TW200831900A - Cancer screen method - Google Patents

Abstract

A cancer screen method includes the following steps. (1) Provide a test sample. (2) Inspect and test the methylation state of a GpG series of at least one target gene in the DNA genome of the test sample. The target gene is composed by SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1. (3) The condition if the target gene is or is not in the methylation state determines whether the sample has or has no cancer or pre-cancer pathological changes. The methylatized state test methods can involve the use of methylation-specific PCR (MSP), quantitative methylation-specific PCR (QMSP), bisulfate sequencing (BS), microarrays, mass spectrometer, and denaturing high-performance liquid chromatography (DHPLC).

Description

200831900 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD The present invention relates to a method for cancer screening, and more particularly to a method for cancer screening using methylated DNA as a biomarker. [Prior Art] Cervical cancer is one of the leading causes of death among women in the world and Taiwan. According to the 2nd World Health Organization shoulder (TOO), the child is the third in the world for female miscellaneous death. In breast cancer; regular sub-prediction is the most expensive method to test the uterus. There are two main ways to use the Wei-Cai screening. One of the most common Pap smear tests (pap sme rainbow), the other is Human papillomavirus test (HPVtesting); Pap smear is the removal of the secretions of the uterine neck, microscopic observation of the towel off the epithelial cell towel ', whether money is generated to detect uterine neck ulcers early, and HPV In the test, the reverse transcHpti〇n polymemse chain reaction (RT_PCR> is used to check whether there is a human papill〇 ma virus (HPV) virus gene. However, Because of the Pap smear, it is necessary to rely on the doctor's sampling and the examiner/pathology to determine 4 smears. In addition to the high faise negative rate and the delay in the diagnosis and treatment of precancerous lesions. Moreover, the labor cost required is too high, which is a problem for many children in the development of the country. On the other hand, the human papillomavirus test (Hp^ Degree 'but it is easy to cause high false positive rate (High faise p0Sitive rate), not only worry patients, but also waste many medical resources in the tracking test of false positive patients; therefore, how to improve the accuracy of cervical cancer test methods Sexuality and convenience are one of the important topics to promote cervical cancer testing. In the etiology of cervical cancer, human cancer virus-producing human papillomavirus (HPV) is the most significant risk factor of 200831900; zurHausen in 2002 The report shows that "high risk, E6/E7 oncoprotein (〇nc〇pr〇tein) produced by human papillomavirus (HPV) and tumor suppressor genes; ? 53 / ^ Fan 8 effect, resulting in abnormal cell cycle regulation In fact, human papillomavirus (HPV) DNA can be detected in all cases of cervical cancer. However, infection with human papillomavirus (HPV) is a necessary condition for cervical cancer, but it is insufficient. To cause cervical cancer Occurrence; approximately 60% of low-grade squamous intraepithelial lesions (LSIL) will regress, • 30% will persist (Persist), and 5-10% will develop into high squamous Intraepithelial lesion (high-grade)

f squamous intraepithelial lesions (HSIL), only less than 1% will become cervical cancer. The persistent infection of HPV and the viral load may be the determinants of the development of highly squamous intraepithelial lesions (HSIL) and cancer; however, the molecular mechanisms of cervical cancer development remain to be confirmed. Other factors, such as environmental and genetic changes, may also play an important role in the deterioration of cervical keratinocytes; and whether or not initiated by HPV, genetic changes that cause genomic instability have long been considered cervical An important mechanism of carcinogenesis, cytogenetic studies have shown that there are non-ran〇m chmmosomal changes in cervical cancer cells; in addition, several molecular genetic studies have identified Some often occur in locations of loss of heterozygosity (LOH), which may be associated with tumor suppressor genes (TSGs) when cervical cancer occurs. 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-hit theory), indicating that mutations or deletions accompanying some homologous tumor suppressor genes may cause or cause cancer; however, other information affecting phenotype may exist in the modified base 5-曱In 5-methylcytosine, 5-mercapto-cytosine was found in mammalian cells in the palindrome 6 200831900 sequence 5'-CpG-3', except in some mammalian cells called " Outside the region of CpG islands, (CpG islands, CGIs), most CpG dinucleotide pairs are methylated, and CpG islands refer to a large number of GCs in a region of approximately 1000 base pairs (1 Kb). - and CpG-, usually located near the gene, and found near the promoter of a widely expressed gene. Methylation of cytosine occurs after DNA synthesis, from a thiol donor s-adenosine thiolamine Acid (iS^aden Osylmethionine, SAM) transfers the monomethyl group to the position of the fifth carbon in the cell. The enzyme reaction is carried out by DNA methyltransferase (DNMTs). DNMT1 is a mammal. The main methyltransferase, which is responsible for post-replicative restoration to permethylation, is called maintenance methylation; otherwise, DNMT3A and DNMT3B are considered Mainly responsible for the new position of methylation 'to carry out a step called heavy methylation (% (10) V0inethylation).

The disappearance of methylation of CpG dinucleotide (i〇ss methylation), meaning the first epigenetic abnormaiity in cancer cells in general low-methylation; however, in the past few years Studies have shown that site-specific hypermethylation at specific locations (eg, some tumor suppressor genes) is associated with loss of function, which may provide selective advantages in cancer production; on promoter regions High methylation of CpG islands can be caused by histone modification followed by successive gene silencing to cause chromatin remodeling; in addition to chromosomal deletions and gene mutations Epigenetic silencing of tumor suppressor genes caused by hypermethylation of promoters is also common in human cancers. 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 methylation cycle, the enzyme gene The multitype (genetiC P〇lym〇rphisms) has also been reported to be involved in the development of intradermal lesions on the cervix 7 200831900; as with the concept of supergene evolution, the study of the association between DNA methylation and cervical cancer is also Prevalence, the DNA methylation study of cervical cancer and the increase, the use of methylation as a possibility of screening for cervical cancer; due to the nature of genetic and environmental interactions, the degree of methylation of tumor suppressor genes is different The genes vary from group to group, and different diseases also have different thiolation expressions ^methylatorphenotypes); however, the pro-Wei-based phenotype and its association with HPV genotypes are still unknown, but in uterine head cancer What are the specific 'genes that will be methylated, and how many genes are needed to meet the needs of clinical applications? These issues are still issues that need to be identified in the future. There are still many defects in the method of cervical cervix. It is not the designer of goodness, but it needs to be improved. ° The inventors of this case have made improvements and innovations in view of the shortcomings derived from the above-mentioned uterus-screening method. After many years of hard work and painstaking research, we finally succeeded in researching and developing the method of screening for cancer. [Invention] The object of the present invention is to provide a method for screening cervical cancer as a screening for the first line-cervical cancer. (cancer screen). The second-purpose of the present invention is recorded in the branch that provides the screening of the _Seed Palace, and the % method can be used as the first-line uterus (four) test. Human papillomavirus test (HPVtesting) to achieve a more accurate uterine cancer screening effect. Another object of the present invention is to provide a method for the diagnosis of the disease, except for the financial method (6) in the detection of cervical cancer, Can also take into account other aspects such as: (4) cancer, liver _ test = diagnosis of auxiliary abnormalities. The method of cancer screening that can achieve the above object is to detect the subject's careful 200831900 target gene thiolation. status As a screening indicator for the presence or absence of cancer, the method comprises the following steps: Step 1 provides a test subject; Step 2 detects a CpG sequence thiolation state of at least one target gene in the genomic DNA of the test subject 'The target The gene line consists of S〇Xl, PAX1, LMX1A, NKXW, WT1 and ONECUT1; and step 3 determines whether the sample has cancer or precancerous lesions based on the presence or absence of methylation status of the target gene. The test body is Zilu neck smear, ascites, blood, urine, feces, lean, oral mucosal cells, gastric juice, bile, sub. cervical epithelial cells. The methylation status of the CpG sequence of the target gene is methylated-spedfic PCR (MSP), quantitative methylation-spedfic PCR (QMSP) ), sulfite sequencing (BS), microarrays, mass spectrometer (mass spectr〇meter), denaturing high-perfo surface liquid Chmmat (), DHPLC Wherein the target gene SOX1 has a nucleotide sequence as shown in SEQn 3: i. Wherein the target gene PAX1 has a nucleotide sequence as shown by SEqID No: 2. Wherein the target gene LMX1A has a nucleotide sequence as shown in SEq ID No: 3. Wherein the target gene NKX6-1 has the nucleotide sequence as shown in SEQ ID No: 4. Wherein the target gene WT1 has a nucleotide acid sequence as shown in SEq ID No: 5. Wherein the target gene ONECUT1 has a nucleotide sequence as shown in SEq ID No: 6. This I-month feed-seed Lv-neck cancer screening method is to detect the methylation status of the target gene in the test cells in the test, as a screening indicator for the presence or absence of cervical cancer, and the method comprises the following steps: Step 1 provides a test subject; Step 2 detects a methylation status of a CpG sequence of at least one target gene in the genomic DNA of the test subject, which is composed of SOX, PAX1, LMX1A, NKX64, WT1, and ONECUT1. Composition; and step 3 determines whether the specimen has cervical cancer and precancerous lesions based on the presence or absence of the methylation status of the target gene. The test subject is a Pap smear, blood, urine, cervical epithelial cells, and the like. The test subject is an abnormal Pap smear. The test subject is a cervical cell sample that is positive for HPV testing. The methylation status of the CpG sequence of the target gene is methylated-specific PCR (MSP), quantitative methylation-specific PCR (QMSP) ), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (DHPLC) ° wherein the target gene SOX1 is There is a nucleotide sequence as shown in SEQ ID No: 1. Among them, the ό海目& gene PAX1 has a nucleotide acid sequence as shown in SEQ ID No: 2. Wherein the target gene LMX1A has a nucleotide sequence as shown in SEQ ID No. 3. Wherein the target gene NKX6-1 has a nucleotide sequence as shown in SEqh) No: 4. Wherein the target gene WT1 has a nucleotide acid sequence as shown by SEqID No: 5. 200831900 wherein the target gene 〇NECUT1 has a nucleotide acid sequence as shown in SEQ N〇: 6. The present invention further provides a method for detecting a secret cancer, which is a method for detecting the state of methylation of a target gene in a test subject cell as a screening index for the presence or absence of a nest cancer, and the method comprises the following steps: Step 1 Providing a test subject; step 2 detecting a sequence methylation status of at least one target gene in the genomic DNA of the test sample, the target gene consisting of S〇xi, ρΑχ卜LMX1A; and step 3 according to the target The presence or absence of the methylation status of the gene determines whether the sample has ovarian cancer or precancerous lesions. The test subject is ascites, gold liquid, urine, and the like. The method for detecting the methylation status of the CpG sequence of the target gene is methylation-speciflc PCR (MSP), quantitative methylation-specific PCR (QMSP), Bisulfite sequencing (BS), microarray (microarrayS), mass spectrometer analysis (mass 叩 加 (10) (4), denaturing high-performance liquid chromatography (DHPLC), coke filling acid sequencing (pyrosequencing) wherein the target gene SOX1 has a nuclear ridge sequence as shown in SEQ ID No. 1. wherein the target gene PAX1 has a nuclear phytic acid sequence as shown in SEQ ID No: 2. wherein the target gene LMX1A The invention has a nucleotide sequence as shown in SEQ ID No: 3. The present invention further provides a method for screening for liver cancer by detecting the state of methylation of a target gene in a test subject cell as a screening for liver cancer presence or absence. The indicator comprises the following steps: Step 1 provides a test subject; 11 200831900 Step 2 detects at least one target in the genomic DNA of the test subject The target gene is composed of SOX1 and NKX6-1 due to the methylation status of the CpG sequence; and step 3 determines whether the sample has liver cancer and precancerous lesions according to the presence or absence of the methylation status of the target gene. The test body is ascites, blood, urine, feces, gastric juice, bile, etc. The methylation-specific PCR (MSP) of the methylation-specific PCR (MSP) of the CpG sequence of the target gene is detected. Quantitative methylation-specific polymerase chain reaction (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denatured Nanyi solution Denaturating high-performance liquid chromatography (DHPLC), pyrosequencing, wherein the target gene SOX1 has a nucleic acid sequence as shown in SEQ ID No: 1. The target gene NKX6-1 is There is a nucleotide sequence as shown in SEQ ID No: 4. [Embodiment] Example 1 Materials and Methods 1. Test Materials Test materials contain a series of complete Cervical lesions, including normal samples (n = 45), low-grade squamous cell intraepithelial lesions (LSIL, n = 45) 'Highly squamous intraepithelial lesions (HSIL, n 58), squamous cell carcinoma (SqUam〇) Us cell carcinoma,SCC,η = 109); The test material also contains a complete series of ovarian tumor samples, including ovarian benign tumor samples (n = 36), ovarian marginal tumor samples (η = 6), ovarian malignant tumor samples (η = 122); All cervical samples and ovarian samples 12 200831900 were taken from the gynecological tumor tissue bank of the Taipei Military General Hospital. The genomic DNA of each sample was extracted with the Qiagen DNA kit and quantified by pic〇Green fluorescence absorption method. DNA, and the quality of DNA was detected by gel electrophoresis. In addition, hepatocyte samples contained normal liver cell samples (n = 13), chronic hepatitis (n = 15), cirrhosis (n = 40), liver cancer (hepatoceiiular carcinoma, HCC, η = 54); all livers The cell samples were taken from the general surgical tumor tissue bank of the Taipei Three Military General Hospital. The genomic DNA of each liver cell sample was also extracted with the Qiagen DNA kit, and the DNA was quantified by PicoGreen fluorescence absorption method. The DNA quality was detected by gel electrophoresis. . 1. Differential Methylation Hybridization (DMH) using CpGisland mieroarrays (DMH) Take 30 cervical cancer tissue samples and mix them together, and take 1 normal cervical cervix The DNA of the smear sample is mixed together, the DNA of the sample is digested with a restriction enzyme, ligated to the linkers, and then you are methylation-sensitive restriction enzymes. All and the injured iUI were digested, and the DNA was amplified as a PCR template for 20 cycles and labeled with a fluorescent dye. The DNA of the normal Pap smear sample was fluorescently stained. The Cy3 label, the DNA of the cervical cancer tissue sample is labeled with the fluorescent dye Cy5; the labeled sample DNA is used as a probe, and the CpG island microarray (CpG island microarrays) containing 8,640 CpG island tags (CpG island tags) The hybridization reaction was performed to identify the selected CpG islands using the CGI database (http://dertab.med.utoronto.ca/CpGIslands/). The microarray data was characterized by the circular feature pattern of the GenePix 6.0 software (circuiar-features m〇(je) analysis, labeling duplicated selections (done), and filtering to remove unacceptable features; Cy5 vs. Cy3 ratio ( Ratio) A locus greater than 2.0 is a gene with a high 13 200831900 degree methylation in a mixed cervical cancer tissue sample, and therefore accepts a quinoa potential with a ratio greater than 2.0. III. Sulfite modification (Bisulfite) Modification), methylation-specific PCR (MSP) and bisulfite sequencing (BS)

