttttttgt tattgtgagg atttgagtta gaagaaagtt taaatatagt tattgagttg gaaagagtgg aaagagaagt aaagaggggg aagttgtagg aaggaygaag ttatttttaa gatatatggt tattgtttat attaagtaag ttgttttggg aaygtttttt tygagtagtt agaatgttta gtagtggaag atatttttat ttttgtaggy gagttttggg aagttggtta atttgtaaat gttaattttt agtagtgagt tyggtttayg tgtaaattaa gatttgggga aagagtaggg tgggtggtat ggttgataat gttattagtt tttttttttg atttttgtgg tygtgttttt atttattttt atttagttat attttatttt yggatttgtg atggaygttg ggtttttagt aattatagta agtgtttttt tygtattttt ttttttttta tttttatttt tatttttaat tattatttta gygatggagt ttattttgtt ttaagtygty gttaagatty ggagaagygg aattttattt tgaaattttt ttgtttygtg agggtyggyg ttgggtatgt ttagtagtyg yggygttgtt gttgggttgt tgggttggyg yggagtttat tttgtygttt tygttttggt ttttgggygt ttagaaggtt aggtatttgt ygtttttgag ygtttttgtt ttttttatty gtaatttttt attgtttttt tttttttttt tttttaggga ggttgaagtt ggtgttggtt tttgtyggyg ttatagattg aaattttagt ygaggatttg attgttttgt aatttyggag attagaagat ttttggyggt ggtttttttt aatagtattt tatttgaagt ggggtygtgg tggagttttt tttttatttt ttaatgtaaa tattatgygg agagtagttt gtttttttgy gttgtgtttg ttttttaatt tttaygttgt aggtaagggg ttgttaggtt tagagtygga gttgtgtatg agatgggaaa ttgtatatgt ttaaggattt taggaaaagt ttgttttgyg aagaaagttt ttttaaaaag gtaaaatagg atagtatttg atagggaagg ggtgagggag tygtgtattt gttggaaaat 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 126 0 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 18

201038739 tgaggtygaa aatttaattt aaaattagtt tttgattttt ttttatttta taygaaaata gtgaatattt ttaatatgaa tataggtttg tttttttttt ygtttttttt tygtttagat tttttgtttt tgtttttagg ttatgtattt aattgttaat tttttaggag agggataaga tattttgtta gatgtaattt tttttattgt agyggttatt atatttataa ggttttttgg tttagygagy gtttatagga aggtattggt tgtaatttga tggattatat tggtatatta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa tttygtttaa aagatygaaa tatatggttt atattagtga tgttgtatta ggtttttttg gtttttgttt ttgttattyg aattttttaa gttaataaga ggaagaaaag gttttaaata gaattttttt gtttttttta gtatygtggt tagygttygt ataattatag ttttaaagtt gataattttg ggttaggtat tgtttttygg atagatttga taaggtgtaa ttaagagtaa tagtgagatt gtttttaaat ataaatagat ttgaggtatt agaattaata ttaagtttyg tgaggaagta aattttggga agtatttgag agatattata tgtggtgttt ttgttttttg tatatatagt atttttattt aatgtagata gatttgttag gttgaatttt ttattattgt tatatatata tgaattaaaa taaaatatgg tagggagatt ttagaaattt tattgtgata gtagttgttt atgtagtagt tttgtaataa ttagagayga ggtatttttt ttgagtgtgt tttttattta attatatata tttatttatt tttttaagta atatttgttg agtgtatatt atgttttagg ggtaatgagt aaaaataatt gttttgaaat tataattttt gtagtatttt ttttaaaaga gtgggtgaat tttatttttt gtgtataata gtaggagtga aatatggtat ttttttattt tagttaaygt tattatttaa atgtattatg aattttttga gtaaataatg tataggatta ttaaagttat tattaaatat atatgttaat tgttataatt atgatgttat attgtggagt ttttaatagt tttttggtat attttgagaa gtagaaaata gttatgattt gattattaaa ttatttgtaa atagttgtag tgaattattt ygttattata tataattttt taatgttgaa atgtgggatg aaaatatata tattgggttg agttttattt atagtattgt ttaataatag attgatataa atatttatta tagaaaaatt ttaattttta ttaaggtagt aatgatttag ttggtttgtt aataatatta gtgtagttat ttttttaaaa ttaatatata aaaaagttta aagattatat ttgatatatt ttgagtaatt atattttaaa agttataaat aaattgaagt atgtgaaaaa tatgttttga tttattgtag atgaatatty gggatgagaa aaatgaggat ttttgtttat tagatgagaa atttagaagt ttgtagtttt agattttatt tggagttttg ttaaagggta tattattaaa gttaatttaa tttattttta