CN112391466A - Methylation biomarker for detecting breast cancer or combination and application thereof - Google Patents
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Abstract
The invention relates to a methylation biomarker for breast cancer detection, and a combination and application thereof, wherein the methylation biomarker for breast cancer detection is selected from at least one of genome fragments of 26 plasma cfDNA methylation modification patterns. The invention also relates to a kit for detecting the methylated biomarker or the combination thereof. The invention jointly analyzes the co-methylation characteristics of a plurality of methylated cytosines on at least 1 genome segment as a biomarker for judging the breast cancer onset, has the accuracy far higher than that of other plasma biomarkers, and can reduce the occurrence of false positive and false negative in the breast cancer detection.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a methylation biomarker for detecting breast cancer, and a combination and an application thereof.
Background
According to the display of the Chinese tumor registration annual report: the incidence rate of breast cancer of women is lower at the age of 0-24 years, gradually rises after 25 years, reaches the peak at the age of 50-54 years, and gradually falls after 55 years. As for susceptibility genes of breast cancer, Europe and America have made a lot of researches, now BRCA-1, BRCA-2, p53, PTEN and the like are known, and the breast cancer related to the gene mutation is called hereditary breast cancer and accounts for 5% -10% of all breast cancers. The breast cancer 21 gene test not only provides recurrence risk prediction after 1-5 years and 5 years, but also can be used as a unique polygene test to predict the benefit degree of chemotherapy and endocrine treatment of estrogen receptor positive invasive breast cancer patients. The breast cancer 70 gene detection plays an important role in early breast cancer diagnosis. Recent research results found that 72 new common genetic variations would lead to an increased risk of women suffering from breast cancer, which increased the number of genetic mutations currently known to be associated with breast cancer to 177. Many people may carry common variant genes, with relatively little risk of carcinogenesis due to mutation of a single gene, but the more variant genes that are relevant in women, the greater the risk of developing breast cancer. Currently, the detection means of breast cancer genes belongs to non-invasive detection, but needs to be based on the detection of tissues taken out during the original operation (breast tumor resection operation, mastectomy operation or core puncture biopsy).
Early screening and early diagnosis of tumors can discover early cancers more probably, thereby improving the five-year survival rate and reducing the death rate. The current situation of Chinese breast cancer screening: breast cancer 5-year survival: 100% of carcinoma in situ, 84-100% of stage I, 76-87% of stage II and 38-77% of stage III. Chinese multicenter research shows that 15.7% of stage I, 44.9% of stage II, 18.7% of stage III and 2.4% of stage IV are used for the initial diagnosis of breast cancer. Breast cancer image screening means: 1. molybdenum target X-ray: chinese women have less age (45-55 years old) of breast diseases, dense breast glands compared with western women, easy missing of MG, and sensitivity of MG to diagnosis of dense breast lesions of only 30%. B ultrasonic: the positive predictive value of simple MG is 55.5%, and the positive predictive value of MG combined with B-ultrasound is 43.3%. 3. Magnetic Resonance Imaging (MRI), MRI is insensitive to microcalcification foci and has clear contraindications; therefore, early screening and early treatment of tumors have great significance for improving the five-year survival rate, and the key of early screening and early treatment of tumors is to establish an effective detection model and search for molecular markers capable of being used for screening and diagnosis. cfDNA (cell-free DNA) is a small fragment of DNA free from peripheral blood, derived from normal or tumor cell metabolism and apoptosis, and contains genetic information such as somatic mutation and DNA methylation. A technology for grasping the occurrence and development of diseases by detecting disease-specific cfDNA fragments is called Liquid Biopsy (Liquid Biopsy), and compared with the traditional tissue Biopsy, the technology has the advantages of rapidness, convenience, small injury and the like. In 2014, a 640-tumor-type study from the group of Bert Vogelstein and Kenneth Kinzler found that more than 75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, melanoma, hepatocellular, and head and neck cancers detected the presence of ctDNA. ctDNA is thus a sensitive and specific biomarker with broad applicability and can be used in the clinic and research of a wide variety of different types of cancers. Lucinstead of biopsy, the technical and theoretical feasibility of liquid biopsy was demonstrated by cfDNA whole genome methylation sequencing in 2015 by professor ruyu; zhang 40525in 2017, professor team described the ctDNA profile of tumor burden and tumor origin using methylation quantification of ctDNA. The Turner team searches for breast cancer specific somatic mutation sites by using high-throughput sequencing, monitors the dynamic change of the breast cancer specific somatic mutation sites, and proves that the ctDNA detection can find the recurrence and metastasis of the tumor earlier than the CT detection. Recent studies have found that combining ctDNA mutations in blood with other analytes can provide an earlier and better diagnosis of 8 common and surgically resectable cancers, ovarian cancer, liver cancer, gastric cancer, breast cancer, prostate cancer, esophageal cancer and colorectal cancer. More and more clinical researches show that plasma ctDNA can be used as a biomarker to be applied to early diagnosis, screening, prediction and treatment response of tumors, monitoring of tumor size and recurrence and the like. At present, the international research direction is to integrate multiomics/multiple molecular markers and multigenes/multiple sites to improve the sensitivity and specificity of the detection technology so as to meet the clinical requirements on detection products.
Disclosure of Invention
It is an object of the present invention to provide a methylated biomarker or a combination thereof that can be used for breast cancer detection.
The technical scheme for achieving the aim comprises the following steps.
A methylated biomarker for breast cancer detection, or a combination thereof, selected from any at least one of the following methylated biomarkers:
cg23035715, cg26371731, cg04541368, cg13973436, cg16304215, cg20072171, cg08402365, cg21501525, cg22778178, cg08599259, cg25566568, cg15634980, cg07458308, cg01348584, cg14140881, cg25756435, cg00594560, cg08279008, cg09760908, cg18087672, cg14868703, cg17632299, cg18786873, cg20631750, cg 25926, cg 24556128, cg 22978855, cg23524195, cg 208607, cg 3522435300, cg 254443, cg 143528, cg 8245528, cg 254543, cg 25067946, cg 227946, cg 797746, cg 7746, cg 2357946, cg 2087946, cg 20846, cg 7246, cg 640426, cg 7746, cg 640426, cg 6783, cg 677746, cg 640426, cg 6783, cg 677779, cg 6783, cg 640426, cg 6427, cg 640426, cg 6427, cg 7279, cg 64048, cg 640435, cg 727946, cg 727947, cg 640435, cg 64047747, cg 64047775, cg 640435, cg 640447, cg 727947, cg 640435, cg 727947, cg 72798, cg 727968, cg 722048.
In some embodiments, at least one of any of the following methylated biomarkers is selected from:
cg23035715, cg26371731, cg04541368 and cg 13973436.
A methylated biomarker combination for breast cancer detection comprising the marker cg23035715 and at least one of cg26371731, cg04541368 and cg13973436 selected from the following markers.
In some of these embodiments, the methylation biomarker combination for breast cancer detection comprises marker cg23035715, and three of the following markers cg26371731, cg04541368 and cg 13973436.
In some of these embodiments, the methylation biomarker combination for breast cancer detection further comprises at least one biomarker selected from the group consisting of: cg16304215, cg20072171, cg08402365, cg21501525, cg22778178, cg08599259, cg25566568, cg15634980, cg07458308, cg01348584, cg14140881, cg25756435, cg00594560, cg08279008, cg09760908, cg 180672, cg14868703, cg17632299, cg 86873, cg20631750, cg25924096, cg 245528, cg22889755, cg 24195, cg 208607, cg 224300, cg25504443, cg14351528, cg25824543, cg23413809, cg 247187, cg 26717715, cg 00487771274, cg 610661356135504443, cg 1435150528, cg25824543, cg 138234234, cg 64047746, cg 6777047727, cg 6777927746, cg 674178, cg 227768, cg 67417946, cg 927768, cg 67357946, cg 67357947, cg 9235793, cg 67357947, cg 6773, cg 22779241797, cg 22779, cg 2277314135, cg 924135, cg 640435, cg 25041357947, cg 70479, cg 640435, cg 640447, cg 640435, cg 64047775, cg 640435, cg 64047775, cg 640447, cg 64047775, cg 640435, cg 640447, cg 64047775, cg 640435, cg.
In some embodiments, the methylation biomarker combination for breast cancer detection comprises markers cg23035715, cg26371731, cg04541368, cg13973436, cg16304215, cg20072171, cg08402365, cg21501525, cg22778178, cg08599259, cg25566568, cg15634980, cg07458308, cg 01853484, cg 40881, cg25756435, cg00594560, cg08279008, cg09760908, cg 180878787703, cg14868703, cg 32286899, cg 18718718773, cg20631750, cg25924096, cg 24619728, cg 229755, cg 24195, cg 612086135350043506300, cg 1242242247943, cg 257746, cg 67357943, cg 257746, cg 67357946, cg 6746, cg 7946, cg 640426, cg 6427, cg 679235798, cg 72357947, cg 6427, cg 7235798, cg 72357946, cg 6427, cg 64047746, cg 6427, cg 64047746, cg 64045040922022, cg 6427, cg 64048, cg 64047740798, cg 64047747, cg 6427, cg 64047747, cg 6427, cg 64047747, cg 6427, cg 64047747, cg 6427, cg 640435, cg 64047747.