Using a DNA modification kit (Chemicon, Temecula, CA) from Chemicon to perform sulfite modification. Take 1 sample of genomic DNA (Senomic DNA) and chemically modify genomic DNA with sodium sulfite. In single-stranded DNA, all unmethylated cytosines undergo deamination and turn into urinary squeaks, while thiolated cytosines are not modified and remain in the state of 5-methylcytosine; Finally, the reacted sample DNA was dissolved in 70 μl of 55 ° C in TE buffer for methylation-specific PCR (MSP) &* < Normally, the normal blood of human peripheral blood was taken. The DNA was subjected to sulfite modification as a control group with a non-methylated promoter sequence; and human normal DNA was treated with SssI methyltransferase (methyltransferase, New Engl - 〇1 (4), ma) To obtain a positive control group with a methylated dual gene. ^ Take lpg sample genomic DNA after sulfite modification, and control group and positive control DNA 'Methylation-specific PCR amplification with MSP primer. The MSP primer is divided into two types, one of which can be specifically identified. The unmethylated gene sequence]^;§1> primer (u), and the other is the MSP primer (M) which can specifically recognize the methylation gene sequence, and the MSp primer sequence of each target gene is as shown in Table 1. The total volume of the methylation-specific PCR reaction is 25 μl, containing 1 μl of modified template DNA, 15 pm per primer, 2 dxps, and j her G〇w (4) DNA polymerase (AppHe(j Bi) 〇systems, F〇ster CA); The mixed reactants were placed at 95 ° C for 5 minutes, followed by densification at 95 ° 3 〇, with appropriate primer bonding (Augmenting) 14 200831900 Temperature bonding 30 seconds, 72 ° C The synthesis was carried out for 30 seconds, and the dissociation, adhesion and synthesis steps were repeated for 35 cycles, and then reacted at 72 ° C for 5 minutes. The amplified product was 2.5% agar colloid containing ethidium bromide (EtBr). Electrophoretic analysis was carried out and observed under ultraviolet light. Table 1 methylation The sequence of the MSP primer used by the PCR (MSP) gene name primer type primer sequence r~" 1ΤΓ'1 ' I,··1 --------------- - - -- -- - --

Μ SOX1 ——u

Μ LMX1A — U M ONECUT1 ~-

Positive stock (F·) Anti-share (R) Stock (F) Anti-share (R) Stock (F·) Anti-share (R·) Stock (F) Anti-share (R·) Stock (F·) anti shares CRD 5r CGTTTTTTTTTTTTCGTTATTGGC 3f 5r CCTACGCTCGATCCTCAACG 5 / TGTTTTTTTTTTTTTGTTATTGGTG 3r 5f CCTACACTCAATCCTCAACAAC 3r 5, TTTAGAAGCGGGCGGGAC 3r 5, CCGAATCCAAACACGCG 5, GAGTTTAGAAGTGGGTGGGATG 3r 5, CAACCAAATCCAAACACACAAAAC 3f 5r TTGTAGCGGCGGTTTTAGGTC 3f GCCAAACCCTTAACGTCCCG 3f (SEQ ID No: 7) (SEQ ID No: 8) ( SEQ ID No: 9) (SEQ ID No: 10) (SEQ ID No: 11) (SEQ ID No: 12) (SEQ ID No: 13) (SEQ ID No: 14) (SEQ ID No: 15) (SEQ ID No: 15) ID No: 16)

U positive stock (F') 5A GATTGTAGTGGTGGTTTTAGGTTG 3, anti-share (R) 5, CACCAAACCCTTAACATCCCAATAC 3r ϋ PAX1

正股(F') 5r TATTTTGGGTTTGGGGTCGC 3r Anti-share QRQ 5,CCCGAAAACCGAAAACCG u Stock (F') 5r GTTTATTTTGGGTTTGGGGTTGTG 3, Anti-share 5r CACCCAAAAACCAAAAACCAC 3f NKX6.1 u Stock (F') 5f CGTGGTCGTGGGATGTTAGC 3, Anti-share (R' 5, ACAAACAACGAAAAATACGCG 3' positive stock (F) 5f GTGTGGTTGTGGGATGTTAGTG 3r anti-share (R') 5f CAACAAACAACAAAAAATACACAAC 31 WT1 positive stock (F丨) 5f TGTTGAGTGAATGGAGCGGTC anti-share (R1) 5, CGAAAAACCCCCGAATATAAACG 3r

U-strand (F') 5f GTTGTTGAGTGAATGGAGTGGTTG 3, anti-strand (R) 5, AATTACAAAAAACCCCCAAATATAAACAC 3 丨 (SEQ ID No: 17) (SEQ ID No: 18) (SEQ ID No: 19) (SEQ ID No: 20) (SEQ ID No: 21) (SEQ ID No: 22) (SEQ ID No: 23) (SEQ ID No: 24) (SEQ ID No: 25) (SEQ ID No: 26) (SEQ ID No: 27) (SEQ ID No: SEQ ID No: : 28) (SEQ ID No: 29) (SEQ ID No: 30) The primer type M represents an MSP primer which can specifically recognize a methylation gene sequence. The primer type U represents an MSP primer which can specifically recognize a non-methylated gene sequence. 200831900 All samples were subjected to at least two independent sulfite modification and methylation-specific PCR, and the same sample was used in the PCR reaction using MSP primer (Μ) which can specifically recognize the methylation gene sequence. If the PCR product cannot be synthesized more than twice, the sample is considered to be non-methylated; the PCR product amplified by the MSP primer (M) which can specifically recognize the methylation gene sequence is selected to the pCR4-T0P0 vector (lnvitr) In 〇gen, Carlsbad, CA), at least 5 independent strains (clones) were selected for sulfite sequencing (BS), sulfite sequencing (BS) The primers shown in Table 2 as "automatic sequencer using 377 instrument (Applied Biosystems, Foster City, CA) for ethylene

Sulfate sequencing.序列 Table II Name of the primer used in the sulfite sequencing (BS) primer name introduction sequence ''' ........................ .....·Γ_,ι.—__.丨丨...丨,丨, — S0X1 正股(F')5' 反股(R')5'