ttttaatttt ttaatttgta aaataaagat aaaaagtaag attaattaat taattttaat ttaataagaa aatgtattta aggatatttt tatattttaa ttgtaagata ttatttattg tatttatatt taatatagta aaatatattg gtgttttatt atagtagatt gtaagtttaa tattatagtt tttttgttaa ttttatttta aatgtatttt aattttaaat agtaatttgt gggttataag ttgttattag attaaaattt attatagtta tttaataata ttgttagggt tttttattag aattaaaata aatatagttt tttggtttaa gtttttatat atttggagat taagaaaata agtggatggg atgataaaga ttaagaaatt aagatattaa tttttatttg ttgaaggtgt gagtttgtta tttttatgga agattttttt tttagaatag aggttgattt tataaagaag ggaaatgaat tttatatatt taggtttttt taattgtaaa gttaattgat ttattttggt taygtttagg gtattagtaa ttgttttatg ttggtggaat tgatgatatt ttataatgtt Ttagagatgg gaggaatgtg ttaaygggat gtaagtgttt tttttagtta &lt;210> 24 &lt;211&gt; 5000 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5000 19

201038739 &lt;212&gt; DNA ' <213>Unknown &lt;220> &lt;223>ZNF582 sequence by sulfite sequencing &lt;400&gt; 24 gaygattttg aatgattttt gttttttagt atttatattt ttgtgtagtt tttatttatt tagatttata gttgattttg tgattaagag tataatggtg tatgatttta gaggtttggt ttagagttag aagtggttat tttattattt tttaaatttt tttttggtag attaatgaat gtgagaaagt aaaaatggta tagaagaaag ttagtttgag agatggttgg ttttgtattt gtttggtttt tttttttttt tttttttata tatttaatat agtaaatatt gaygtatgta tatatagata tatttttttt tttatggata aaaaaaaaaa agtgggatgt taaagggaaa ttagagttat tygttttttt ttaagattag agaatatagg aaagatgatg attttttgta tttatttttt ttttttgaat tygtaatatt gttttgaatt ataaataagg ttaagtagga taaatttata attgtttttt taattgttyg ittataagtg tgaaatttaa ttatttttgt tgaagttgtt ttttaatatt agtaattagt aataattaat ttttgagttt tgtttatatt attaaattat tttatttgat ttttaggata aagttgtaag tttagtatta aataaaataa aatygagata Ggtaygaaga aattaataaa tggttgaaat gttatttgaa tttatatttg tttaatttta aaagtgtgtt ttttattata ttatygtatt tataaggaaa atgtagtttt tatttttttt tgaagaatgt gt aaagaggt aatatggtgt gttttggaaa aagtattttt gtagaaagaa agtattagtt ttagttatat tagtattttt atttagtagt agtagtatta ttttatatat tagttatttt tttttaataa tttaatgaat tagatgttat aattattttt atttttaaat gtggagatyg aggtatattt ttaagattgg agtttaggta ttttggtttt agttttagag atggttaagg gtttatattt ttaattattt attatattat agagtttatt aggtttgagg gaaataggat taaattaaaa gagttattta ggattttagt ttttatttaa ggataaattg ttttatttyg gatagggaga gttttygtat tttgagattt agtataatag gttttgatyg gtatttggta ttyggatttt taattatatt ggattatatt ggttygggat gtgtaaagtt tagggttttt tatatttgat gatattaaag tygtttaaaa ataagagaga attaataatt atttayggyg gtttgatatt tgtttaagag atgtygtttt ataaaatttt tttatatttt tataaygttt ttattttgyg ttttttttta taatttatat ttaatttatt atagatgtaa tgtttaaaat tagttattag ataaattttt aygtttttaa attttaaggt tttttygaaa ttttttggta aaattgttgt tttayggaaa tgggaaygta ayggatgagg taatttttta tagtygtata tagttgtgta tttatygtta aayggtttta gttatatatt taaygattta ygyggagtta gaagttatta ttatatattg ttaaaattay gtatatatag tgayggtttt ttgtttatty ggttattygt t tataatttt tygttagaga attataygta gatagtatat ttagtattat agattttagg aagtaattta gggattygaa tataygaata etatttttty gygtattgyg taggtaygtt tgygttyggt ttattttgaa atatygygag attyggtttt aaggtygggt tgttgttttt aygtttaaag attatgtttt tyggaagata ttgyggygty ggttttatta tggygtagta tyggtgtgtt ttgtgygttt gygttatttt tyggttgygt ayggygaatt tatyggtaty gtggtggaag ygygttttgg gttgtygggg gygyggtygy ggtggtattt ggattygagg aggyggtagg tgagaggttt yggagttttt taggygtttt ggggtttagt aggagttggt gttygggtyg gttgggtttt aggtttgaga agaaygtaga ygtttygttt tatygtygtt ttgtggtttt atyggygtga Gattatattt ttygtyggtt ttygyggygt gygttttttg gygttttttt tttgttttta gttttatagt 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 20 Ο