In some embodiments, the marker cg23035715 is represented by SEQ ID No.1, cg26371731 is represented by SEQ ID No.2, cg04541368 is represented by SEQ ID No.3, cg13973436 is represented by SEQ ID No.4, cg16304215 is represented by SEQ ID No.5, cg20072171 is represented by SEQ ID No.6, cg08402365 is represented by SEQ ID No.7, cg21501525 is represented by SEQ ID No.8, cg22778178 is represented by SEQ ID No.9, cg08599259 is represented by SEQ ID No.10, cg25566568 is represented by SEQ ID No.11, cg15634980 is represented by SEQ ID No.12, cg 458308 is represented by SEQ ID No.13, cg 01344084 is represented by SEQ ID No. 141799, cg 09799 is represented by SEQ ID No.9, cg 25649 is represented by SEQ ID No.16, cg 257619 is represented by SEQ ID No.16, cg 079 is represented by SEQ ID No.16, cg 94079 is represented by SEQ ID No.4, The sequence of cg18087672 is shown as SEQ ID NO.20, the sequence of cg14868703 is shown as SEQ ID NO.21, the sequence of cg17632299 is shown as SEQ ID NO.22, the sequence of cg18786873 is shown as SEQ ID NO.23, the sequence of cg20631750 is shown as SEQ ID NO.24, the sequence of cg25924096 is shown as SEQ ID NO.25, the sequence of cg15321298 is shown as SEQ ID NO.26, the sequence of cg22889755 is shown as SEQ ID NO.27, the sequence of cg23524195 is shown as SEQ ID NO.28, the sequence of cg 61607 is shown as SEQ ID NO.29, the sequence of cg 35224300 is shown as SEQ ID NO.30, the sequence of cg25504443 is shown as SEQ ID NO.31, the sequence of cg 51528 is shown as SEQ ID NO.32, the sequence of cg 254543 is shown as SEQ ID NO.33, the sequence of cg 6150234504443 is shown as SEQ ID NO. 13835, the sequence of cg 678735, the sequence of SEQ ID NO. 1238735, the sequence of SEQ ID NO. 2667223, the sequence of cg 6735 is shown as SEQ ID NO. 267735, the sequence of SEQ ID NO.46, the sequence of SEQ ID NO.35, the sequence of SEQ ID NO. 2671223, The sequence of cg27111970 is shown as SEQ ID NO.40, the sequence of cg22851944 is shown as SEQ ID NO.41, the sequence of cg23228540 is shown as SEQ ID NO.42, the sequence of cg26225694 is shown as SEQ ID NO.43, the sequence of cg07790615 is shown as SEQ ID NO.44, the sequence of cg14825633 is shown as SEQ ID NO.45, the sequence of cg11901043 is shown as SEQ ID NO.46, the sequence of cg 2404927 is shown as SEQ ID NO.47, the sequence of cg21962423 is shown as SEQ ID NO.48, the sequence of cg 35814 is shown as SEQ ID NO.49, the sequence of cg01070209 is shown as SEQ ID NO.50, the sequence of cg 1484141828 is shown as SEQ ID NO.51, the sequence of cg04947764 is shown as SEQ ID NO.52, the sequence of cg 0191141 is shown as SEQ ID NO.53, the sequence of cg 62004397756, the sequence of SEQ ID NO. 6256, the sequence of cg 2716757, the sequence of SEQ ID NO. 2508357, the sequence of SEQ ID NO. 2508359, the sequence of SEQ ID NO. 25057, the sequence of SEQ ID NO. 2508359, the sequence of SEQ ID NO.4 is shown as SEQ ID NO.48, The sequence of cg03355998 is shown as SEQ ID NO.60, the sequence of cg19019849 is shown as SEQ ID NO.61, the sequence of cg22009488 is shown as SEQ ID NO.62, the sequence of cg15012484 is shown as SEQ ID NO.63, the sequence of cg20817483 is shown as SEQ ID NO.64, the sequence of cg21254450 is shown as SEQ ID NO.65, and/or the sequence of cg23134869 is shown as SEQ ID NO. 66.
The invention also relates to application of the methylation biomarker combination in preparing a kit for detecting breast cancer.
Another objective of the invention is to provide a kit for detecting breast cancer.
A kit for detecting breast cancer comprises a reagent for detecting the methylation state of the methylation biomarker or the combination thereof in a sample to be detected.
In some of these embodiments, the breast cancer is a breast cancer of different stages, including stages 0, I, II, III, IV, depending on TNM.
In some embodiments, the kit employs reagents for detection using pyrosequencing, bisulfite conversion sequencing, methylation chip method, qPCR method, digital PCR method, second generation sequencing, third generation sequencing, whole genome methylation sequencing, DNA enrichment detection, simplified bisulfite sequencing techniques, HPLC method, MassArray, methylation specific PCR, or combinations thereof.
In some of these embodiments, the test sample is plasma, serum, or blood.
The invention discloses a plasma gene methylation marker or a combination thereof for early diagnosis of breast cancer, and particularly researches on obtaining a genome fragment with obvious abnormal methylation modification patterns in plasma cfDNA of the breast cancer, namely cg23035715, cg26371731, cg04541368, cg13973436, cg16304215, cg20072171, cg08402365, cg21501525, cg22778178, cg08599259, cg25566568, cg 1563434881, cg07458308, cg 348584, cg14140881, cg25756435, cg00594560, cg08279008, cg 0976090768, cg18087672, cg 68703, cg 32217632299, cg 18773, cg20631750, cg 251486, cg 615528, cg 6106799755, cg 611808787927746, cg 67357946, cg547746, cg 747746, cg 3227746, 747746, 747946, 74798, cg 1878688, cg 1874546, cg 187747746, 747746 g, 74798, cg 18773, cg 6773, cg547746 g, cg4095, cgc, cg, 747746 g, 747779 cge, 747779 cg547779 cg, 747779 cg54778, 747779 cge, cgc 048, cgc, cge, cga, cgc 047779 cgc, cgs 35747779 cgs, 747779 cgc 048, 747779 cgs, cgc, cge, cgc 047779 cg, cgc 0435, cgc 048, cgc, cgs 35747779 cgc, cgs 35747779 cga. By studying the methylation modification differences of the genome fragments of the plasma cfDNA methylation modification patterns in breast cancer patients and healthy people at different stages, the combination of the plasma genome fragments can be used as an early diagnosis marker of the breast cancer. Furthermore, the inventors have found through their creative studies that the first methylation marker (cg23035715) or the first 3 combinations of methylation markers (cg26371731, cg04541368, cg13973436) have diagnostic power similar to the 26 combinations of methylation markers selected by the two algorithms LASSO and Random Forest. The accuracy of the combined analysis of the co-methylation characteristics of a plurality of methylated cytosines on at least 1 genome segment as a biomarker for judging the breast cancer onset is far higher than that of the detection of other plasma biomarkers, and the occurrence of false positive and false negative is reduced.
Drawings
FIG. 1 is a ROC curve for the 26 methylation marker combinations in the training set of example 2.
FIG. 2 is a graph showing the ROC curve for the 26 methylation marker combinations in example 2 independently validated for sensitivity and specificity for different stages of breast cancer.
FIG. 3 is a ROC curve for the combination of 42 methylation markers from the LASSO screen and 50 methylation markers from the Random Forest screen in the independent validation set of example 2.
FIG. 4 is a graph of the sensitivity, specificity of the 42 (a) and 50 (b) methylation marker combinations of the Random Forest screening of the centralized LASSO screening in example 2 independently validated combinations at different breast cancer stages.
FIG. 5 is a ROC curve (a) of the independent validation set of the marker combinations of SEQ ID Nos. 1 to 26 and X of the randomly selected additional markers of SEQ ID Nos. 27 to 66 in example 2 and the sensitivity and specificity of the different breast cancer stages (b).
FIG. 6 is a set of independent validation sets of the combined ROC curves for the methylation markers of SEQ ID NO.1 or SEQ ID NO.1-4 in example 3.
FIG. 7 is a graph of the sensitivity and specificity of the different breast cancer stages in example 3 independently validated randomized combinations of the methylation markers of SEQ ID NO. 1-4.
FIG. 8 is a ROC curve (a) of the markers of SEQ ID No.1 in the set with randomly selected X of the markers of SEQ ID Nos. 5-26 and the sensitivity, specificity (b) of different breast cancer stages independently verified in example 3.
FIG. 9 is a graph of the ROC curve (a) of the pooled SEQ ID NO.1 markers versus X of the randomly selected SEQ ID NO.27-66 markers and the sensitivity, specificity (b) of different breast cancer stages independently verified in example 3.
FIG. 10 is a ROC curve (a) of the markers of SEQ ID Nos. 1-4 in the set with randomly selected X of the markers of SEQ ID Nos. 5-26 and sensitivity, specificity (b) for different breast cancer stages independently verified in example 3.
FIG. 11 is a ROC curve (a) of the markers of SEQ ID Nos. 1-4 in the set with X of the randomly selected markers of SEQ ID Nos. 27-66 and the sensitivity, specificity (b) of different breast cancer stages independently verified in example 3.
Detailed Description
The experimental procedures of the present invention, without specifying the specific conditions in the following examples, are generally carried out according to conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.
The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
In some embodiments, the invention relates to 66 methylation biomarkers cg23035715, cg26371731, cg04541368, cg13973436, cg16304215, cg20072171, cg08402365, cg21501525, cg22778178, cg08599259, cg25566568, cg 34980, cg07458308, cg01348584, cg14140881, cg25756435, cg00594560, cg08279008, cg 09768, cg18087672, cg14868703, cg17632299, cg18786873, cg20631750, cg 254096, cg 245528, cg22889755, cg 24195, cg 61607, cg 61300, cg 352242586843, cg 25357943, cg 6792357943, cg 67927746, cg 640447, cg 6446, cg 64048, cg 64047740922027746, cg 6446, cg 64047746, cg 6446, cg 64047746, cg 6427, cg 64048, cg 64047747, cg 640447, cg 64048, cg 640447, cg 6427, cg 64048, cg 640435, cg 640447, cg 64047747, cg 640447, cg 64048, cg 640447, cg 640435, cg 64047747, cg 64048, cg 640435, cg 64048, cg 6404.
In some embodiments, at least one of any of the following methylated biomarkers is selected from: cg23035715, cg26371731, cg04541368 and cg 13973436.
The invention relates to a screening method of ctDNA differential methylation markers (markers) for predicting breast cancer, which specifically comprises the following steps:
1. screening out methylation markers specific to breast cancer according to TGCA data
1.1 downloading data of methylation450 k chip (Illumina HumanMethylation450Beadchip) of breast cancer tissues and tissues beside the breast cancer tissues from TCGA;
1.2 preprocessing the data: filtering the deletion value, completing the deletion value, removing batch effect, removing unstable CpG sites, and selecting 3288 methylation sites with breast cancer specificity as a final marker set;
1.3 grouping of samples: the samples were divided into training and test sets: the patients are grouped randomly, and the clinical stages, the ages, the follow-up visit time and the like of the patients in the two groups of samples are ensured to be distributed consistently.