LMX1A positive stock (Ff) 5' anti-share (R) 5' 0NECUT1 positive stock (P) 5, anti-share (R〇5' ΡΑΧ1 ΝΚΧ 6.1 WT1 BS1 positive stock (Ff) 5, anti-share (R〇BS2 positive Shares (F1) S' Anti-Shares (R) 5' BS1 Stocks (P) 5, Anti-Shares (R,) 5' BS2 Stocks (F1) 5, Anti-Shares (R) 5' Stocks (F) 5 , anti-strand 5' GTTGTTTTYGGGTTTTTTTGTGGTTG 3, (SEQ ID No: 31) ATTTCTCCTAATACACAAACCACTTACC (SEQ ID No: 32) TAGTTATTGGGAGAGAGTTYGTTTATTAG 3r (SEQ ID No: 33) CTACCCCAAATCRAAAAAAAACAC 3,_(SEQ ID No: 34) GAGTTTATTTAAGTAAGGGAGG 3r (SEQ ID No: 35) CAACTTAAACCATAACTCTATTACTATTAC 3f (SEQ ID No: 36) GTGTTTTGGGAGGGGGTAGTAG 3f (SEQ ID No: 37) CCCTCCCRAACCCTACCTATC 3,_(SEQ ID No: 38) GATAGAAGGAGGGGGTAGAGTT 3f (SEQ ID No: 39) TACTACCCCCTCCCAAAACAC 3f_(SEQ ID No: 40) GGTATTTTTGGTTTAGTTGGTAGTT 3r (SEQ ID No: 41) AATACCCTCCATTACCCCCACC 3f_(SEQ ID No: 42) GGTGGGGGTAATGGAGGGTATT 3r (SEQ ID No: 43) CCTAAATTATAAATACCCAAAAAC 3f (SEQ ID No: 44) —-~ ........ 1 ----------- _ - A GTGTTGGGTTGAAGAGGAGGGTGT 3r (SEQ ID No: 45) ATCCTACAACAAAAAAAAATCCAAAATC 3/ (SEQ ID No: 46) IV. Treatment of methylated genes in cervical cancer cell lines by treatment with 5'_aza-2'-deoxycytidine 16 200831900 First in In the HeLa cervical cancer cell line, the methylation-specific pcR (Msp) test may have a hypermethylated gene methylation status and select a methylated gene. The cervical cancer cell line was treated with 10 μM DNA methyltransferase inhibitor 5'-aZa-2'-de〇XyCytidine (Sigma Chemical Co.) for 4 days to induce methylation in the cell line. Genes that are not expressed can be re-expressed and analyzed for gene expression by RT_PCR; total RNA (total RNA) is extracted using -Qmgen RNeasy kit (Qiagen, Valencia, CA), and -DNase 1 is added to remove DNA contamination; One sample of 1 pg total RNA was synthesized by Superscript II reverse transcriptase and random hexamer (Invitrogen); the synthesized cDNA was amplified by PCR master mix reagents kit (Applied Biosystems). Increasing, and placing in a thermocycling reactor (thermal cycler, GeneAmp 2400 PE, Applied Biosystems), the amplified cDNA was analyzed by RT-PCR, and the RT-PCR primers used in each target gene are shown in Table 3. Shown. Table 3 Sequence name of MSP primer used for RT-PCR Gene name Primer type Primer sequence S0X1 Positive strand (F·) 5, AGACCTAGATGCCAACAATTGG 37 (SEQ ID No: 47) Anti-strand (R') 5, GCACCACTACGACTTAGTCCG 3f (SEQ ID No : 48) LMX1A positive strand (F) 5f GCTGGTTCTGCTGCTGTGTCT 3f (SEQ ID No: 49) anti-strand (R:) 5, ACGTTTGGGGCGCTTATGGTC 3r (SEQ ID No: 50) 0NECUT1 positive strand (F·) 5, CAAACCCTGGAGCAAACTCAA 3r (SEQ ID No; 51) Anti-strand (R〇5r TGTGTTGCCTCTATCCTTCCC 3; (SEQ ID No: 52) PAX1 positive (F') 5f CCTACGCTGCCCTACAACCACATC 3f (SEQ ID No: 53) Anti-strand (RD 5, TCACGCCGGCCCAGTCTTCCATCT 3f (SEQ ID No: 54) NKX6.1 positive stock (F*) anti-strand (R〇5, CACACGAGACCCACTTTTTCC 3f CCCAACGAATAGGCCAAACG 3, (SEQ ID No: 55) (SEQ ID No: 56) WT1 positive stock (F·) 5, GCTGTCCCACTTACAGATGCA 3f (SEQ ID No: 57) Inverse (R) TCAAAGCGCCAGCTGGAGTTT 3; (SEQ ID No: 58) 17 200831900 V. Detection of human papillomavirus (HPV) by LI consensus PCR and reverse line blot Detection of the presence of human papillomavirus (HPV) DNA in squamous cell carcinoma (SCC), If there is more than the analysis range of the hybridization technique, the sequence of the novel human papillomavirus (novel HPV type) is confirmed by DNA sequencing. VI. Statistical analysis Data analysis by statistical software SAS version 9.1, methylation of genes and each The relationship between clinical parameters (including HPV status) is calculated using the f test (X2 test) and Fisher's exact test (and calculated by the logistic regression model) and adjusted for age and The Odds ratios (ORs) of HPV infection and the 95% confidence interval (CI), the alpha level of statistical significance are set; 7 = 〇·〇5; and the calculation uses HPV and Methylation markers are used to diagnose the sensitivity and specificity of cervical lesions. Example 2 Screening of cervical cancer methylation index genes Screening for cervical squamous cell carcinoma (sec) by differential methylation hybridization (DMH) by CpG island microarrays The gene with high thiolation in the CpG island microarrays showed that there were 216 points in the cervical cancer tissue sample and the normal Pap smear sample with differential methylation, and the sequence was removed. After the repeater, 'pay to 26 gene promoter regions CpG islands (promoter CGIs). These gene promoters were sequenced and analyzed, and six genes were selected, including S0X1 (SEQ Π) No: 1), PAX1 (SEQ ID No: 2), and LMX1 A (SEQ ID No: 3). ), NKX6-1 (SEQ ID No: 4), WT1 (SEQ ID No: 5), and 0NECUT1 (SEQ ID No: 200831900 6), the details of which are shown in Table 4; as shown in Table 4, these six genes It is an important transcription factor in the development process. SOX, PAX1, LMX1A, NKX64, and wti are important for the development of brain, nerve, bone, islet, and genitourinary tract. 0NECUT1 is for the liver and ▲ The performance of dirty genes is important, but few studies have shown that these genes are involved in cancer.

UniGene --BE_^ Chromosome localization gene name S0X1 _〇〇5986 13q34 PAX1 NM—006192 20pll.2 Gene full name Known gene function Sex determining DNA binding region Y-box 1 Transcription factor activity Paired box gene DNA binding LMX1A NM_177398 Iq22- Q23 LIM homeobox Transcription factor activity transcription Zinc ion binding ---_____factor 1 alpha NKX6 1 NM—006168 4q21.2-q22 NK6 Transcription factor activity transcription factor related

ONECUT1 _____locus 1_ NM 004498 15q21 · 1 -q21.2 One cut domain Transcription factor activity family member Transcriptional activator _ ___1_activity_ NM_024426 llpl3 Wilm?s tumor 1 Transcription factor activity

CpG sequence analysis was performed on each of the above 500 bp nucleotides before and after the transcription start point (+1) of each gene. As shown in Fig. 1, the CpC} sequence in each gene was indicated by Γ |" and designed for each gene. Its MSP primer (as shown in Table 1) and sulfite sequencing (BS) primer (as shown in Table 2), each target gene for methylation-specific PCR (MSP) and sulfite sequencing (BS) The position of the synthesized fragment is also indicated in Figure 1. 19 200831900 Next, methylation-specific PCR (MSp) was performed on mixed cervical cancer tissue samples (mixed with 3 samples) and mixed normal Pap smear samples (mixed samples) to confirm these 6 genes. Whether the methylation phenomenon is different in different tissue samples, as shown in Figure 2, the methylation of the six tissue-linked uterine tissue samples is shown in Figure 2 There is no methylation in the mixed normal cervix smear (as shown in Figure 2). The test is performed on individual cervical cancer tissue samples and 4 cervical cancer tissues are taken. Sample - Ben (D1, D3, D4) and 4 normal samples (Nl, N2, N3, N4) for methylation specificity (PCR(MSP)' can specifically identify non-methyl The MSP primer (U) of the gene sequence and the MSP primer (M) which can specifically recognize the methylation gene sequence are subjected to methylation-specific pCR (Msp), and the results are shown in Figure 3. The cervical cancer tissue samples all have methylation (as shown in Figure 3, Bu, 3, 5, and 7 lions), and the related genes are positive. The sample towel could not detect the occurrence of methylation (as shown in column 9, paragraph 9, 13 and 15 of Figure 3). According to the above results, the six genes were used as screening indicators for methylation of cervical cancer. Gene X. Correlation between DNA methylation and gene expression in cervical cancer