201038739 ttaggtgtat ggatttttta ggtgggtgyg taggggtttt ygattttttg aaattgygga tgttttgtat ttattgttat gygtgttttt tttttttttt aatttgagag gaaattttgt tttatgaggt ttttagagag ttataagatt ttagaagatt aaatgataat attgygaata tagaatygtt agtttagaaa aatttatatt tgggaatatt ttatatgttt aaattttatg tttagtaatg gggaaatatt tgagtggatt ataggttatt ttttgtaaat aggatatygt gtagttaata taaataatgt ttataaaagt ggttataata aggaaaagtt gtttattata ttatattaag taatatttat aattatttta tttataygtt atttagaatt aaatgtttgt aatgtagtag gtattttata tattttttta tagagtattt tttgtttaat agtgagttta agaattggtt tttgggtgtt ttttttttta atttttttgt tttgttttgt ttttagtttg tgtgttagtg agtttgattt ttttttgttg aatgttttta aaggatttag tagttaatat ttttgtttgt tagtttttga tagttatttt tgatagttat ttggtatttt attatatggt tgtgttaaaa atgatttaat tttttggtta tttgtggatt tgtttattta ygtattaatt tttagttttt ttatttagta aatattattg gatatgtagt tttgtgtatt gttgtgtttt tatggaagga tatatttatg tgttgataat ggagtattgg gygtgttgtg tgtaatttgt tttttattgt tttttttaat tttattttat ttgttgatgt aaggtatgtt ttatgaagtg gtatgtataa agttttagtt tgggttgtag agttttttgt atatttggyg gttgggaaaa gagttaggta gatttttaaa ggtgttatat ttgttgtagg agagagttaa atggtttagt tttggaaagg ttttatttta ggttggggtt atgtaaatga tgtaggtttt ttatgttttt tggtttgagg gaagtttttt ttgtttttga ggtaatatgt gggttttagt tattagaata gtttttagta tttttgtttt agatttgtat ttgtagtaaa atagaattat tggatgttta ggggaaatgg gagggaaagg attattggtt tttttttgtt ttgtagtagt taaagttggt tttttggggt tgggtgagga tttttttgtt tttttttttt ttttattttt ttggtgggta gagggtaagt aggtggggat ttgtaatttg gtattttagg aattgtgagg taagtttagt tttttatgtt tgtttttttt ttttttagtt ttatygtygt aggattttgt ttttttttaa gagaggaatt agaaggagga tttattagtt tttgaaattt taaaagttat gttttttgtg agtttttaaa ttttataaat atttgttttt tttattttga aattttttat ttgggtttta tttagaaatt tggtttttag atttttttat tttagggaat aaaaggatta ggtaggagtt ttttttttta ttgttttttt ttttttgata aatgtttata gatttatttt ttatagtatt ttgtattttt tgatggtttt ygtttgttag gaggagaaat ttattttttt gtatgaaatg agagtatttt ttgtaagaat tgaagtttga agagatggaa tgaatgttta ggagtaatta tatatagaaa agtaggggag gtggtgttgg ttggttttag ttttagygta tggtatagtg ttttgttttg ggaatgatta atgattggat atgtaattga ggtttggttg ggaatttatt aaaaatttga tgttagaatt aaagtttagt ttgaygttgg atagagtatt tgaatttggg atatttttta ttagtttttt tagtttaaga ggaagaaaag tatagagttg aggatggtga gygatgtagt aagtgagatg attatttagg tgggtgagtg gttagggttt ggaaaagttt tagagatttg atttaattty gggatttaaa ataagaggat gtttgagtta gttgttttat ttgatagaaa attgtgtatt taataaaagt tatttttatg gtttttttta tgaagaaaaa tgtttgtgtt atgttttaat ataatgatga atttttattt ttaagaaatt tttgatttat atgaaaattt tgtatttaag atagaaaata tgtttttata attttaaaaa ataaaatatt tggggaaaat ttattagaaa tattttttgt tgttattgtt attttaaaag atagaagtga taggttttat tttgagaatt tattgtatgt taagtataga gtgggtagtg tgttttatta tattgttttt taataataag gaaaatttta tttagtttga ttttaatttt tattttagat ttgtatatag agtattgatt tgtagttaaa aggaatgttt gagaataatt tgattatttt gttttattta tttttatttt ttttgttatt ttttatattt agttttattt tttttgttta 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3 360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 21 201038739 ataaaataat ttttttttty gtaaagattt gttttatttt tttttatttt attgtgaatt 4980 attgaaatat atatatatta 5000 o