1.4 validation 3288 markers in paired tissue and plasma samples in consistent methylation, and further screening, filtration confirmed the final 1996 methylation markers.
1.51996 significant methylation sites were used for the construction of the classifier model later.
The consistency of methylation levels was further verified by 40 pairs of paired tissues and plasma using 3288 formazan biomarkers from TCGA screening, and further screening for 1996 genetic methylation biomarkers associated with breast cancer diagnosis. The TGCA source screen 3288 and 1996 methylation markers after filtration were clustered in normal and breast cancer samples at the site data, and the 1996 methylation markers after screening were at the most significant sites of aberrant methylation in normal plasma and breast cancer samples. The 1996 methylated biomarker, hereinafter referred to as site or marker. The 1996 methylated biomarkers were further screened using two algorithms, LASSO and RandomForest, wherein the two algorithms overlapped by the 26 methylated markers described in Table 1.
Genomic sequence of 66 regions of markers methylation
TABLE 126 methylation markers
TABLE 2 additional 40 methylation markers associated with breast cancer
Example 1
1. Plasma cfDNA or tissue extraction and methylation database construction
1.1 extraction of blood cfDNA or tissue DNA.
The specific procedures for plasma DNA extraction were carried out according to the protocol of the MagMAX TM Cell-Free DNA Isolation Kit from Life. The Tissue DNA extraction step was carried out according to the DNeasy Blood & Tissue Kit protocol of QIAGEN;
1.2 transformation
The extracted cfDNA (10ng) or tissue DNA (50ng) was subjected to bisulfite conversion to deaminate unmethylated cytosine in DNA to uracil while methylated cytosine remained unchanged to obtain bisulfite converted DNA, and the specific conversion was performed according to the EZ DNA Methylation-Lighting Kit instruction of Zymo Research.
1.3 end repair
1.3.1 the 17ul sample after conversion was reacted by adding the following reagents:
1.3.2 the reaction was carried out in a PCR apparatus according to the following procedure:
37℃ 30min
95℃ 5min
105 deg.C thermal cover
1.3.3 when the second step of the PCR reaction (95 ℃) reaches 5min, the sample is immediately taken out of the PCR instrument, directly inserted into ice, placed for more than 2min and then subjected to the next operation
1.4 connection I
1.4.1 the following reaction solutions were prepared
1.4.2 the reaction was carried out in a PCR apparatus according to the following procedure:
37℃ 30min
95℃ 5min
10℃ hold
105 deg.C thermal cover
1.5 amplification of I
1.5.1 the following reaction solutions were prepared
1.5.2 the reaction was carried out in a PCR apparatus according to the following procedure:
1.6 purification of I: 166ul of Agencour AMPure Beads (which need to be balanced at room temperature for half an hour in advance) diluted by 6 times at a ratio of 1: 166ul are added to purify a product after the Amplification I reaction, 21ul of EB is used for elution, and the purification steps are as follows:
1.6.1 taking the reaction product in the previous step, centrifuging, adding 166ul of Agencour AMPure Beads diluted by 1:6 times into each sample, and blowing and mixing by using a pipette.
1.6.2 incubate for 5min at room temperature.
1.6.3, centrifuging, and standing for 5min on a magnetic frame.
1.6.4 the supernatant was aspirated.
1.6.5 Add 200ul 80% EtOH, let stand for 30s, and suck off the ethanol.
1.6.6 repeat step 5) once.
1.6.7 centrifugation, the PCR tube was placed on a magnetic rack and the remaining ethanol was aspirated.
1.6.8 the beads were dried for 2-3min with the lid open, taking care not to overdry.
1.6.9 adding 21ul EB for elution, fully and uniformly blowing by a pipette, and standing for 3min at room temperature.
1.6.10 centrifuging, placing the PCR tube on a magnetic frame, and standing for 3 min.
1.6.11 aspirate 20ul of supernatant into a new PCR tube.
1.7 connection II
1.7.1 the following reaction solutions were prepared:
| components | Volume (ul) |
| Reaction volume of the |
20 |
| |
4 |
| |
8 |
| |
2 |
| |
2 |
| |
2 |
| |
2 |
| |
40 |
1.7.2 placing in a PCR apparatus to perform the reaction according to the following procedure
| Temperature of | Time of day | Number of cycles | |
| 37 | 30min | 1 | |
| 95 | 5min | 1 | |
| 10 | Hold | 1 |
1.8Indexing PCR:
1.8.1 the following reaction solutions were prepared:
1.8.2 placing in a PCR apparatus to perform the reaction according to the following procedure
1.9 purification of II
Adding Agencour AMPure Beads (needing to be balanced at room temperature for half an hour in advance) to purify a product after the exponential PCR reaction, and eluting the product by using 41ul EB, wherein the specific purification steps are as follows:
1.9.1 the reaction product from the previous step was centrifuged, and 71ul of undiluted Agencourt AMPure Beads were added to each sample and pipetted and mixed well.
1.9.2 was incubated at room temperature for 5 min.
1.9.3 centrifuging, and standing on magnetic frame for 5 min.
1.9.4 the supernatant was aspirated.
1.9.5 Add 200ul 80% EtOH, let stand for 30s, and suck off the ethanol.
1.9.6 repeat step 5) once.
1.9.7 centrifugation, the PCR tube was placed on a magnetic rack and the remaining ethanol was aspirated.
1.9.8 the beads were dried for 2-3min with the lid open, taking care not to overdry.
1.9.9 adding 41ul EB for elution, fully and uniformly blowing by a pipette, and standing for 3min at room temperature.
1.9.10 centrifuging, placing the PCR tube on a magnetic frame, and standing for 3 min.
1.9.11 aspirate 20ul of supernatant into a new PCR tube.
1.10 quant quantification:
1.1 ul of the library was quantitated using the Qubit dsDNA HS Assay Kit.
2. And (3) carrying out oligonucleotide probe capture enrichment on the sample after the library is built to obtain the final on-computer library in a specific region. The hybridization capture kit is xGen Lockdown Reagents of IDT company, and is specifically operated according to the instruction.
3. Sequencing the sample after hybridization capture by adopting a sequencer of Illumina company to obtain a sequencing result.
4. Analysis of the data:
performing conventional bioinformatics analysis processing on off-line original data of a sequencer, filtering low-quality (low QC, short length, too much N and the like) read lengths (reads) through fastp, then removing adapters, common sequences and PolyA/T at two ends of the reads to obtain an ideal insert sequence (target interval), comparing the reads with corresponding positions of hg19 by using a bismark, removing the reads according to UMI to obtain real reads data (bam file) obtained by capturing each sample by a probe, and counting and analyzing the bam file to obtain methylated data for subsequent data reanalysis.
As used herein, the co-methylation ratio of a methylated marker is the sum of the individual methylation ratios of all methylation combinations of a particular region of the marker. The P-vlaue is obtained as wilcoxn test, and the P <0.001 has significant statistical significance; we also calculated the individual AUC (area under the curve) for all 26markers, which is the area under the roc (receptor Operating characterization) curve, to judge the ability of this combination to diagnose breast cancer, with AUC closer to 1 indicating better.
Example 2
The embodiment discloses a methylation specific biomarker combination for diagnosing breast cancer, which utilizes 74 molybdenum targets, plasma samples of normal people without color ultrasound abnormality and plasma samples of 155 breast cancer patients, screens 66 methylation biomarkers related to breast cancer by utilizing methylation level difference of different groups, and verifies independent data sets of a differential methylation combination model in normal and breast cancer plasma samples, and comprises the following specific processes: marker screening and model building were performed on training and test sets using LASSO and Random Forest methods (Random Forest) respectively, including plasma cfDNA samples of 52 healthy persons and 108 breast cancer patients. 42 methylation markers are screened out by using an LASSO method, 50 methylation markers are screened out by using a random forest method, and finally, a total of 26 methylation markers are further screened out. Finally, we validated the diagnostic ability of 42(SEQ ID NO.1-42), 50(SEQ ID NO.1-26, SEQ ID NO.27-SEQ ID NO.66) and 26(SEQ ID NO.1-26) methylation markers, respectively, in an independent validation set of the plasma cfDNA composition of 22 healthy people and 47 breast cancer patients.
The 66 methylated biomarkers (sites or markers hereinafter) are: cg23035715, cg26371731, cg04541368, cg13973436, cg16304215, cg20072171, cg08402365, cg21501525, cg22778178, cg08599259, cg25566568, cg15634980, cg07458308, cg01348584, cg14140881, cg25756435, cg00594560, cg08279008, cg09760908, cg18087672, cg14868703, cg17632299, cg18786873, cg20631750, cg 25926, cg 24556128, cg 22978855, cg23524195, cg 208607, cg 3522435300, cg 254443, cg 143528, cg 8245528, cg 254543, cg 25067946, cg 227946, cg 797746, cg 7746, cg 2357946, cg 2087946, cg 20846, cg 7246, cg 640426, cg 7746, cg 640426, cg 6783, cg 677746, cg 640426, cg 6783, cg 677779, cg 6783, cg 640426, cg 6427, cg 640426, cg 6427, cg 7279, cg 64048, cg 640435, cg 727946, cg 727947, cg 640435, cg 64047747, cg 64047775, cg 640435, cg 640447, cg 727947, cg 640435, cg 727947, cg 72798, cg 727968, cg 722048. The specific sequence is shown in SEQ ID NO. 1-66.
In a training set of 52 healthy persons and 108 breast cancer patients, 26 methylation biomarkers (markers) are detected by intersection of the two algorithms, and under the specificity of 98.07% (51/52), the detection sensitivity of 0 stage is 60% (3/5), the detection sensitivity of I stage is 80% (32/40), the detection sensitivity of II stage is 95.45% (42/44), the detection sensitivity of III stage is 94.74% (18/19), the overall detection sensitivity is 87.9% (95/108), and the AUC is 0.9839, and the result is shown in detail in FIG. 1.