To confirm whether the expression of the cervical cancer methylation indicator gene is regulated by DNA methylation, the HeLa subunit is treated with 10 μM DNA methyltransferase inhibitor 5, _aza_2, _de〇xycytidine (AZQ (Sigma Chemical Co'). The cervical cancer cell line was tested for 4 days, and the methylation-specific PCR (MSP) was used to examine the demethylation of the above six gene promoters; respectively, the MSP primer (9), which can specifically recognize the unmethylated gene sequence, and The MSp primer (Μ), which specifically recognizes the methylation gene sequence, was subjected to methylation-specific PCR (MSP). The results are shown in Figure 4A, and the untreated 5'-aZa-2'-de0XyCytidine (AZC) HeLa In the cervical cancer cell line (AZC_), all of the six target genes have methylation (as shown in the first block of Figure 4A), and the unmethylated gene 200831900 is not detected (Figure A, the second block) In the HeLa cervical cancer cell line (AZC+) treated with s^^^deoxycytidine (AZC) for 4 days, it can be _ to the unmethylated target gene (as shown in Figure 4A, Block 4) 'Showing the above 6 target bases in cervical cancer cell lines treated with methyltransferase inhibitor 5, strip 2, '〇χ_- (AZC) The methylation was partially removed. The expression of these 6 genes in HeLa cervical cancer cell lines was analyzed by RT-PCR. The results are shown in Figure 4B: '5'_aza_2': de〇xycytidine (Azc) In the treated sylvestris, the mRNAs of the six target genes were extracted (as shown in Figure 6 b, block 6), while the cells not treated with Γ 5'-'2'-deoxyc_ine (AZC) In the strain, the mRNA of any target gene was not detected (as shown in _B 5). From the results, it can be seen that these six target genes are indeed regulated by DNA methylation in cervical cancer cells. Gene expression, when the gene has methylation, the gene's performance will be inhibited, and after the methylation is removed, the target gene will begin to perform. The sulfite sequencing (BS) will be used to analyze the target gene in the HeLa cervix. Whether there is hypermethylation in cancer cell lines, the results are shown in Figure 5, and there are no high-methylation samples of target genes in cell lines (5) and 5(5) of (4) Township-(10)(10). It is more than a cell strain treated with 5, J'-deoxycytidine (AZC) (Fig. 5B). Cervical squamous cell carcinoma (scc) and normal samples were also analyzed by sulfite sequencing (BS). The results are shown in Figure 6 in a sample of cervical squamous cell carcinoma (scc) (Fig. 6A). The number of samples with high methylation of the target gene was also significantly higher than that of the normal sample (Fig. 6B). Example 4 Methylation analysis of target genes in clinical cervical samples, see Table 5, normal samples, low-grade squamous cell intraepithelial lesions (lsil), highly squamous intraepithelial lesions (HSIL), and squamous cell carcinoma The average age of the (scc) samples were 5i bauxite 200831900 11 ·3, 39.7 soil 9·6, 46·4 ± 14.4 and 53.3 ± 1〇·9 years old (ρ<0·05); high-risk pjpv DNA presentation in the sample The positive rates were 21.4% for normal samples, 47.7% for low-grade squamous cell intraepithelial lesions (LSIL), 59.3% for highly squamous cell intraepithelial lesions (HSIL), and 88.9 for squamous cell carcinoma (SCC) samples. %. The results showed that patients with HPV were more likely to have cervical lesions of different severity (the odds ratios of LSIL and HSIL 'SCC samples were 3.1, 5.2, and 29.9 respectively; 95% of the letters and latitudes were 1.1- 8.3, 2.1-13.0, 11.5-77.7). - methylation-specific PCR (MSP) analysis of the methylation status of the target gene, methylation status of the target gene, and human papillomavirus in a sample of cervical lesions of different severity The results of the analysis are shown in Table 5. The six genes of SOX, PAX, LMX1A, NKX, 1, Α\ΓΓ1 and ONECUT1 have high frequency methylation in squamous cell carcinoma (SCC). The proportion of methylation in the squamous cell carcinoma (see) samples was 81.5%, 94.4%, 89.9%, 80.4%, 77.8%, and 20_4〇/, respectively. The proportion of methylation of each gene in normal cervical samples is: 2.2%, 〇%, 6.7%, 11.9%, ιι·1%, and 〇% 2$〇〇〇1); Compared with normal cervical samples, these 6 genes were significantly more methylated in squamous cell carcinoma (scc) samples. . The frequency of NKX6-1 gene thiolation was 53.3% in the LSIL sample and 55.1〇/ in the biliary sample. In the SCC sample, it is _%; the statistical results show that patients with gene thiolation have a higher risk of squamous cell carcinoma (SCC) (the odds ratio is 29,85% and the confidence interval is 1〇· 4_85·2). ° ΡΑΧ1 based gm is 3% in LSIL sample towels, 42.1% in ancestral samples, and 94.4% in SCC samples; statistical results show that patients with ρΑχι gene methylation phenomenon Squamous cell intraepithelial lesions L) and squamous cell carcinoma (scc) winds 22 200831900 High risk (hsil 0·1 -> 999·9) The odds ratios for both sputum and SCC samples are >999·9 95% confidence interval is < X and ONECUT1 three genes in the precancerous lesions (precancer〇us) samples of methylation frequency is very low, but the frequency of methylation in the positive samples and scc samples is large Nakata increased, respectively, 9 3% and & catch, secret and 7 · general and run %; Tongmu. For example, patients with s〇Xl, (10) or (10)^(3) methylation of sputum gene have a higher risk of squamous cell carcinoma (SCC) (the odds ratio of the three is 200.2'124.5, respectively). 7.3 ; Γ 95% confidence interval is 25·8_999·9 ' 33.G-47 (U, 2 hunger 9). The frequency of WT1 gene methylation increases with the severity of the lesion, WT1 gene in normal samples The frequency of methylation is n 1%, which is [% for muscle sample towels, 42.1% for HSIL samples, and 77:8% for scc samples; statistical results show that there is a medical gene methylation phenomenon. The risk of patients with 'high squamous cell intraepithelial lesions l' and squamous cell carcinomas) was the same (the odds ratio of the two was 6.7, 27.9; the jump confidence interval was a·, 9.8 respectively). -78.9) Diagnostic performance of DNA methylation indicators Analyze the sensitivity of DNA methylation (_out_) and specificity (specifid(7) to determine the target k gene as a biological indicator of high-grade uterine lesions and uterus , analysis, ". As shown in Table 8, the sensitivity and specificity of the squamous cell carcinoma (SCC) with HPV test to check the sample were 83.1%. 5% private (the 9 state letter ships are 77 6·88 5 and 79.6-91.4 respectively); and the methylation status of the $ genes of S (m, LM LMX1A, ship 61 and wti are analyzed for screening The presence or absence of squamous cell carcinoma (SCC), the sensitivity of each gene methylation status to squamous cell carcinoma (see) is 77.8%_94.4%, and its specificity is 881%_1〇〇%: when simultaneously combined When Qing and individual methylation indicator genes are used to detect diseases (_bined pa· 23 200831900 testing, CPT), it means that as long as the test results of any of the methylation indicator genes are positive, the test sample is identified. Cervical cancer screening results were positive, with a sensitivity of 97.2 〇 / 〇 -98.2 〇 / 〇 ^ # ^ ^ ^ 66 · 7 〇 / -79.5 〇 / 〇; testing, CST) HPV test and individual methylation indicators At the time, it means that the test is first carried out, and the samples that are positive for the HPV test are tested on the basis of each index. The sensitivity is between 69.4% and 85,0%, and the specificity of all tests. 100%. - # simultaneously with high-grade squamous cell intraepithelial lesions L) and squamous cell carcinoma (scc) diagnostic criteria, test to check the sample for HSIL The sensitivity and specificity of SCC are 75.0% and 85.5% respectively (the 95% confidence interval is 7〇2-79·8 and 79 6 91 4 respectively); and the analysis of SOX, PAX, LMX1A, ΝΚΧ6-1 and The methylation status of the five genes of WT1 to screen samples for HSIL or SCC: the sensitivity of methylation status of each gene to cerebral palsy or similar is between 57.4% and 76. 2%, and its specificity is between 881%•lion%; when combined with Hpv test and individual methylation indicator genes to detect disease (cpT), the sensitivity can be increased to 85.8%-94·9〇/. When the sequential combination (CST) book (10) was tested with individual methylation indicator genes, the specificity of all tests was 100. /. When the combined methylation (CPT) Hpv test and S〇xl, P PAX and LMX1A methylation status tests to screen samples for squamous cell carcinoma- (SCC), the sensitivity can reach 100%. When the sample was screened for HSIL or SCC by the same method, the sensitivity was 93.4%. In the results of screening for squamous cell carcinoma (scc) with individual methylation index genes, the sensitivity of squamous cell carcinoma (scc) was the highest in the detection of PAX1 gene thiolation status. Up to 94.4% (95./. confidence interval is 90.0-98.8). Similarly, the sensitivity of screening for samples with PAX1 gene methylation status of HSIL or SCC can reach 76.2% (95% 彳g赖The interval is 69.7-82.7), while the specificity of both tests is. 24 200831900 HPV 9/42 (21.4%) 21/44 (47.7%) 3.3 (1.3-8.6) 3.1 (1.1-8.3) 32/54 (59.3%) 5.3 (2.1-13.3) 5.2 (2.1-13.0) 96/ 108 (88.9%) 29.3 (11.3-75.8) 29.9 (11.5-77.7) ^<0.0001 ONECUT1 0/45 (0%) 3/45 (6.7%) 4/54 (7.4%) 2.3 (0.5-10.8) 2.3 (0.5-10.8) 22/108 (20.4%) 7.4 (2.1-25.7) 7.3 (2.0-25.9) Corpse = 0.001 WT1 5/45 (11.1%) 9/45 (20.0%) 2.0 (0.6-6.5) 2.7 ( 0.8-9.3) 24/57 (42.1%) 5.8 (2.0-16.9) 6.7 (2.2-19.8) 84/108 (77.8%) 28.0 (10.0-78.8) 27.9 (9.8-78.9) /?<0.0001 NKX6-1 5/42 (11.9%) 24/45 (53.3%) 8.5 (2.8-25.5) 9.6 (3.1-30.4) „ 27/49 (55.1%) 9.1 (3.1-27.0) 9.6 (3.2-29.1) 86/107 ( 80.4%) 30.3 (10.6-86.5) 29.8 (10.4-85.2) p<omoi LMX1A 3/45 (6.7%) 6/45 (13.3%) 2.2 (0.5-9.2) 2.2 (0.5-9.7) 8/50 (16.0 %) 2.7 (0.6-10.7) 2.7 (0.7-10.9) 98/109 (89.9%) 124.7 (33.1-469.9) 124.5 (33.0-470.1) Corpse <0.0001 PAX1 0/41 (〇%) 1/44 (2.3 %) .24/57 (42.1%) >999.9 (<0.1 - >999.9) >999.9 (<0.1->999.9) 101/107 (94.4%) >999.9 (<0.1 - &gt ;999.9) >999.9 (<0.1- >999.9) <0.0001 SOX1 1/45 (2.2%) 2/45 (4.4%) 2.0 (0.2-23.4) 3.1 (0.3-36.7) 5/54 (9.3%) 4.5 (0.5 - 39.9) 5.1 (0.6-45.9) 88 /108 (81.5%) 193.5 (25.2-1000) 200.2 (25.8-999.9) ^<0.0001 Sample normal (η = 45) LSIL (η = 45) Winning ratio (95% confidence interval) Winning ratio* (95% trust Interval) HSIL (η = 58) Winning ratio (95% confidence interval) Winning ratio* (95% confidence interval) SCC (η= 109) Winning ratio (95% confidence interval) Winning ratio* (95% confidence interval) Statistics^ Male ^1-Umbrella ^Chr AdH^t^ik* 200831900 Highly squamous cell intraepithelial lesion (HSIL) / squamous cell carcinoma (SCC) Specificity (95% confidence interval) (79.6-91.4) (93.0-100.0 (63.3-89.1) (100.0-100.0) (100.0-100.0) (66.8-92.2) (100.0-100.0) (85.1-100.0) (57.8-85.1) (100.0-100.0) (80.7-99.3) (55.8-84.2 ) (100.0-100.0) (78.3-97.9) (52.4-80.9) (100.0-100.0) Winning ratio 85.5 97.6 76.2 100.0 100.0 79.5 100.0 92.9 71.4 100.0 90.0 70.0 100.0 88.1 66.7 100.0 Sensitivity (95% confidence interval) (70.2-79.8 (49.8-65.0) (80.4-91.2) (42.9-58.3) (69.7-82.7) (85.0-94.3) (58.0-72.5) (59.3-74.0 (84.5-94.1) (50.8-66.2) (65.4-79.5) (91.4-98.3) (51.3-66.7) (58.2-72.7) (85.8-94.8) (46.3-61.5) Winning ratio 75.0 57.4 85.8 50.6 76.2 89.6 65.2 66.7 89.3 58.5 72.4 94.9 59.0 65.5 90.3 53.9 Specificity (95% confidence interval) (79.6-91.4) (93.0-100.0) (63.3-89.1) (100.0-100.0) (100.0-100.0) (66.8-92.2) (100.0- 100.0) (85.1-100.0) (57.8-85.1) (100.0-100.0) (80.7-99.3) (55.8-84.2) (100.0-100.0) (78.3-97.9) (52.4-80.9) (100.0-100.0) Squamous cells Cancer (see) odds ratio 85.5 97.6 76.2 100.0 100.0 79.5 100.0 92.9 71.4 100.0 90.0 70.0 100.0 88.1 66.7 100.0 Sensitivity (95% confidence interval) (77.6-88.5) (74.2-88.8) (95.6-100.0) (63.8-80.7) ( 90.0-98.8) (95.6-100.0) (78.3-91.8) (84.3-95.6) (95.7-100.0) (73.3-88.1) (72.9-87.9) (95.6-100.0) (62.4-79.6) (69.9-85.6) ( 94.1-100.0) (60.8-78.1) Winning ratio 83.1 81.5 98.1 72.2 94.4 98.1 85.0 89.9 98.2 80.7 80.4 98.1 71.0 77.8 97.2 69.4 Test method Individual test Individual test CPT CST Individual test CPT CST Individual test CPT CST Individual test CPT CST Individual test CPT CST Biomarker HPV SOX1 PAX1 LMX1A NKX6-1 WT1 VIXm+ Ρ·ε8-8Ί75) Γ69 (ε·卜 6 丨 inch CK8) inch·£6Ρ·ε8_00·κ) S9 (ooosooi) 0001 Idur—ίχνΐ IXOS+