twenty two

Claims (1)

201038739 VII. Scope of application for patents: 1. A method for screening cancers by detecting the state of methylation of a target gene in a test subject cell as a screening indicator for cancer presence, the method comprising the following steps: Step 1 provides a test subject; step 2 detecting a methylation status of a CpG sequence of at least one target gene in the genomic DNA of the test subject, the target gene consisting of DBC1, PDE8B, PIPRR and ZNF582; and step 3 according to the The presence or absence of the methylation status of the target gene, whether the sample has cancer or precancerous lesions, or as an indicator of prognosis. 2. The method of screening for cancer according to claim 1, wherein the test sample is Pap smear, ascites, blood, urine, feces, sputum, oral mucosal cells, gastric juice, bile, sub- Isolated samples of cervical epithelial cells, cancer tissues after surgery, etc. 3. The method for screening cancer according to claim 1, wherein the CpG sequence thiolation state detection method of the target gene is methylation-specific polymerase chain reaction (MSP), Quantitative methylation-specific polymerase chain reaction (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing HPLC The method of screening for cancer according to claim 1, wherein the target gene DBC1 has SEQ ID No. The nucleotide sequence shown in Figure 1, the target gene PDE8B has the nucleotide sequence shown in SEQ ID No: 2, and the target gene PTPRR has the nucleotide sequence shown in SEQ ID No: 3, the target gene ZNF582 has the nucleotide sequence shown as SEQ ID No: 4. 23 201038739 '5' Seed Green Screening for Green Screening detects the methylation status of the target gene in the test specimen as a marker for the presence or absence of cervical cancer. The method includes the following steps: Step 1 provides a Test sample; step 2 detects the methylation status of CpG sequence of at least one target gene in the genomic DNA of the test subject', the target gene line consists of DBC1, PDE8B, pTpRR and ZNF582; and step 3 according to the target gene The presence or absence of the thiolation state, whether the specimen has a cervical cancer lesion or as an indicator of prognosis. 6. For example, the method of screening the uterus of the uterus according to the fifth paragraph of the patent, the towel is the smear, blood, urine, cervical epithelial cells, cancer tissue after surgery, etc. Isolated samples. 7. The method of screening for cervical cancer according to claim 5, wherein the subject is an abnormal Pap smear. 8. The method of cervical cancer screening according to claim 5, wherein the test subject is a cervical cell sample positive for HPV testing. Q 9. The method for screening for cervical cancer according to claim 5, wherein the methylation-specific PCR (MSP) of the CpG sequence methylation state detection method of the target gene is methylation-specific PCR (MSP) ), quantitative methylation-specific polymerase chain reaction (QMSP), bismuthite sequencing (BS), microarrays, mass spectrometer analysis (mass spectr() metei&lt; Denaturing high-performance liquid chromatography (DHPLC), pyrosequencing (Pyrosequencing), such as the method of cervical cancer screening according to claim 5, wherein the target The gene DBC1 has the nucleotide sequence shown in SEQ ID No: 1, the target gene pde8B is 24 201038739 has the nucleotide sequence shown in SEQ No. 2, and the target gene PPRRR has SEQ ID No: 3 The nucleotide sequence shown, the target gene ZNF582 has a nucleotide sequence as shown in SEQ ID N::4. 