Further, in 22 tissue samples of normal persons without abnormality of molybdenum target and color Doppler and 47 plasma samples of breast cancer patients, 26 methylated biomarkers (markers) were independently verified and analyzed, and at a specificity of 98% (22/22), the detection sensitivity at stage 0 was 66.67% (2/3), the detection sensitivity at stage I was 75% (9/12), the detection sensitivity at stage II was 95% (21/22), the detection sensitivity at stage III was 100% (8/8) and the detection sensitivity at stage IV was 100% (2/2), and for all breast cancer samples, the overall detection sensitivity was 89.37% (42/47), and the overall AUC was 0.9816, as shown in FIG. 2. In addition, we also performed independent validation analysis on 42(SEQ ID NO.1-26 and SEQ ID NO.51-66) and 50(SEQ ID NO.1-SEQ ID NO.50) methylated biomarkers (marker) respectively screened by LSSAO and RF, and at 98% specificity (22/22), the detection sensitivities at stage 0 were 66.67% (2/3) and 33.33% (1/3), respectively, the detection sensitivities at stage I were 66.67% (8/12) and 58.33% (7/12), respectively, the detection sensitivities at stage II were 95.45% (21/22) and 63.63% (14/22), respectively, the detection sensitivities at stage III were 100% (8/8) and 75% (6/8), and the detection sensitivities at stage IV were 100% (2/2) and 0% (0/2), respectively, for all breast cancer samples, the overall sensitivity of the assay was 87.23% (43/47) and 59.57% (28/47), respectively, and the overall AUC was 0.9709 and 0.9497, respectively, as shown in detail in fig. 3 and 4.
To further validate the importance of the combination of 26 methylated biomarkers (markers), we performed independent validation analysis of 26markers (SEQ ID No.1-26) in combination with cg22889755, cg23524195, cg20861607, cg22889755, cg23524195 and cg20861607 were 3 randomly selected from the other 40 methylated markers ((SEQ ID No.27-66)), at 98% specificity (22/22), detection sensitivity at stage 0 was 100% (3/3), detection sensitivity at stage I was 83.33% (10/12), detection sensitivity at stage II was 86.36% (19/22), detection sensitivity at stage III was 87.5% (7/8) and detection sensitivity at stage IV was 0% (0/2), overall sensitivity of detection was 82.98% (39/47) for all breast cancer samples, overall AUC was 0.9797, see fig. 5a-5 b. The overall sensitivity is reduced, but the sensitivity for early breast cancer is higher than that of the combination of top1-26 methylation markers.
Example 3
Using the information of 26 methylation markers (SEQ ID NO.1-26), carrying out random combinatorial testing on 22 normal persons without abnormality of molybdenum target and color ultrasound and 47 plasma samples of breast cancer patients (3 cases in 0 phase, 12 cases in I phase, 22 cases in II phase, 8 cases in III phase and 2 cases in IV phase), carrying out modeling analysis by using a logistic regression model, further screening 4 markers (cg23035715, cg26371731, cg04541368 and cg13973436), wherein the specific data analysis method is consistent with that of example 2, Top1(cg23035715) and Top1/2/3/4(cg23035715, cg26371, cg04541368 and cg13973436) are at 98% specificity (22/22), the detection sensitivity of the breast cancer in 0 phase of the verification set is 100% (3/3) and 100% (39 3/3), and the detection sensitivity of the breast cancer in I phase is 91.91.91.13967% (11/12) and 11/12) (11/12), the detection sensitivity of the breast cancer at the II stage is respectively 90.90% (20/22) and 90.91% (20/22), the detection sensitivity of the breast cancer at the III stage is respectively 87.5% (7/8) and 87.5% (7/8), the detection sensitivity of the breast cancer at the IV stage is respectively 50% (1/2) and 0% (0/2), the overall sensitivity of the detection is respectively 91.49% (43/47) 87.23% (41/47) and the overall AUC is respectively 0.9395 and 0.9796 for all the breast cancer samples, and the combination of the loci is highly related to the diagnosis of the early breast cancer. See fig. 6 and fig. 7a-7b for results.
The 4 biomarkers: cg23035715, cg26371731, cg04541368 and cg13973436, wherein AUC 0.9395 of the single marker cg23035715, as shown in fig. 6, and fig. 6 is a comparison of the combination of 4 markers (cg23035715, cg26371731, cg04541368 and cg13973436) of the 26 co-methylated markers with the marker1(cg23035715) in identifying breast cancer, as shown in fig. 6, the AUC of the marker of 4 methylated markers has been very close to the AUC of the combination of 26markers, and the AUC of the marker of marker1 has been 0.9395, indicating the importance of these 4 methylated markers, particularly the marker of marker1, has been very strong in diagnosing early breast cancer.
7c-7d, the detection sensitivity of the Top1/2/3 and Top1/2/4 methylation marker combination in phase 0 of the validation set was 100% (3/3) and 33.33% (1/3), respectively, the detection sensitivity in phase I was 100% (12/12) and 66.67% (8/12), respectively, the detection sensitivity in phase II was 81.82% (18/22) and 63.63% (14/22), the detection sensitivity in phase III was 87.5% (7/8) and 75% (6/8), respectively, the detection sensitivity in phase IV was 50% (1/2) and 0% (0/2), respectively, and the overall sensitivity of the overall assay was 85.11% (40/47) and 61.7% (29/47), respectively, at 98%. In FIG. 6c, top1/2/3 represents cg23035715, cg26371731 and cg04541368 in combination, top1/2/4 represents 3 markers (cg23035715, cg26371731 and cg13973436) in combination, and so on.
Further verifying the importance of top1 alone, at 98% specificity, top1(SEQ ID NO.1) in combination with cg23035715, cg16304215 and cg08599259 in the random SEQ ID NO.5-26 markers and in combination with randomly preceded cg01167274, cg07790615, cg23134869 and cg01832036 of the other remaining 40 markers (SEQ ID NO.27-66), the detection sensitivities in phase 0 of the validation set were 100% (3/3) and 100% (3/3), respectively, the detection sensitivities in phase I were 100% (12/12) and 83.33% (10/12), the detection sensitivities in phase II were 95.45% (21/22) and 95.45% (21/22), respectively, the detection sensitivities in phase III were 100% (8/8) and 87.5% (7/8), the detection sensitivities in phase IV were 0% (0/2) and 0% (0/2), respectively, and the overall detection sensitivities were 93.6361.6361) (29/47) and 29/47), see fig. 8a-8b and fig. 9a-9 b.
Further verifying the importance of the top1/2/3/4 combination, at 98% specificity, top1-4(SEQ ID No.1-4) combined with cg16304215, cg20072171 and cg08402365 of randomly selected SEQ ID No.5-26 markers and with cg22889755, cg23524195, cg14135814, cg15012484 and cg2081748 of the remaining 40 markers (SEQ ID No.27-66), the validation set showed 100% (3/3) for detection sensitivity at phase 0, 91.67% (11/12) for detection sensitivity at phase I, 90.91% (20/22) for detection sensitivity at phase II, 87.5% (7/8) for detection sensitivity at phase III, 0% (0/2) for detection sensitivity at phase IV, 87.23% (41/47) for overall detection, and 0.9739 and 0.9797 for AUC, respectively, see fig. 10a-10b and fig. 11a-11 b.
As a result of the above-described examination of 22 molybdenum targets and blood plasma samples of normal persons without abnormality of color Doppler and 47 breast cancer patients, cg23035715 exhibited excellent diagnosis of early breast cancer, the combination with cg26371731, cg04541368 and cg13973436 exhibited diagnosis similar to that of top1-26, and cg23035715 exhibited excellent diagnosis in combination with cg23035715, cg16304215 and cg08599259 of the random SEQ ID NO.5-26 markers, and cg01167274, cg07790615, cg23134869 and cg01832036 of the remaining 40 markers (SEQ ID NO. 27-66). In addition, the combinations of top1-4(SEQ ID NO.1-4) and top1-26(SEQ ID NO.1-26) with cg22889755, cg23524195, cg14135814, cg15012484, cg2081748 and cg22889755, cg23524195, cg20861607 of the other remaining 40 markers (SEQ ID NO.27-66), respectively, also exhibited very stable diagnostic ability, and the AUC of the top1/2/3/4 marker was also above 0.75, e.g., the AUC of cg 23071, cg26371731, cg04541368 and cg13973436 was 0.9395, 0.8020, 0.7701 and 0.7501, see Table 3.1, demonstrating that the sites selected by this method have a very high correlation with the diagnosis of breast cancer at an early stage.