As 200831900 Example 5 Methylation analysis of target genes in ovarian tumor samples

Methylation-specific PCR (MSP) was used to analyze the methylation status of target genes in ovarian tumor samples. The results of the county-based recordings were as shown in Table _, and the SOX1, PAX1, and LMX1A in each ovarian tumor sample were 3 The methylation status of the genes showed that the three genes, SOX, PAX1 and LMX1A, were not methylated in all of the _ nest benign tumors and _ nest marginal tumor samples; in the (iv) malignant tumor samples, The frequency of methylation of these three genes increased significantly. The frequency of methylation of S0X1 gene was $55.7%, the frequency of methylation of ρΑχι gene was 49.2%, and the frequency of methylation of LMX1A gene was 32 ships. (i Table 7 Analysis of methylation status of target genes in tumor samples

SOX1 PAX1 1 *-------- !P good color gj color (n=36) 0/36 (0,0%) 0/36 (0.0%)

Example 6 Methylation analysis of target genes in hepatocyte samples. Methylation-specific PCR (MSP) analysis of methylation status of target genes in hepatocyte samples. As shown by the researchers, in the normal hepatocyte samples, the frequency of SOX1 gene methylation was 7 7%. Underneath, the frequency of SOX1 gene methylation in abnormally abnormal lesions was significantly increased in chronic hepatitis samples. In the liver cirrhosis samples and liver cancer samples, the frequency of methylation of S〇X1 gene was 33.3%, 27.5%, and 53.7%, respectively. In addition, the frequency of methylation of the brain (6) gene in the normal liver cell sample was also significantly lower than the frequency of methylation of the NKX6-1 gene (57%) in the liver cancer 27 200831900 sample.

The cancer diagnosis method provided by the invention is compared with the aforementioned conventional technology (four), and more

1. The cancer screening method provided by the present invention is a diagnostic indicator for the presence or absence of cancer in the methylation process of the vitamins, and compared with the Pap smear and the human papillomavirus test (HPV = ng). The cancer prescription (4) of the present invention is superior to the above two aspects. 2. The method for screening cancer provided by the present invention may be combined with screening for the first-line uterus, or may be combined or Auxiliary human papillomavirus test (HpVtesting) test, as a second line of cervical cancer screening, in order to achieve a more accurate uterine screening test. 3. The cancer diagnosis method provided by the present invention can be applied to the detection of other cancers (such as W liver cancer) in addition to fine detection in the uterus, to assist in the diagnosis of abnormal samples. The detailed description above is a detailed description of one of the possible embodiments of the present invention, and the embodiment is not intended to limit the scope of the invention, and the equivalent of the invention is not deviated from the spirit of the invention. For example, the equivalent 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 this case. "The method of cancer diagnosis provided in this case is indeed innovative, and it can be used in comparison with the above-mentioned multiple functions of the uterus cancer. It should have fully complied with the novelty and progressiveness of the legal patent for the month. The law filed an application, and requested the f bureau to approve the invention patent towel request, in order to invent the invention, to the sense of virtue. [Simplified illustration] ( ® - is the target of the cancer screening method of the present invention _ 〇 ^ sequence For analysis, each of the f-containing CPG sequences is indicated by "I"; the position of each gene MSIM scorpion synthetic fragment is indicated by "_"; the position of each gene sulfite sequencing (BS) primer is indicated by "(a)"; Figure 2 is a diagram showing the target genes used in the cancer screening method of the present invention, in a mixed cervical cancer tissue sample (mixed with 3 samples) and a mixed normal Pap smear sample. Results of a basic-specific PCR (MSP) analysis; a mixed normal Pap smear sample (10 samples mixed), and a second column of mixed cervical cancer tissue samples (9) "mixed", 3 blocked as a negative control group (neg The ative amplitude rol), the fourth block is the positive control group (four) control), the fifth block is the blank control group (water); the third figure is the target gene for the cancer (four) test method of the present invention, and the individual sells the neck. Methylation-specific pcf (MSP) analysis was performed on cancer tissue samples and individual normal Pap smear samples. T1, T2, T3, and T4 represent 4 individual cervical cancer tissue samples, milk, sputum 2 Ν3, Ν4 represent 4 individual normal samples, and the sheds indicating υ indicate the result of methylation-specific pCR (MSp) with MSP primer (U) that can specifically recognize the unmethylated gene sequence. The position indicates the result of methylation-specific PCR (MSP) by the primer (10) which can specifically recognize the methylation gene sequence; 29 200831900 Figure 4A shows the target genes used in the cancer screening method of the present invention, in the absence of Treatment of 5'-aza-2'-deoxycytidine in HeLa cervical cancer cell lines (AZC-, 1, 2), and HeLa cervical cancer cell lines treated with 5'-aza-2'-deoxycytidine ( AZC+, columns 3 and 4), the results of methylation-specific PCR (MSP) analysis; The ^^ field indicates the result of methylation-specific PCR (Msp) with a MSP primer (U) that specifically recognizes the unmethylated gene sequence, indicating that the stop of the sputum indicates that the methylated gene sequence can be specifically identified. Results of methylation-specific PCR (MSP) of MSp primer (μ); Figure 4B shows the target genes used in the cancer screening method of the present invention, without treatment of 5'-aza-2'-deoxycytidine HeLa cervical cancer cell line (AZC-, column 5), and HeLa cervical cancer cell line treated with 5'-aza-2'-deoxycytidine (AZC+, 6th stop), RT-PCR analysis Results; Figure 5A shows the sulfite sequencing (Bs) in the HeLa cervical cancer cell line without treatment of 5'-aza-2'-deoxycytidine for each target gene used in the cancer screening method of the present invention. The results of the analysis; Figure 5B shows the sulfite sequencing in the HeLa cervical cancer cell line treated with 5'-aza-2'-deoxycytidine for each target gene used in the cancer screening method of the present invention ( Bs) results of the analysis; Figure 6A is the target used for the cancer screening method of the present invention Because of the results of sulfite sequencing (BS) analysis in cervical squamous cell carcinoma (SCC); and FIG. 6B is the target gene used in the cancer screening method of the present invention, in a normal sample. , the results of sulfite sequencing (BS) analysis. [Main component symbol description] None 30

Claims (1)