11_ A method for screening ovarian cancer by detecting the state of methylation of a target gene in a test subject cell as a screening indicator for the presence or absence of ovarian cancer, the method comprising the following steps: Step 1 provides a test subject Step 2: detecting a methylation status of a CpG sequence of at least one target gene in the genomic DNA of the test subject, the target gene consisting of DBC1, PTPRR and ZNF582 〇v; and step 3 according to the target gene thiolation state The presence or absence of the test determines whether the sample has ovarian cancer lesions or as an indicator of prognosis. 12. The method of screening for ovarian cancer according to claim η, wherein the test subject is an isolated sample of ovarian cancer tissue, ascites, blood, urine, and post-operative cancer tissue. 13. The method for screening for ovarian cancer according to claim 11, wherein the CpG sequence thiolation state detection method of the target gene is methylation-specific PCR (MSP). ), quantitative methylation-specific polymerase chain reaction (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, high efficiency A method for screening ovarian cancer according to claim 11, wherein the target gene DBC1 has the same function as the method of screening for ovarian cancer according to claim 11 of the present invention. The nucleotide sequence shown in SEQ ID No: 1, the target gene PTPRR has the nucleotide sequence shown in SEQ ID No: 3, and the target gene ZNF582 has the nucleotide sequence shown in SEQ ID No: 4. . 25 201038739 15 .--Colorectal cancer _ method, check __ cell _ methylated edge 'to the large intestine is not _ rehearsal, this face contains the following steps step 1 to provide a test sample; 2 detecting the methylation status of CpG sequence of at least one target gene in the genome of the test subject, which is composed of DBC1, pDE8B, pTpRR(10) ZNF582; and Ο Ο step 3 according to the methylation status of the target gene Whether or not the sample has a colorectal cancer lesion or as an indicator of prognosis. 16. The method of applying for a large intestine touch according to the subject of M15, wherein the subject is an ex vivo sample of ascites, blood, urine, and cancer tissue after surgery. 17_ The method for detecting a large intestine according to claim 15, wherein the CpG sequence thiolation state detection method of the target gene is methylation-specific polymerase chain reaction (MSP), Quantitative methylation-specific PCR (QMSP), bisuiflte sequencing (BS), microarrays, mass spectrometer, denatured sputum A method for screening colorectal cancer according to claim 15, wherein the target gene DBC1 has SEQ ID NO: </ RTI> </ RTI> </ RTI> <RTIgt; a nucleotide sequence represented by ID No: 1, the target gene PDE8B has a nucleotide sequence as shown in SEQ ID No: 2, and the target gene PTPRR has a nucleotide sequence as shown in SEQ ID No: 3. The target gene ZNF582 has a nucleotide sequence as shown in SEQ ID No: 4. 26