TABLE 3.126 expression of methylation marker fragments in breast cancer diagnosis-AUC.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Guangzhou City benchmark medical Limited liability company
<120> methylation biomarker for detecting breast cancer or combination and application thereof
<160> 66
<170> SIPOSequenceListing 1.0
<210> 1
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ctgtccttgc ctttcatggt gtcagcctct tcatgcctcc ccaccattcc aggcactccg 60
ggctttctct tcaaaccctc tttcgacacc tccaagtgct gagcctcctt gcttttctag 120
cccatctgcc agcctcctgc acgtctacac ctgacggctg ggctcctgtc gcagaaacca 180
tgcgccaggc acattggggc ccacataaat gtgcagtgtc cgatacgcca tgtggtcagc 240
acgacttctc cacgtctcct ctttctcttc ttcctgcaga gtgagcccac cggttcacca 300
cgttgctctt gcccctagcc caccaggccc tggccctcag cagcggacct gccgcggttt 360
ctggactagg aaggccctgc tcccgaccga cccggagcgc gctgctgccc tctaccggtc 420
atccgtgcgg ccggacaccg tgtcaggccc gcgaggaggg ctctgccgca gtcccgggga 480
acagcaccca gcagcgccac tgggagagga aactggggtc agggaggtgt gactgggccc 540
caggcagtca cactcaggcc aacaaggtca gggagggagg gcagctcact cgtgcaaacg 600
t 601
<210> 2
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgaaatggga ctgtgactcc tacctgtgcc gactggggtg ctgggggggt gaaatgtcgc 60
ccgaaccagt gaggtctctc tcctcctctt gtatcctctg gtgctggtca gtcctcgcgg 120
ctctagcccc tcgctcaagc ctctccctcc cgcccgtgag gccggctttc cccgggcccc 180
tctgcgcagt gtatggggtt atttttactt tcggttatct agctttatga agactccaca 240
ccactcatac agctagataa ccaaagataa caaccaaccc cgcctcctgg ctgctgtcgc 300
cgcctcttcc acgcagcctc ccggccgccg ccgccgccag cacctccgca gcttcccggt 360
cgcccgtcag cgggagtagg agggaaggga cacgagtgga gttgaggggg agggtgaaga 420
gagaaatgaa gtccgagaca aaacaacaac aaaaacctca gacacggaga tacagacacg 480
acagagaccg aaaaaggcgt ggaaaggacg cgatgacccg tggcgtcgaa gtcggggagt 540
tgaccccgat ccagacccaa aaagtttctg gtgccccatt tcccgctctc ccattcgggc 600
c 601
<210> 3
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctttccaccc tttctcactc cagtcactcc cgaggacttg gggcacgact atcacacgca 60
actgggagat cctggaagac ggaggaaaaa cgaacaaggg gacatggccc tcactgcagt 120
gacagggctt tccttcagtc agtggccaca ataaatttaa ccaaggctaa aggagattaa 180
tttcccagca taatccaatt aaaagatttc taaagtaatc ttttgcgaaa atgaaaagtg 240
cgcgactaaa gaggggactg gttttgatga cagtgattta tcgcctcagc acagcacgca 300
cgggacgctg tctctccaag cgatttgacc agagcatccc gtcctcgcct cctgtccaaa 360
ccttcctctt cctgaaagac agccatctat cacgccaacc tgggcaggag agaatgtgca 420
aggggctggg gcggcttaca agcaccacag acctttttaa gtgctattag atttatggtc 480
tctttttcga cacccatcca gagtaattag cacatatgtt ctaaatagat gatagttttg 540
tgagcaataa agcaattacc catcgttgga gctgacagtt cctccaacta aactccaacc 600
a 601
<210> 4
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cgcgccagcg tgggctgtcg gccccggccc cgcggggcgg ccagagcggc gctgcccagg 60
cgcgtctgcg ccactggaat ctcgtccacg gactctgccg acgccgagtg gtaggcgccg 120
gcagggccag cggcgtgcac agggctggcg tccgcggaat ccgtcgacga gctggagcgg 180
cggctggggg ccctaggccc tgcgcaaggg aagggaacat gagcccagtg gctgcggctc 240
gcttgtggct ctgccttccc ctccctcgag ttcccagagc aggacccgga cagggagagg 300
cgctcacccg acgccggatc cgaggtctca gacgttcgca ctttatacac gcgcctcttc 360
ctctttttct gcagagacac gggggagcct gagggcccca catcgccaaa agaagcagag 420
gcgagcgcca gggtagaggc ttctttctat cccactccac ctggaccgga ggcccagcac 480
agtccctcct acctggggtc ggatccgggg aggcagggcg agagtgtctg gggaagaggg 540
tggtggaggt tccgcttggc cacgggtggc aagaaactgg gagggactga gggggaagca 600
g 601
<210> 5
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
taccactttc ttcctgtggt ttctacccca ttttgcagat tgctgcgtat cccagtgtcg 60
gcgatggata aacaggtctc gtctcttccc agttgcagcc cgtgagctgg tggcacaacg 120
gattaatgag gcaacagggc tctagatggt gagtgggatc aatgagaccc aattgagctg 180
ttagcggtct taggcggaga gcatctctca ggaggaaagg aaggcacagc gagaagacct 240
aaggccgaaa gtcgctgtgc agcacccacc caaggctgga ctcgggggag gtagacggga 300
cggtgtctgt gactaaacgg ttcccacacc ttacacgccg caccgggatt atgttttgaa 360
aggtgtctta taaaatcaag accggttcct aacaacctgc aagtgccagt gaatcccgaa 420
atgtttgttt gaggagaggg agtgtgaggg aaggagcaga aaaaagaaag agggggagga 480
ttgcccagta gaatttcaat agaaaatgtg actaccagaa tggtttctga atctaggatc 540
tgctcaggca caggcggaaa agaacagctg ttaaagaact aaatattata gaaataatcg 600
g 601
<210> 6
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
caaaggcttt cgccaggcca gcacgctctg caggcacaaa attatccaca cccaggtacg 60
tggcccccgg gtcgggccta gcccgcgcgc aacccccaga atcttagtga taacaccggg 120
gatacgatgg ccaaagatta tcccttaccc tctagggttg agtgggggtg ggggggatgt 180
cccttccagg tgttcccgag gtggcctctc cttactgtgc actcgcccct ttcctcagga 240
aaagccacat aaatgcaacc agtgcggcaa agcgttcaac cgcagctcca cgctcaacac 300
gcatatccgc atccacgcgg gctacaagcc cttcgtctgc gaattttgcg gcaaaggctt 360
tcaccaaaaa ggtaacgtgc caggcgaggc cttctcttct cacctcacct caggactcgg 420
gtcgcggctg gctggcagga aggcaaagag ggatctggag agagaaggcg aaatctgcag 480
gcgcgggcgc agcatttctt tagaatcggg ttgtgcctgg tgtaggggga agctctccta 540
gcggggttag tagaccacgc tgtatgcagg ctgggtgcac tgagagactc cgaattcgag 600
a 601
<210> 7
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gttcggattt ttcggcagac gtgcaaagtt cagatacaaa gatcactgta actgtcagag 60
aggtaaggac ctctgataca atggatttaa gaggtttttt actccaacag tcagggcaag 120
ggaaagagaa aaccgtgctt ctcttggaat ctcttccaac aagggggttt cctccagtcc 180
cctccagatg cgttgaccgc gctgggaaac cgagtccagg gccaggattt gtttttccaa 240
agccgcgcga ccccagcagg gcttcagatc cgagttccca gttgctcttc agtctccccc 300
gcgttcttcc aaacgctatc aggagccacg caggttaccg caccaccggt catttccctg 360
tttcttctaa aagttctcca gccaaatttc agcaatttat ttaaaaaggc accaggtcac 420
cgcgaccttc gggcggctcc tcaggcctca tggggtagtt tggtcaggat ctgcgcgccc 480
ctcgacgtga tcagaaccgt gtgctcgaac tgcgccgacc tgtaacacag cacccagccg 540
tcagaacgcg cacgcaagct ccggccgggc cacaagcgcg ccgtcggacc aatcttcctt 600
t 601
<210> 8
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
caactctgaa gagtggggtg aaaagatttc tacacagtgt acattctaca gaattctagt 60
ttctccgcgg gcgcagactt ttccgccctc tagtctagcg tgaaaacagt tcgaccactg 120
gatgtgggcg gcgaagcccg caccctagag cgcggtgtct ccctgcagga gggagctcca 180
gcccgcgccg ccgcggccgc ttctggcctc ctcgggaacc gaaccgagca tacctcagtg 240
ctctcctaga agcggctcct tttccccttc ttactcgcga atggcgaaat gaatagccgc 300
gcgggatttt acccgtttgt gtagtggact ctgagggggc accaggtacc gagacggaaa 360
tctttgcggt aagaataaag tcaggtaaaa cgcctggtac agccgccctg cagatgtccc 420
aagagtaggt cccccgtaca ctcttcccat taagttctta gggggaggac agggaaggcc 480
ctttggtctg gctggattga tcagggaggt atgcgcatat gtttgccctc tggacatttg 540
gggaggggtt atgggggagt tgaaggcaag aggcaaaatc aggtagaaca aacaactaca 600
g 601
<210> 9
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cggagaatgg gcagtctgaa gagacctgcg tcccccctct ccgatttgac ttttcctgaa 60
atttggaatt attgcgcctc tttttctctt tctgatcgag catttagtgc tggcgcaagc 120
cgggcagccg actggttact gagcccacgg ccagcccggc cttgtgcttc catttgcgct 180
cacctcagca agcccctgag cgctttctca gggattggag ttccccctca tttcccaaat 240
aaccctggag aagctcccgg gggaggagga ggggagaggt cttggggtca gcaccacttc 300
gatcccagca ctgcagtggg gctccccacg cccgtctccc gctccaccat cggcgacgct 360
ctcccggagt cttctttgca ttacttgcaa atttcagcct ctgctcagga aaagtctatt 420
tgagattagc tgggatgttt tatgcacaca ctaaacatgt taaaaacaaa actaggaaaa 480
attcttggga cacggggtgg ggggacggag gaagagagag agagacagag agagagagag 540
agagaaaacg gttaacaaaa aatgaggcgg ttttgagggt gtcttggagg caggctgcgc 600
c 601
<210> 10
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