200831900 X. Patent application scope: A method for screening cancer 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 a subject to be tested; gamma step 2 privately measuring the sequence methylation status of at least one target gene in the genomic DNA of the test subject, the target gene consisting of S0X1, PAX1, LMX1A, NKX64, WT1, and ONECUT1; Step 3 determines whether the specimen has cancer or precancerous lesions based on the presence or absence of the methylation status of the target gene. 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- Cervical epithelial cells, etc. 3. The method for screening cancer according to claim 1, wherein the methylation status of the CpG sequence of the target gene is methylated-specific polymerase chain reaction (MSP), quantitative A Quantitative methylation-specific PCR (QMSP), bisulfite sequencing (BS), microarray (infracroys), mass spectrometer, denatured liquid chromatography (Deauturing high-performance liquid chromatography, DHPLC), pyrosequencing, and the method of screening for cancer according to claim 1, wherein the target gene s〇xi has the SEQ ID No: 1 is a nucleic acid sequence. 5. The method of cancer screening according to claim 1, wherein the target gene MX1 has a nucleotide acid sequence as shown in SEQ ID No: 2. 31 200831900 6. If you apply for a patent scope! The method of screening for cancer according to the invention, wherein the target gene swarm has a nucleotide sequence as shown in SEQ ID No: 3. 7. If you apply for a patent scope! The method of cancer screening according to the invention, wherein the target gene has a nucleotide sequence as shown in SEQ ID No: 4. The method of detecting a cancer according to the invention of claim 1, wherein the target gene has a nucleotide sequence as shown in SEQ ID No. 5. 9. The method of claim 1, wherein the target gene p 0NECUT1 has a nucleotide sequence as shown in SEQ ID NO: 6. 10·—Seed cervical cancer screening method is to detect the methylation of the target gene in the test subject cells, as a screening indicator for the presence or absence of the cervical cancer, the method comprises the following steps: Step 1 provides a a test subject; step 2 detecting a thiolation state of a CpG sequence of at least one target gene in the genomic DNA of the test subject 'the target gene is composed of SOX1, PAX1, LMX1A, NKX6], wti, and ονεοιγι; Step 3 determines whether the specimen has cervical cancer and precancerous lesions based on the presence or absence of the target gene thiolation state. 11. The method of cervical cancer screening according to claim 10, wherein the test corpus callosum is a smear, blood, urine, cervical epithelial cells, and the like. 12. The method of screening for cervical cancer according to the first aspect of the invention, wherein the test subject is an abnormal Pap smear. 13. The method of screening for cervical cancer according to the first aspect of the invention, wherein the test subject is a cervical cell sample positive for HPV testing. 14. The method of screening for cervical cancer according to the first aspect of the patent application, wherein the 32 200831900 CpG sequence methylation state detection method of the target gene is methylation-specific polymerase chain reaction (methylation-specific PCR) , MSP), quantitative methylation-specific PCR (QMSP) 'bisulfite sequencing (BS), microarray (mictOarrays), mass spectrometer analysis (mass spectr〇meter ), denaturing high-performance liquid chromatography (DHPLC), pyrosequencing (pyrosequencing). The method of cervical cancer screening according to the invention of claim 5, wherein the target gene ζSOX1 has a nucleotide sequence as shown in SEQ ID No. 1. The method of cervical cancer screening according to the first aspect of the invention, wherein the target gene PAX1 has a nucleotide sequence as shown in SEQ ID No. 2. 17. The method of cervical cancer screening as described in claim 1 (), wherein the target gene LMX1A has the nucleotide sequence as shown in SEQ ID No: 3. 18. The method of cervical cancer screening according to claim 1G, wherein the target gene NKX6-1 has a nucleotide sequence as shown in SEQ ID NO: 4. The method of cervical cancer screening according to the invention of claim 1, wherein the target gene WT1 has a nucleotide sequence as shown in SEQ ID No. 5. The method of screening for cervical cancer according to the first aspect of the invention, wherein the target gene ONECUT1 has a nucleotide sequence as shown in SEQ ID NO: 6. 21. The method for detecting nest cancer _ detection is to detect the state of methylation of a target gene in a test subject cell, and to determine whether or not the vaccination cancer has a _ test index, the method comprising the following steps: Step 1 provides a test Step 2: detecting the CpG sequence 歹丨 methylation status of at least one target gene in the genomic DNA of the test subject, which is composed of soxi, PAX1, LMX1A group 33 200831900; and step 3 according to the target The presence or absence of the methylation status of the gene determines whether the sample has imprinted cancer and precancerous lesions. 22. The method of screening for a nested cancer according to the second aspect of the invention, wherein the test subject is ascites, blood, urine, or the like. 23. The method for reading a nested surface test according to claim 21, wherein the method for detecting the methylation status of the CPG sequence of the target gene is methylation-spedfic PCR (MSP), Quantitative methylation-specific polymerase chain reaction (QMsp), sulfite sequencing, BS), microarrays, mass spectrometer (mass spectr〇meter), high efficiency Liquid chromatography (denaturing hjgh-perf_ance ehr_t()graphy> DHPLC), pyrolysis (pyroseqUencing) 〇24. The method of screening for ovarian cancer according to claim 21, wherein the target gene SOX1 is There is a nucleotide sequence as shown in SEQ ID No: 1. The method of screening for ovarian cancer according to claim 21, wherein the target gene PAX1 has a nucleotide sequence as shown in SEQ ID No. 2. The method of screening for ovarian cancer according to claim 21, wherein the target gene LMX1A has a nucleotide sequence as shown by SEqID No: 3. 27. The method for screening for liver cancer is to detect the state of methylation of a target gene in a test subject cell, and to serve as a screening indicator for the presence or absence of liver cancer, the method comprising the following steps: Step 1 provides a test subject Step 2 detecting the methylation status of at least one target gene wCpG sequence in the genomic DNA of the test subject, the target gene is composed of SOX NKX6-1; and 34 200831900 Step 3 according to the methylation status of the target gene Whether or not the sample has liver cancer and precancerous lesions. 28. The method of screening for liver cancer according to claim 27, wherein the test subject is ascites, blood, urine, feces, gastric juice, bile, or the like. 29. The method of screening for liver cancer according to claim 27, wherein the methylation-specific PCR (MSP) of the CpG^ sequence methylation status of the target gene is methylated-specific PCR (MSP) Quantitative methylation-specific polymerase chain reaction (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturation A method for screening for liver cancer as described in claim 27, wherein the target gene s〇Xl has a method of decoupling high-performance liquid chromatography (DHPLC) A nucleotide sequence as shown in SEQ ID No: 1. The method of screening for liver cancer according to claim 27, wherein the target gene NKX6-1 has a nucleotide sequence as shown by SEqIDN〇:4. 