aagcgcatga cccctgcggc cggcgcgggc cacccgccca tggacgatgt atacgcgcct 60
ggggagctag ggcctggcgg gggcggcgca tcgccaccct ccgcgccccc accgcccccg 120
ccggcggcgc tgcaccacca ccaccaccac acactgcccg gctcagtgca gtgaccccgc 180
gggccgggcc cccgccggcg cgctgcaggg cgcgggcgcg cagcccgccc gcgcggcctg 240
gactcttttt gttcggttgc tttggatttt acaaaaaaaa aaaaaagccc agaaactttc 300
gaacaaaacc aaacacccgg acgaccctct ccggtgagcg gcgaagacag cccggtaggc 360
tgcgtcgccc gagccccggg agagaggagc gaagatccgg aactcgccaa ctcaccccgg 420
gcatcctgcc cgccgcctgc tctctgcccc catcccctcg acgacccccg cccctgcccc 480
cccgggctgt tcggggccgg atcccggcag cggcctcccc aaagcgacag gtaacgcagt 540
gcggggttag ggattccccg ggagagagga cgaagcgagg agagttctcc atcccctctc 600
c 601
<210> 11
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
cttgtcccag gcagtttccg gtggccgcag tatcgacccc aggccattgg ggtgggtgtg 60
agggaagggt ggagggggaa tctccgaggc ccaatgtttt ctgggacttg agggaggctg 120
aattctccct cgctgcctag gacttggggt ctgaaggttg cagaccgagg cgatccgcag 180
cgccctcttc cggcggccgt ctggagaact gcagctttag agcggtctcg gggaagcttt 240
gctactagtt tccggctagg gaaaactggt ggtggtcgga gactggtagt acatcatcca 300
cgcattcgct cattctaact gtccgcccga ccatacgtca ggctgtctat cggtccaccc 360
aaagccaata taagttatga ttgactcttt gaatcaccca gtctccttgc acctgctatt 420
tgaaagagcc caggtgtaga tcagggcttg gaagctgctc ttgtacctaa aaccctcttt 480
taattatgct ttgcttcgga attttgttat gcgaatgtcc tgtccaagtc agagggcatt 540
tggaggcata tttggtgacc aatggagtag cttgcacttt ctgcatgtcc tttctggagg 600
a 601
<210> 12
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tacccctgca gtgtagggca tagaaataaa atggtaaata aaccagggtc actgctcctc 60
ggaggctggc tgtcactgtg aggataaact gagataatag gtgggaaagc ctgtttaggg 120
aaacgattgt tgttaagatg ataacaatta ttattacgtt ggggtagggt attcaagata 180
tacccacagg aaaatgcaga ttacaattca gtctgcaatt agggaccaga tagcagccaa 240
ctctcaggtg atccatgcag ggattctgag cattcgtcgg cgcctcccca ggggctgctg 300
cgccccctgc ttcccgcgcg cccaccacgc acgctgctct gggagcaggg ccggcggcgc 360
cgccgcctcg cagcgattgg ttgaaccgga ggttgttgct aggctaccag tgcgccctga 420
gcctggggcc ccgcagtccc atcctctgtg gcagatccat ccctcactgc agacctaatt 480
ccggtaccct gtgaacggca tcctcagcag cttaaattat cagccccaac tgcccgcctt 540
tctatgattt ttcatttcgc aagaggccat gtggagttgg ggaagagaag ccttctgttt 600
t 601
<210> 13
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
cctaggctgc ctctctggca ggttaggaaa ttgactcatt aggatttcat ccaccttttc 60
actggctacc tctcccgctc cccacccccg ccctagactg cctcactaag tcaacactgc 120
aatctctctg gctcagctaa gtggtcccta gtgaggagca ggcctgagcg ccgggtaacc 180
gaggaactcg gaggcacctg gccgccggca tgctcacctg cagcgctcta ggatggggtg 240
cgcagtgggc agccaagcgc cccccaccca gacggaggag gggcgggagg ccctgttccc 300
gacctgtcaa tcactgcccg gcccgcagct gcgctctccg cagccccagc gcgctgttcc 360
tctgagggag ataaacatcg agaaatccaa tccagcgccg cttcagagat aaacagatgt 420
gcggccctct tcggtcagga gataacgccc ctgccccgcc ccacgcgccc gcccggcctg 480
cacctgcctg aggcgacagg gacgcgcctc tcgggaggca ggttgcgtcc cccagcctgg 540
agaaatggtc tccagagctg cgggtggggg tggggttgga gaagaaagaa gctcggtttg 600
t 601
<210> 14
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ggtgaaaaaa ggtttggggt tgttttgctt ttagccagac cacatacaat gaaggtttcc 60
cacggtgact ctgtcattgg gaatccactg gaagggaagt cccagttaag tggcagtgag 120
ttattcccat gtaaaaagag gaaaagcaat cctttcacct ccactgtgct aagataagag 180
ttattttcaa gtcagagtca tagcggaaga aaaatatttc aaatgaaaac aaccacaaag 240
aggtttgcgg acaagggagg acagaactca cggcaccccg tcggcagagg aaggcagcgg 300
cgcttcccgc gtgcacacaa cagtccctgc ggcccgcaca cccctggctg tcatcgggga 360
ggccgagtcc tgctacctgt cctgagtgac aaggcgggac gacaaatgca aactcagagc 420
tggcactcag cgacaacaca gacctgccac ccgcctgcca gctccatcgc agcactgggg 480
gcaggagggc agcccaccac gccgccaggt ggccagggct gcaggttcct ctgtcgctca 540
agccacatcc acactcctgg acggccacac catccccaca ccaccctccc acacacacac 600
a 601
<210> 15
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gaaagcgaat gtgtaagagg ggggtttcgt gtgggaagcg aaccccacat ggaggtatgg 60
ctgtaccggg gcatgggggc ccttacccgg ccgaaagaaa aggagggcgc tggtgaggta 120
gctaaccacc tccttgatct gatgcttttc caaatactcc gcggcttgga gctccctgct 180
gctggtctcc atcgctccgc gtcccgctgt tgctaggcga ctgcctggcg tctcagccgt 240
gcgactctgt ctccctctac ccggcgcggt cacgtgacac gcctgactgc cctgtggctg 300
cgacccggac ctggaatttc tagagctcac ggttcagggc aagcctgggg catgtagcag 360
ggcagagagg gcaggtgagg tgtggaccaa cgtcactgtc cgcgatccag gcttagggct 420
gggcccaggt gcctttttcc atctctctct agcacctagc tcggtacttg ttgatccaat 480
gattaacaag gcttgaagtg ctggcatcac aactcagcat gaactaataa cagaaggaac 540
ttgtgcaatt ttgagggtgc ccagtggaga gttcttttta ttattattat ttaagacctt 600
t 601
<210> 16
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cgtttctcca gaaaacatca gagaaacctc tctgcctccc ccagtttcac caaaggccca 60
tagaaaattc atttaaggac accccagaaa ccctaagaga cccttggaaa gaaacctgag 120
attcctcccc ttgagaaaca gccctagtcg tcctccccca cctcggggac caagagtccc 180
tcgataggca tcgcgcaatc tgcgcttgcg cacgtcatct tggctgcccc gtcctgttgc 240
tatggaaaca cctggccagg agagcaaggc tcggagagac taagaccccg cgcccagggc 300
gcacgcgctt tcgacctgcc gcaaagggag ccccaccgcc ttccccgttc cagatcggcg 360
attggacccc gccttcttat aggccccgcc ccgaaggcta ggcgctcctt cccgctgaga 420
ccttggggca ctcgctggtc ccctcaggcc ccagctcggg tgcaattagg aggagaacag 480
atggcggccc agacagtggt tggggagggt ctgcccaggg ttctggggcc ctggagggga 540
gggactcacc ttcgggagga ggcaaacgag aagtgggcgg gggtgttggg ggagaagttt 600
c 601
<210> 17
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ttatcgtttt agccctgttt aaaaaaatgg ctgattaagt tgagtgcgca tgtctttgtg 60
tgtctattcc cgatatgcac tctgggaatg agagaggaga gcgagggaga gggagaggga 120
gaggggggag agagaggtgt aattagtgag gtgatcaaca ttccccagac tgcaaatgac 180
gcgcagcccg gactcagagc agctccggag cctcacggct tccaaagctc ccagcgctgc 240
ggctggagcc ccggatgcgg cgaccgcgga gaggacaggg gatggggacc tccgggtctc 300
gcaccctacg cgcccctgcg cgcgactccc aaacttcatt agacacacac gcgctcttac 360
acacaaacac agacacacac agagcaagaa ggggaaagaa ataaaacata gataccgcca 420
aagcatggcc ctttaaccat tcgagatttt ttttttattt taatgaggca aagtaattat 480
tatcggacca gagatttgtt tactagggag ctttaagcag aaatatgagt tagggtaatg 540
aagagctttt ctctgcctag tttattctgc tgtgcacatg aatgcacttt aatggcgaaa 600
t 601
<210> 18
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ccctgattcc cccagttggg gatgccttct gccttccctg cctcttttgg tgaccaggac 60
ttgtggagaa cgtaggaggg aaggaggtga gaaggcagaa tgaatggctg gtgagtaagt 120
ggtaggcatg tgaccagtgt gccagttttc ctggaggtgc aatttgccct tctatggttt 180
tgccaagagc aggggaacta acggcccaca gtgactgcct tctcaaggtt gtgcagctca 240
cctgaatgta gcaacagaaa gggaacagga ggggcagggg cagagaagcc tcccgtccca 300
cgtaaataat tacaaacaga gcacatgacc cctggcggtt tctgaacgcg cctggcaaca 360
gctccaccac ctgctgtttg gaaagtcaga ttcacagaag ctacaattac agactgtcag 420
ctgggtcttt tcatggctgg ggaccggagg ccaaaactta cagcccctaa ctcctagctc 480
agtgctcttt ccacttcatc tcccctgcca cctgccagac aatttacaca aacagcccat 540
gtgatgggtg ccagtgcaga gatctggggg gccttctacg cttgtatatc tctacctagg 600
g 601
<210> 19
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
aatgagaatg cattctggag gaagaaggga aaaaaaaaaa aaaaaaaaaa acaagcgctg 60
attgtccctt taagccattt caatcacagt ctcgcagtta aaataatgct cacctactgc 120
gcttccaact gggccatcta ccttttccgc cgggtcaggc ccagtcaagc ctgaggaggc 180
ggccgcggcc ctcgtgagcc cgctctaggc ccggttacac ctgtttatca ccaagtcgct 240
tcatttccct tcccctcctc cctgcttgaa aaggagcatt aattaaaccg gtaaacaagc 300
cggctcccat tctaaatcag agctgcaggc aaaggagaga taacttaggc tccggagaag 360
agggattttc agttaattta tggaatccac cgtcacactc tctccgagca gccagctccc 420
cgcttaacgg ggaaattgaa gcagacagcc tttgtctaaa cacttctttt gcccagaata 480
tcttaatttt cctatttgaa tgtttaataa ggtttggggt gcagcagctt ccttttaatt 540
gtgacggtgc ggccgcttgg gcgtgatccc ttggctgggg ctgcaggggg cccgtcctcc 600
a 601
<210> 20
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
tctccgtttc tctatatttt tactgtggag gactcagaag agagctgagg ctattgtccg 60
gggaggaaag gaattcgggc aaatttgtgg gtaggggccc agacccaaga cccgtgtttc 120
tcctgcgtgg gttggagtct gtctcaggtc gctccaggga catcaagagc ccgcgccgcc 180
gagagcccgc gccgccgtcc agcgggaagc agcggaccca caggggcctc cagccgcctc 240
cccgctcccg ccccgtgttt ctcctgggcc tccagctccg tggagagagc tgagagttct 300
cggcccgcgg gcttcctcac caaatcccca aaaaccgacg caggcacaga gggctgactg 360
tgttttgagt aatgcacgcg aggcagtcca atccggcgag atggcccgaa gcggggccca 420
gcggtcgggg gtgtgggtct ggagagagag ggtctcccca cttccttcct ccggctgctc 480
ggtcacccat cgactacccg ggcggaagcg gggcgcagag gggcgcagag ggaggcattg 540
ccctccagga gtatctattc ccatcggggt atggtgaatg ccatctaggc ccatgctcca 600
t 601
<210> 21
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
agctctctcc atgagaaagg cccggctggg ccccagcccc gtgtatgaca aaaagaagcc 60
ggggccggct ccaggagccg ggagaaccgc gctgacgcag cttcagggcg cagagggggc 120
gcgggggtgg gggaatccaa tctgggctgg ctgggctgga gaaggggagc gaagtgtggg 180
gcgtggggca acttctgcaa gttccaggga tggcgaaggg ggcaggggta cctcccggac 240
taggccttgg aacccgtgac ccccaaatat ttgtgaggag aaagagagag gaagagagac 300
gcaattttcg tttcgcgctg cccccgcccc gtcagcccca aactggaagg taaatactta 360
cttagcctcc gaaccaggta caagctaata ctcaacaata ctgatgcctt gttttttttg 420
ctctgtccgg acagcaacgc tgtagccaat ttagatatgc tataaattta agaggttgcc 480
atggccacgg tgcgcccatt ggccgctggg ccccctacgt gcagcgccac gtcaccaaat 540
ctgaataagg atgcgcgaat tacgcggcga ccagacaaag atgaggatcc ggaccgcttg 600
a 601
<210> 22
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tggctaggtc acaaaaaaac cttctgaaag gcaaaaattc cttcccaatg ggtctgaata 60
tgggtctgaa tattcctcca ctatctttgt cctggatttt aaacccacag cctttcttag 120
cagggacagg ccttagagat cgtccagttc cattttttcc cttcaggacg agtacactga 180
ggccaaaagg tgacgtgcct tccccacggt cgcgccactg cttagtggga gatgagccga 240
gaacggcctt tctcggaccg tccactacat tctcccctac tctacgcttc aatcttctcc 300
cgtgaaacct tcacttgctt ttctacaacg ttgacctaag gcggaaagcc gcgtgccaga 360
gtgggatggg agaggggagc tcacgctggg cccgaggggc ccgccggcag ccgcgcgccc 420
ctcgccggcc cgcgctcggg ctccccctag gggcaccatg ggcgacacgg gggtccccgc 480
gcgccgccct ctggacttac gtgactgtcg ctccccttcc agaagtagaa ggccccgatg 540
gccccaaaga gcagcagcac agctcccgaa atgaggacca cggctcccac cttgagcagc 600
c 601
<210> 23
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gaatatatca aaatgagaac aagggggtaa gaaaattaac gatttcagtt gaacggagga 60
caggccaaac gaaagggagg caggcgggtg cgagcagagc ctggtagaga tcctggcggc 120
cgctagcctc tggcgctctt gccggtgttc aacgggctgc cagcgtccct gcggcagagc 180
ggacctcggc gcagcgcggc gcgcggtgga gcctggctgc ttgaggacag tcagccccca 240
gggccgcaca gcctacttgg tgggaagtgg cgggcgaaga gggaacccgc cttatttccc 300
ggtttttaaa ataaccggct cggagcgttt ccagtccaca cgagtgagcg cgcagctgcg 360
ccccatctgc ggcgcgatct ttcacgaggc tgggcagttg cgcgccccag agcctggcgc 420
acctcgctct tctcccgcct gcagtccgcc gcccgcgcag ccccaggccg ccctttgctg 480
agagcgccca gccttgctct gaacccaggc tgcgtgctgg cgctgccagc cactctcgcg 540
ccgtccgcgc ttggctagtc tgtcccgagt ttggctctga cgtcgaaaca cgccctcggc 600
a 601
<210> 24
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
cctcccgcct cagcctccca aagtggtgct gggattacag gcgtgagcca ctgtgccctg 60
ccgctagtct tctattttaa gtatttagtg gtaggtcccg ggccggcaga atctattttc 120
agcatttacc acgtgtggcg cgcaaaccac aggttttggc gattgggttg cgcgggatct 180
cagagctgac gccgcggggg cggctggggg tcccggtttc cgactggagc cgcgacgacc 240
ccggcgacgc gagcctgggg ctgcagcgag ggccggggag ctccccctcc atatgtgcgc 300
gcacattctc cagacttgct caaactaacc ccccggcagc gccagcgcgc tgcgggactg 360
atgatcaaat atttggtttc cgagataaca caccccgata gcgctgtttc ctgagccgct 420
ttcattctac ttgtgtaact tgctgcgaaa acccgaacca agtcaagaca gcaaactcac 480
gcccacgggc gctgtgtcaa catggaaata atgatactga agccccacgc tgggcacctg 540
gggcgtggac tgggggcgcg ggggaagcgc agatccgcct tcatgcttcc ccctcctgat 600
a 601
<210> 25
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
aatctccttt acaataaagc caaggggaga gaattaaaaa tctttgaagt agatcaaggc 60
tcaaaggaat cagccaagta gacttaaagt agtcacttat ttcccaaaac tggtttgtcg 120
gggaacaata aaaggaagta aaatttatgg agaaattata cagtggattt gtcacttaaa 180
atatcgtaac tgtctggagg acaatacccc atgctgagga ttaatggtcc cgccggacct 240
ttgattcacc agtgcctttt cttcgccctt gacaaattgg atttttagga atgggaaggt 300
cgcctggacc attgtgtgct agccattcag cggcctcgat tatgcagggg gctgagggaa 360
ccactccatg tgaccctctc gggtgggact ctgcagctgc ttcgcagcgc aactctctca 420
ccaaactccg cgcccttgcg ctagcggtgc caaaaggctc ccgccccgat tgaaaaggcg 480
cagtgcatgc ccgcccgcgt cactccgcgg gcggaggacg cacgtcgggg cgcggctctc 540
tggctagcgc gcagctccag ctctgtcact cgcgcccttc caaggacctg gagcacccga 600
g 601
<210> 26
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
tcgggacggg accggagctc gggcgcagac ggtgccacgg gagcgcgcag aaccctgagg 60
gatgctgccg ggcggaactg aaacccctcg caatgaactt tgaggaaagg ctttagcggt 120
tgaaatccac aaaagccttt aatatcctcc ctgaggcgcg caagagacaa aaatgacttt 180
agagcgactc cgacccgatt tcatcaccca cagacacaca aaaaccaacc acacagaaaa 240
agtacaaaac ccaatccgtg ctctttgtga aacactcatt taagacaata aagaagcagc 300
gctcagatgg gagaaaagat ttgcaaaaca atgaagtgaa atgatctcct ggaaaggatg 360
ggaagcagga gacaggagga gtgaaacgtc aggacttttc tccgcgcttc ggctccaccc 420
gggtgaccaa agccccacac ggctcagcgg gaagctccgc agtttcccct gcgcggcgaa 480
caccggtgtc ctcggcattc cgtcgccagg tcccggtccc aaaggcgctg gctgagggcc 540
ccacgttgat tcattgcatt ctggctctgc ttctgctgca ggactgtccc tggacggcgc 600
g 601
<210> 27
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ctgcggtttt cacagtgtaa aacggatcag agggaaagct ccagcccctc tcgactttcg 60
cttgcggttt gcaacccctg gggtgggtct gaccccgcgc ggcacttgcc caggcgagct 120
tccctcgtcg cggcccgacc cgcgcgcgga ctcggcttcc ccaggtcagg acccactagg 180
tgggccgctc ggccccaagc ctggcagggc ggagcctagg ccgcgcgccc cggggttgga 240
ggttcccacg gagccgggaa actgcgcgcc ccaggtccgc aggccgcctc ccttccctcg 300
cgccctcgca cccgccctct gctctgccgc tcccggtcct gggtgtgcgg gagtcccggg 360
ggtccctctc ctggacgcag aggaggaatt tggagcaaaa ggaagggaat ttactgcgga 420
tggcaccgga acctgaattg acttgtgcgt ttcctgctct gggcacgtag gtttccccta 480
ggaatctgga gttcgctgag gacacttaat tccaagagat gatgggttca cataaacata 540
ccatttagtt aagaaaaata tttaaaataa agaaaaaaga aaaactgaac ttcgatcttc 600
t 601
<210> 28
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gggcaccgaa gtctacactg ggtcgggaga ccggtctagg agccagccgg cagtgctcgg 60
gatctttgct gatgtccaag ttcatctctc ggggagaccg agtttgaatc aaatctgcgt 120
gcgcccagct gtcaaatctg caaacctatc acgccagaca atgggccgcc gcggaggaca 180
gacaagaatg gctcattttg atcgaggggg gtaaattgaa aagggcacgg gctttggttg 240
aagtttgagg cccgggtgtc agggcgggga gaagaagagg aggaaggagg agggttgggc 300
cgctctccat tcaaccacac atcagccagg cgcgcccctc ggctgcagcg gctgcttgca 360
ggcgggctgc atattgtgtg gagtcgccaa agagggtctg cgcctcgggg cgcgccccaa 420
acccggggga catcctacgg atccgcactt ctcttgtgct gtacgcgtgt tcctccaggg 480
taagtatgag gagacggacg ccctcatcag acacccctcc tccaacacac acacacccat 540
agccacatca ttcagggata ctctagttca gtgtatcagt ttacacttgg ttgttgagta 600
a 601
<210> 29
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gaggcgtctg gctggcgctg gccgcgtcgc tcctgcacgg taaagccact gcctccccgc 60