〇35 200831900 Sequence Listing <110> Lai Hongzheng <120> A method of cancer screening. <160> 58 <210> 1 <211> 2300 <212> DNA <213> (Homo sapiens) <400〉 1 f tctccacaag cactctcatc tcagggtgcc tcggggaggg cagaggagga atacttgggc 60 tcaaaagtcg tctttggacc actttcagat cagtctggtg gaaacggtag tgtgagcgct 120 atgctacttg agactgcgtt tgaaaatctc tttcacgttc ccaaatcaaa gccactttga 180 ggtttaagaa tgataaccac aggtgaatgc cttactcttt ccacgagcca ggcctttcct 240 ttgcatgacg cagaccggcg gctgagaccc gtcctgaggc cccgcctttc attcggttta 300 tgtggccccg cgcagttcac gcagttttct ccgttcttag gcaaccagct cgtgggaaat 360 tcactccaga aaagcgtgcg ccatatcatt attttgcgat tcaacaaact ttttcaactg 420 ttgttcagga actagattcc agatagatct tgttgtgttc ggccttccta gaaattcctt 480 ttccagagga agaagatccg ggttgggaag agtgcgtgac tatggccccg ggctcattga 540 aagactcctc cccacaaaag tttagggctg atactaaacg aagctatgga gccatgccca 600 cacaaatact ccccctttac ccgaattgtg gggcctggac tgtgaaggcc ttcgctgcaa 660 gagcgggcac tggcgaactt cagtgcacgc cgcggccgag aacggatgca gggcggggga 720 tggctgagcc gcctgatcct ctgcaggggc cctcggaggg tatcccctgc 780 gtccaaggga gcgcccctcc ttttcagcac tcgggggaac tgagggcgac gtgccagccc 840 cgcactcaac tttccccttc tgcagagaac cctgcaggca cagagttggc cggcgggggc agaggaggag 900 ctgggtctcc actgcgcccg tttaaacctg gccaggggct gcgtttcctc cccccacccc 960 acgacgatcc tttcttagtc ttcgcttttc aacccaatcg ttaatcattc ggaacgcgcg 1020 ggcggggagc ggcgaggagg gcgagctcgg ggttcgccgc cgccgccgcc gccgcgcgcg 1080 cgcgctcagg aagcggtgtg gctgtcaccc cctcccgggc ctcctccccc ctccttcctg 1140 ctttgctccc cctccttcct cccctcctcc ccgctccgcc gcccgcgccc agtgtatcta 1200 ctccctcccc acgtcactcg ccagcgcgcc atgcaaatca ccgccgccgc cggctcccat 1260 tggccgcggc gcgctcattt aatggcagcc cgggcccggc gtatggctgc tgggccccgc 1320 gcgccgccgg ccccgcgtgc gcctccgctc cgagcgcacg gccccgggca ggcagcgggc 1380 agcccatccc gggctcggcg gccccggctc tccggccctc tccgcgagcc cgcgctcctc 1440. ccgctgtccc cgggcccctc cctggctgca ccgtaatcgc cccctgcagg cccccctgcg 1500 cctccccccc cccgccactg gcgcctggct tcccccgggc acctgggacc agcacatgcc'1560 cagcgcacgc ggcgcgccgc cctgc.tagaa gttgcagcct ccgagttgga ggccgctgag 1620 gaccgagcgc aggaggaagg agacagcgcg cagcggcggc cggcgaggag acagcacacc 1680 Ccgggccggg cccagcgcac cgc tcccggc cccaaaagcg gagctgcaac ttggccacga 1740 ctgcacctgt ttgcaccgct ccgccgaggg cgcctgggct gcggtggcgg cgaagacggc 1800 200831900 gaccccgacc gtcggcctct ttggcaagtg gtttgtgcat caggagaaac tttccacctg 1860 cgagccgaac cggcgccgag tgcgtgtgtt tctgcctttt tttgttgtcg ttgcctccac 1920 ccctccccat tcttctctcc gctaggaccc ccccgccccc gtctcactcc gtctgaattc 1980 ctctccgtct ccctcccacc ccggccgtct atgctccagg ccctctcctc gcggtgccgg 2040 tgaacccgcc agccgccccg atgtacagca tgatgatgga gaccgacctg cactcgcccg 2100 gcggcgccca ggcccccacg aacctctcgg gccccgccgg ggcgggcggc ggcgggggcg 2160 gaggcggggg cggcggcggc ggcgggggcg ccaaggccaa ccaggaccgg gtcaaacggc 2220 ccatgaacgc cttcatggtg tggtcccgcg ggcagcggcg caagatggcc caggagaacc 2280 ccaagatgca caactcggag 2300 < 210 > 2 < 211 > 1200 < 212 > DNA f ≪ 213> person who is wisdom species (Homo sapiens) < 400 > 2 agaaagacga ggccaggcca cctgggatag agtggtgcga gattccaccg ccagggagaa 60 aggaacttgt ccttcagacc tagagctgga gctatgcatt tggcctcccg ccggtcgcgc 120 ttggggacag gagggcgccg gatctatgcg ccccttagca gcagatcggg atccttttgc 180 ctcctgcccc ttctgtcact gcttggagag ggatgagttc tggtggctgg gcctggctgt 240 aggagacagg atttggaccg tgcccctctc gcatcaccga aatcaccccc actattccaa 300 gagtggttgg ctattaaacg tgaagatttc ctgagagaag gattgaggac ctggccagga 360 atgggacaca agttccgcct tgtgtcttcc tgacaggagc cctgcaccgc gctggacgct 420 caccttgaca ctcccagcca gctggggtac tgatcccacc cttccccggc cgctgccccg 480 ggagtgggga ggt ^ gagaga gccacacccg aaacaccttt ccacgataaa cttttattct 540 ctatcttatt attaatggtg gcggaaataa aactaaaacc aaaacgaaaa cgagtactag 600 tactaacaca ctaatcaatt tgagatgact tccccctcat tcccaaagct agaggaggaa 660 gggggctgaa aggggctcag agcagtggaa ggtcccaggc Ccagctgggg ttgggacgtg 720 tgtgtgtgtgt tgtgtgtgtgt tgtgtgtgtgt tgtgtgtgtg tagaggggtg ggttccgggg 780 gggtggtgga agagggccga ggggggagga tagaaggagg gggtagagtt tcagggcggg 840 gaggggggcg ctggggcgca gtgacgggaa ccaatgagct gccaactcgc gcgtctccgg 900 cgtgactgcc gagattgacg tggaggacac gtcaaattga ttcccgcacg ctgcagcctc 960 ccggtcagac gaatttctcc caatcggatg aagttcaccc tgggcctggg gtcgcgggcg 1020 tggagagtgt cctgggaggg ggcagcagcg gcggcggcag gccctggagc gggcggcagc 1080 gcgctccgct gccgcgcaca gcgcgtctcc agcccgcggc tgggccgccg cggctctcgg 1140 ctctcgggcg ccctccctct atgcctctca cgcggcggcg gcggcgccca agctctcccg 1200 < 210> 3 <211> 2350 <212> DNA <213> Homo sapien's 2 200831900 <400> 3 agccccccgc ccagcgcacc ccaccccctc tccgccgccg cgcacgcagc gccgcggccc 60 ctgtcagaag ctgcaggatc cgccccggcg aagcagggcc gactcgcacc caggaccctg 120 ggcctctgcc ttccctccta gccttggaga agcaactggc cctctcctcc cgctgaggag 180 cgacgcgggc tggtaggacg tcccgggaag gccggcagct cgcgaccacg tcccggccca 240 gcctgggcgc gccgaggagc agagccagcg gccggcgttc gctccggctc cctccccggc 300 gctccgaagc cgagggcggc tcctccggct gcagtctcgg gggcgacgcc ttcccgggca 360 gaagcttcca gcagcgctcc gcaacttctc tctgctccag tcactgggag agagctcgcc 420 taccaggtaa gtaaggctgc ccgtgcctag gctgtggctc gggcgggcgt gtttctgaaa 480 gttgacttga aatgcatcca agagtgagcc gggccagccg gggctcctcc ccgaggcgac 540 tcttttgctt ctgcagacat tcccggcact ggccgactgg cgggaggacc tccccgcgcg 600 ccccgcacac cggctcctgc gcgcacccca acagagcgca gcgccaggag tccagaagcg 660 ggcgggacgc cctccgggtc ccttacagtg ccccttctcg acctggggca ggtgagggcc 720 gcaacggggc ggctgggacg cgggattgca aaccccatcg tcccgcgtgc ctggacccgg 780 tcgctcgagg gagggtaccc actctttata tacccaatat accccagtag ctgcgttcct 840 gcagagacgt cccgagggcc caccttcgta taggttgggg cggagtcgga ttcgggatgg 900 aaaacctggg gcaagggatg taggtggggg tgaggggggq aggagaagga gaaacgcagt 960 tggggggcgg aggcctaagt acataacgtg ttgacttcaa gtgaaatcag atcagccaga 1020 gcagttcgct gtgactgatc tctcctccca ccctacattc tcttggctgg accctatcct 1080 cctggctgat tctggtcgcc ctggacactc cctcagttct ttcccaggag tgcggtggct 1140 gctggcgccg agtcccagcg ggcacggacg tcagacgcat cgtttcttct cctctacagg 1200 tcctcccggc ccggcccgaa catgctggac ggcctaaaga tggaggagaa cttccaaagc 1260 gcgatcgaca cctcggcctc cttctcctcg ctgctgggtg agtgttcagg ccgtgcgtcc 1320 tgggcgcact ctctttccgc ttggcgctga gctctggagc cccgctctct gggacctggt 1380 ccgcgatagg gaagctagcg cccctcttca tacactaaat tgagccccat cactatctgt 1440 ccgtcagtgc ttgtgggtcg tccctaccca aataaatcca acaagccgcc ccaggcctca 1500 cgcactgggc accgaattcc ccaaagccgc gaggggcggg cgagcttgtt cgtaggcgtc 1560 tgagtggcaa gtgattaaaa atacccaggg ctggattttt aatctcggag ctgatcgacg 1620 tctcataaat gccgccctct tctcgcggcc tagaggcaat agcatccgag acccgaggcc 1680 tggagcgccc aagttcgagg aggcttc tct cccccaccaa ctccagcccc aatttcagcc 1740 atgggcaagg ccgagagaga cttttctggg ccagtaggca acgcagcgcg gggattagac 1800 cgcgcggctg ggccctaggc tccgcgttaa ttaactggag ctggatgtcg ggtcctgtgg 1860 gtcccccctc acaactcctt tgcagcgaca gaggaggagc agcgagtcaa ggaccccgga 1920 agagtgtcca cacacgggtc ttgcagagct gaagccagag attctggtta cggctccccc 1980 acccactctg cccttggagc ttttcaagtt tgggaagccc gtattttatt ttattttatt 2040 ttatttattt atttttagca ggagggagtt ttccttctgt cctttaaacc tcttgcctct 2100 ttcactcgga accgctagag cggcatgaat gtggaggatg agacagttct gctggagaaa 2160 ccaaatccag tgaggaatct ccccaccccc aacaccctag atgaaacgaa atcacgtgca 2220 ctgagcgccc ctctttgaac ccccctttcg ggaacttttg gacttgccca gcctcggaag 2280 agagcggaag ccagagcagg cagtacccgg gcgggcagcg ggtgcggatt tatgtataca 2340 gcgggcgtcg 2350 3 200831900 < 210 > 4 < 211 > 1250 < 212 > DNA < 213> human genus Homo Sapiens <400> 4 f.. o aaagagcgga ggagccgcgg agagggtcag tttggccaga ggacaggact tagaagaccg 60 aagcctggga agccgcgaag aaaatgccag ag aggagagt caaaggctga gagtgagagg 120 gagagaggga gcgggggtgg ggcgggggtc gccgggcccg tttgcagaag tggactgacg 180 agcggcgccg aaacccagcc gtcagacttt tcacacttag tcttttgttt ttctgtgttt 240 ttcctccccc tttttctttt caatattgca actccagtgc cccgtgggcc agagggcaag 300 gcgggtggag gtgaggacct gggagccgcg gggatccgtg gcactgccct ttctggcgca · 360 gcagcccggg gcagcgtggg cggaggaagc ccgcacagag gctagatctc ccgcgggctg 420 gatgcgcttt ctccccgggc acagtgagcg tcgaatgcga atcagccgcg cgaccgaaag 480 agcagagcat cccagtaaga tcagaggagc gccacgggct gcacaaggcg tcctttgaac 540 ctccccaaag aaagcaagcc acccccaccc tccaacttca aagtggagat tcggcaacta 600 actttgctac aaactctccg gagccagcct gggttttgtt cccgggggca 660 gaagatgaga agtagcgcac tttgaacagc taggaaaagt gaggaagaga gaatagccag 720 ggatcgaatc taggactcgc ggaacgaaag gactgcctag cccgccggga cgcctgcttt 780 tctcggcgag ctgccgcctc ccgcgtggag ggtttggaca tctctgctgc gcagctaggc 840 gagcaactcc cggcagcggc atttttggtt cagttggcag ctcgcctccg ggcgcgccga 900 gtgcctctcc gctcgcgccc tcggcgcttc cggctcctct ttgcttattt Gagccccgc g gggggcacca 960 gccagcgccc tcgctgcaag gctacggtct ccggcgtggc cgtgggatgt tagcggtggg 1020 ggcaatggag ggcacccggc agagcgcatt cctgctcagc agccctcccc tggccgccct 1080 gcacagcatg gccgagatga agaccccgct gtaccctgcc gcgtatcccc cgctgcctgc 1140 cggccccccc tcctcctcgt cctcgtcgtc gtcctcctcg tcgccctccc cgcctctggg 1200 cacccacaac ccaggcggcc tgaagccccc ggccacgggg gggctctcat 1250 < 210> 5 < 211 > 1700 < 212 > DNA < 213 > person who is wise species (Homo sapiens) < 400 > 5 tcactgagca accagaatgg tatcctcgac cagggccaca ggcagtgctc ggcggagtgg 60 ctccaggagt tacccgctcc ctgccgggct tcgtatccaa 120 tccccaaact accctcccct tcacccctcc gggcgccagg atgctccggc cggaatatac gcaggctttg ggcgtttgcc 180 caagggtttt cttccctcct aaactagccg ctgttttccc ggcttaaccg tagaagaatt 240 agatattcct cactggaaag ggaaactaag Tgctgctgac tccaatttta ggtaggcggc 300 aaccgccttc cgcctggcgc aaacctcacc aagtaaacaa ctactagccg atcgaaatac 360 gcccggctta taactggtgc aactcccggc cacccaactg agggacgttc gctttcagtc 420 200831900 ccgacctctg gaacccacaa agggccacct ctttccccag tgaccccaag atcatggcca 480 ctcccctacc cgacagttct agaagcaaga gccagactca agggtgcaaa gcaagggtat 540 acgcttcttt gaagcttgac tgagttcttt ctgcgctttc ctgaagttcc cgccctcttg 600 gagcctacct gcccctccct ccaaaccact cttttagatt aacaacccca tctctactcc 660 caccgcattc gaccctgccc ggactcactg cttacctgaa cggactctcc agtgagacga 720 ggctcccaca ctggcgaagg ccaagaaggg gaggtggggg gagggttgtg ccacaccggc 780 cagctgagag cgcgtgttgg gttgaagagg agggtgtctc cgagagggac gctccctcgg 840 acccgccctc accccagctg cgagggcgcc cccaaggagc agcgcgcgct gcctggccgg 900 gcttgggctg ctgagtgaat ggagcggccg agcctcctgg ctcctcctct tccccgcgcc 960 gccggcccct cttatttgag ctttgggaag ctgagggcag ccaggcagct ggggtaagga 1020 gttcaaggca gcgcccacac ccgggggctc tccgcaaccc gaccgcctgt ccgctccccc 1080 acttcccgcc ctccctccca cctactcatt cacccaccca cccacccaga gccgggacgg 1140 cagcccaggc gcccgggccc cgccgtctcc tcgccgcgat cctggacttc ctcttgctgc 1200 aggacccggc ttccacgtgt gtcccggagc cggcgtctca gcacacgctc cgctccgggc 1260 ctgggtgcct acag cagcca gagcagcagg gagtccggga cccgggcggc atctgggcca · 1320 agttaggcgc cgccgaggcc agcgctgaac gtctccaggg ccggaggagc cgcggggcgt 1380 ccgggtctga gccgcagcaa atgggctccg acgtgcggga cctgaacgcg ctgctgcccg 1440 ccgtcccctc cctgggtggc ggcggcggct gtgccctgcc tgtgagcggc gcggcgcagt 1500 gggcgccggt gctggacttt gcgcccccgg gcgcttcggc ttacgggtcg ttgggcggcc 1560 ccgcgccgcc accggctccg ccgccacccc cgccgccgcc gcctcactcc ttcatcaaac 1620 aggagccgag ctggggcggc gcggagccgc acgaggagca gtgcctgagc gccttcactg 1680 tccacttttc Cggccagttc , 1700 <210> 6 <211> 1850 <212> DNA <213> Human genius (Homo sapiens) <400> 6 gaaagaaagc cgcggcatgt ggcggccggg tgtacgtctc aaactggggg cctccgcaca 60 cgtccccacc aggcccaaag accaggttcg attccagatt ggagcgtgac tgtgggaagg 120 gcgaaattac Tcccgaagct gaattgattt tcaaatctgg aggcgtctct cggggacgcc 180 gggaaaaggg cgtccctagg ggccaagcgg agacccgcgc gccggcgtcc accctgtctg 240 cgtcagtact tctggaaagc aaccgcctcc gggtgcttta gcaggagggc cttgggagta 300 accgcaggga cggcccgagc ctccacgg cc ccaccagcac ccggaagtta acatgggtac 360 cctccagggc actccctccg ctctccctcg cccccctcca caggccgagt catcggcgaa 420 gcggacgcac agccttaatt atgagctgag cgggagaagg agccaggcgg cgggggacag 480 tgaggtatgg cccgaactgg gattcttggc actgattact cctctcccca gggcatggat 540 gagaaagggt gggcaagtat gtatctggga ggacgaaggg tgccgggtca acggccgcca 600 aacggaccca gccctttaag caatctgcac cccaccccac cccaaccccc atcccccaat 660 aggccgtgaa ttcaagggtg ggaaagcgca ctcccagcag ccccccggga aataaagctt 720 agtgggctag agccgaaggg gtgatgacac agtccccagc tccccgggca agctgcaccg 780 200831900 f ggaagcagca actggagaga gaggggcgat gtctccaagc acagcactcc agccgctaga 840 agcccgacac gagcgtcccc gggctgggag gacagagccc actcaagcaa gggaggcgag 900 cgagccaggc gcgagtctcc tgggattgca gcggcggccc caggtcgcgc tctgcgccaa 960 tctttcgcac gtgcccgcag ctccctggcc atccagcgcc gcagggaagg cgctgggccc 1020 cctccttcat ttgtaccggg acgccaaggg cctggcgcgc cgcgacctag ggggcggggg 1080 cgggcctcgc gcatgcgcgc tgcgcctggc gggcgtgagg gcgggccgct gcggcggcgg 1140 cggcggcggc taccgaaccg Cggccacaga gtctgtaaca gtaacagagc catggctcaa 1200 gctggccagc ggggcgggca ggcagcagac gcggcaggcg cgcgggccgc ggcaggggag 1260 ccggagacct cagaatttta agaaagagag gggcgagagg tggccgaggc gggcgggctg 1320 gggcactgcg ctctcccaac ggcgcggatc ctctttggaa attaatatta aaaaaaaaaa 1380 agccgaggac gcagagggga aggtgggggg taagagggaa ggcgagacac acacacacac 1440 acacacgcac acgcacacac ggacacacac acacggagag agagagagag agagacagag 1500 ccccacagtg agaggaagga aggcaacagt cgccagcagc cgatgtgaag accggactcc 1560 gtgcgcccct cgccgcctct gcctggccac atcgatgttg tgtccgccgc ctgctcgccc 1620 ggatcacgat gaacgcgcag ctgaccatgg aagcgatcgg cgagctgcac ggggtgagcc 1680 atgagccggt gcccgcccct gccgacctgc tgggcggcag cccccacgcg cgcagctccg 1740 tggcgcaccg cggcagccac ctgccccccg cgcacccgcg ctccatgggc atggcgtccc · 1800 tgctggacgg cggcagcggc ggcggagatt accaccacca ccaccgggcc 1850 < 210 > 7 < 211 > 24 < 212 > DNA < 213> Artificial sequence <400> 7 cgtttttttt ttttcgttat tggc 24 G <210> 8 <211> 20 <212> DNA <213>Artificial sequence<213>;400> 8 cctacgctcg atcctcaacg 20 <210> 9 <211> 25 <212> DNA <213> artificial sequence 200831900 <400> 9 tgtttttttt tttttgttat tggtg <210> 10 <211> 22 <212&gt DNA <213>Artificial sequence<400> 10 cctdcactca atcctcaaca ac <210> 11 <211> 18 <212> DNA <213>Artificial sequence<400> 11 tttagaagcg ggcgggac <210> 12 <;211> 17 <212> DNA <213> artificial sequence Ο <400> 12 ccgaatccaa acacgcg <210> 13 <211> 22 <212> DNA <213>artificial sequence <400> 13 gagtttagaa Gtgggtggga tg <210> 14 <211> 24 <212> DNA 200831900 <213>Artificial sequence <400> 14 caaccaaatc caaacacaca aaac <210> 15 <211> 21 <212> DNA <213 〉Artifical sequence<400> 15 ttgtagcggc ggttttaggt c <210> 16 <211> 20 <212> DNA <213> artificial sequence<400> 16 gccaaaccct taacgtcccg o <210> 17 <211><212> DNA <213>Artificial sequence <40 0> 17 gattgtagtg gtggttttag gttg <210> 18 <211> 25 <212> DNA < 213 > artificial sequence <400> 18 caccaaaccc ttaacatccc aatac <210> 19 200831900 <211> 20 <212> DNA <213>Artificial sequence <400> 19 tattttgggt ttggggtcgc <210> 20 <211> 18 <212> DNA <213>Artificial sequence <400> 20 cccgaaaacc gaaaaccg <210> 21 <211 〉 24 <212> DNA <213>Artificial Sequence<400> 21 gtttattttg ggtttggggt tgtg <210> 22 <211> 21 <212> DNA <213>Artificial sequence<400> 22 cacccaaaaa ccaaaaacca c <210> 23 <211> 20 <212> DNA <213><400> 23 cgtggtcgtg ggatgttagc 200831900 <210> 24 <211> 21 <212> DNA <213>Artificial sequence <400> 24 acaaacaacg aaaaatacgc g 21 <210> 25 <211> 22 <212> DNA < 213 > 213 > artificial sequence <400> 25 22 gtgtggttgt gggatgttag tg <210> 26 <211> 25 <212> DNA <213>artificial sequence <400> 26 caacaaacaa caaaaaatac acaac 25 <210> 27 <211> 21 <212> DNA <213>Artificial sequence <400> 27 tgttgagtga atggagcggt c <210> 28 <211> 23 <212> DNA <213><400> 28 10 200831900 cgaaaaaccc ccgaatataa acg <210> 29 <211> 24 <212> DNA <213>Artificial sequence <400> 29 gttgttgagt gaatggagtg gttg <210> 30 <211> 29 C <212> DNA <213>Artificial sequence <400> 30 sattacaasd adcccccaea tataaacac <210> 31 <211> 26 <212> DNA <213>Artificial sequence <400> Gttgttttyg ggtttttttt tggttg <210> 32 <211> 28 <212> DNA <213>Artificial sequence <400> 32 atttctccta atacacaaac cacttacc <210> 33 <211> 29 <212> DNA <213 〉 artificial sequence 200831900 <400> 33 tagttattgg gagagagtty gtttattag <210> 34 <211> 24 <212> DNA <213> artificial sequence<400> 34 ctaccccaaa tcraaaaaaa acac <210> 35 <211> 22 <212> DNA <213>Artificial sequence<400> 35 gagtttattt aagtaaggga gg <210> 36 <211> 30 <212> DNA <213> <400> 36 caacttaaac cataactcta ttactattac <210> 37 <211> 22 <212> DNA <213>Artificial sequence <400> 37 gtgttttggg agggggtagt ag <210> 38 <211> 21 200831900 < 212> DNA <213>Artificial sequence <400> 38 ccctcccraa ccctacctat c <210> 39 <211> 22 <212> DNA <213>Artificial sequence <400> 39 gatagaagga gggggtagag tt <210> 40 <211> 21 <212> DNA <213>Artificial sequence <400> 40 tactaccccc tcccaaaaca co <210> 41 <211> 25 <212> DNA <213>Artificial sequence <400> 41 ggtatttttg gtttagttgg tagtt <210> 42 <211> 22 <212> DNA <213> artificial sequence<400> 42 aataccctcc attaccccca cc 200831900 <210> 43 <211> 22 <212> DNA <;213>Artifical sequence <400> 43 ggtgggggta atggagggta tt <210> 44 <211> 24 <212> DNA <213>Artificial sequence <400> 44 cctaaattat aaatacccaa aaac <210> 45 <211&gt ; 24 <212> DNA &lt ;213>Artificial sequence <400> 45 gtgttgggtt gaagaggagg gtgt <210> 46 <211> 28 <212> DNA <213>Artificial sequence <400> 46 atcctacaac aaaaaaaaat ccaaaatc <210> 47 <211> 22 <212> DNA <213><400> 47 agacctagat gccaacaatt gg 200831900 <210> 48 <211> 21 <212> DNA <213>Artificial sequence <400> 48 gcaccactac gacttagtcc g <210> 49 <211> 21 <212> DNA < 213 > artificial sequence <400 > 49 gctgcttctg ctgctgtgtc t <210> 50 <211> 21 <212> DNA <213><400> 50 acgtttgggg cgcttatggt c 〇<210> 51 <211> 21 <212> DNA <213> artificial sequence <400> 51 caaaccctgg agcaaactca a <210> 52 <211> 21 <212> DNA <213> artificial sequence 200831900 <400> 52 tgtgttgcct ctatccttcc c <210> 53 <211> 24 <212> DNA <213>artificial sequence <400> 53 cctacgctgc cctacaacca catc Ο <210> 54 <211> 24 <212> DNA <213>Artificial sequence <400> 54 tcacgccggc ccagtcttcc atct <210> 55 <211> 21 <212> DNA <213> Sequence <400> 55 cacacgagac ccactttttc c <210> 56 <211> 20 <212> DNA <213>Artificial sequence <400> 56 cccaacgaat aggccaaacg <210> 57 <211> 21 <212&gt DNA 200831900 <213> Artificial sequence <400> 57 gctgtcccac ttacagatgc a 21 <210> 58 <211> 21 <212> DNA. <213> Artificial sequence <400> 58 * tcaaagcgcc agctggagtt t 21 Γ) o 17