cctccactcc tccgtgggat cccgggcaca tcccgggcgc ctctgtgcgc cccgcgcctg 120
ggccaggttt gggatctccc cgccgccggg gaggggcagc gggggcgctg cgggggctgc 180
ttgtctgggc tgcaccgggt gggcggcggg ggacgccggc aggagggagt cgggggtacc 240
cccgccggcc tgccctgagc cccctgcccg ggtcttctct ccttaagtgt ccctgcaagg 300
cgagttccag aggaagcttt acaaggagct ggtcaagaac tacaatccct tggagaggcc 360
cgtggccaat gactcgcaac cactcaccgt ctacttctcc ctgagcctcc tgcagatcat 420
ggacgtggtg agtcccgcct ggctacaggg ctgccctctc cccttcctgg gctccgaggg 480
gctttttaga cagcgtcggg cggccaggcg gtggagctcg gctggggcac tctagttggc 540
cccaagctag gcagggccat gctctgagtc tgtccccagc ctgccctctc ctgagtgtct 600
c 601
<210> 30
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
atcttcgtgg tgggggatta tggggtggaa atgcactctc ccagtcggat cacatcccac 60
tactcaccag cgttatatcc cgtcgcggct tcacatatat tacttacctg gcccgggtgg 120
cattttactt tgcatccttg attgggagtc tcaggggcta cgaacaaacg ttttaccccc 180
gggtagcagg cgggtgagga gcgagtattc gcggagcggg ataagtgggg atgtcgctgc 240
gccctcgtcc cttcatctcc gggggacggc cactctcagg gttcccgagg agccggctcc 300
gtgcacctag gccctaggtc ggctcccaag cccgggcccg ccgctacacc agccgtttct 360
tacccgcagc tacaaatctg acacagacac cagctcctca tactcttggc tcccatgctt 420
tcctccaccg cgcctcagca cttgcagctt ctccactccc acagcgcccg ccgtagtcaa 480
ggcaacagag ggatgcccgg tgacagactc ggcgccggct tccggcggcg tcagagacgt 540
gcgggtgaaa ggtcgagcga cagccggccc cgccccctgg cgcgcgggcg tgtcggaccc 600
g 601
<210> 31
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
gaattatttg cagcatatat tattgactcg aattgcctcg atataaaaaa gcactttttt 60
tttttcgaaa ctctattgca caagctggag tgcagtgagg caatctcggc tcactgcaag 120
ctccgcctcc cgggttcacg ccattctcct gcctcagcct cctgagtagc tgggattaca 180
ggcgcccacc gtgcgggttt caccgtgtta gccaggatga tagtctcgat ctcctgacct 240
cttgatccgc ccgcctcggc ctcccaaagt gctggtatta caggcgtgag ccaccgcgcc 300
cggcctattg tcagactctt aatcaagcct gacttttggg tttagctctg gctccagcag 360
agcaactctg gcggttggtt ctttctcttc cttatctcct tgtccccaag catccaccag 420
aggacgcagc tcccccaaaa ctttccaccc agtgcttggc tggaggaact cagtccacgc 480
ttctctgcta gtctaggccg gcggttaagt caatgtaacc ggagacccgg ctgcccgttg 540
ccatggggac ggaaagctat gatgtcacca ccgtccgggt gggtgtgctg gggttcaccc 600
t 601
<210> 32
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
cctataatgg taaatgttga attaaaaact tacagtgctg gcgaacaggg acttagcgtg 60
cctaggaact caagtgcaaa aacaaaacaa catccacccc caaaaattgc cctttttttt 120
tttcgaagtt tgcagttata ctattctgtg acttaaaagg gaggggaaag aaagtatgca 180
attcagattt cgcccccaac gcaaaactac cgaaacgaaa atctccccag gaaaatgtgg 240
cttgatttat agacgaaaaa gaaaaatatt gttcctcttg gcaaagtctt tttgcatcac 300
gtgtatttgc agcctagtat aaacttgctt tgccgtgtgt ggatgtgtga gtgagaggga 360
acgagagtaa gagaaagaaa gaagtgaggg gatgtaaact cgaataaatt tcaaagtgcc 420
tccgagggat gcaacgggca aaaactgaac tgttcaggct tcagattgta actgacgatc 480
tgaggaaaaa tgaggtgctc gatgaatttt cgtttgtatt ttttggcgag gcgggggagg 540
tgttgagatt tttttttttt cccctcgggg tgggtgcgag ggggatgcat cctagcctgc 600
c 601
<210> 33
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gccgagaaat ccctttatca cgcaccccca cccccaacaa gcccctcatc actcccccaa 60
agccagccgc cccttcctga ccctacagaa cccccactgc taatattccc aatcacttca 120
gcgtccagta agcgacatag gcttagatcc acttaacaac agaagctaaa tggtcgctct 180
aggccacttc cggtttgggt tacattcccg ttaggatttc cgtaggcgct gttgaaccgc 240
gcgcatgccc actaacactt ttgaagctac gctccgccaa cagttatcat ggcgtataac 300
ggcgtacctt tattccgcgg ttcattcaac ttcagaaact tggtagggag gacggcatgg 360
ccgcacccat acggattatg atattggagg ccgccatcgt caaagggctt ttattccgta 420
cagccaagga caaatggaat gtcacagagt aaaagtcttg ggtcctacat tggcagggga 480
cctattcaat acttgtgtat atatatttct aggagaaact gacatgtatt ctgaaaaata 540
ctttttctct atatttttcc ctctgattta aagacttttt tttcctgaat tgctgaacaa 600
c 601
<210> 34
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gttccttaac ctgctagccc cacatctacc cagcaaacct ggcttgtgga gcctagagtg 60
atctgaaaat ccaggcatcg tctcttcctc ccggctcacc ccacaacttc agggaaccct 120
gttctcctgc acctctgttc ctgaaaagaa atattgggga aggggggaaa gaggaaatat 180
agaaaagata ttgatgagat tgacagcatg agaggtgggg acgataaggg cagaggggca 240
agagaaattg ggtgttgaga gatggagaga acgggcagaa aggagaaccc agaccaggtc 300
gaggaaatgg tagaaaccga aaacacccgc aggccacaag cccgggatct gtctcccgag 360
gctggtggct ggctctgact gtgactgaga tggcgctggg tggtctcgat gtccctgctg 420
accccgctgc tttgctcccg cacttgcagg gagaacaacg agaactccaa ttctaacagc 480
cacaacccgc tgaatggcag cggcaagtcg gtgttaggca gctcggagga tgagaagact 540
ccatcgggga cgccagacca ctcatcatcc agccccgcac tgctcctcag cccgccgccc 600
c 601
<210> 35
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
taagtcccat attggaataa tacactaaat gacaataata tgattatata agatagaata 60
ctgatatata aataagtata caaaatatta taggaaataa taaaatgatg gcacctattc 120
ccagcatagt gtttttgttg tttataatga tggcacctat tcccagcata gtgtttttgt 180
tgtttataaa gtacattcat gtttgtgact ttagtggagc tgcttatagt tcagtaacat 240
caggtggcag cgtcgtcctt tccgcagagc ggggctcgcc cgcgaagcag tgaaggctgc 300
gtactagagc tacctcgcgc ctctcgtgga gagacagcgc cctctcgtgg tggaagaagg 360
aagcggccgg cccctgtgac ccaacagagt gattcaccta atgctgtaaa attatctagt 420
gataaatgta tttatggaat accttcttgg aattttgaat tctatttttg gacaaaagat 480
gttccctcac agacattata tgcacgtata gaaagatttc ctatgtagaa acaaggtatt 540
gttattgagt accttagaac aacaacaaaa atgtttaagt cttgtttgcg cacgacgcac 600
t 601
<210> 36
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
gggagacatc cattatcagc gaggcagcgg agctcctctg ccgccctccg gctccaagga 60
ccaggtggga ggtggtggcg aattcggggg ccacgacaag cccaaaatca cggcgtggga 120
ggcaggctgg aacgtgacca acgccatcca ggtaagcgcg ggattcccag ttctgcctgt 180
cctcccccct cccagctcag cgtgccgggc tctgcccccg acagtcgccc ggtgatctcg 240
gcctggagac cccctcctgt acccaggaat ctccctttct ccatccctcc cagccctgcg 300
cggggaccta cgcccccagg cggtgttttc cgccctaacc cacgctccct cccaacggca 360
ccagctgcaa gaccgctagg ctgaagttcg gtctgagaca cctgtccgga gacactgcaa 420
aagtgaagga aatgggggga gggagcagga agcgatgaga aagaaagaaa atcaggattg 480
gagggcacgg tttggtcttg gactctggaa cggattcaca gctgcatttt tgggaggaaa 540
gaagaagggg aaatcgctga ggtcggagtc tcctcccccc gcacacacgc atagacacgc 600
a 601
<210> 37
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
cccagcccct gagctggccc cagagctctg gcctggagcc tgatcaccct gaggccctcc 60
ctgctgcccc acactggggg caggtgccct cctcattctc caaggccctc cctgctgccc 120
cacaccaggg gcgggtgccc tcctcatcct ccagctctgg acatccccca tctaagggcg 180
ggcgggtccc cttaccaaga cctaaggtgc aaaacccaaa gctgaggctg cttaggaatc 240
cacagcccca caggcctctc agaaccttgc atcagagtcg cccttgacct caaccctgcc 300
cgagaaggaa gtgctggtgc aaccccagcc cagctgcagg aagtggtacc tgtcaccaag 360
tcccctgcac aggggaagca gggggctgag ggggcaggac cagggggacg aggtcatcct 420
tccccggaga acccctcagg ggccgggcgc ccttccctcc cgggcaccaa atggacacag 480
ctaacaagaa ccacacttca cggtgagcaa agctctgaac attcaggctc tgagacacga 540
aagcggcgtc agaagagacc tgcagcagat ccgggccaaa tcacacacac cctttcacag 600
a 601
<210> 38
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
agagctgtgt ttacatgtgg gtagggttcg ttcggaggcc ttcctggaag tggtagccaa 60
actcccacac cagactaaat cccgtcaggg ctggcgctga aaagtgggat cctgggtggc 120
ttccaatgcc caccccccac cccccacccc tcccaccacc caacctcttc tccttccttc 180
ctttccaaag gaagggcctc aggctataag tttgatcttc ctttagactt tcgaagtaag 240
cttccaattc ctttacctca ctctcccccc tcctcacttt tttctgcttt gccaacatcc 300
gaatccaaga aaatggctca aataggatgc agattaactt catagttccc attccaaagt 360
ctgggaagcc acggtgatca cctcagtttg tgtgttcctc attcctaaga aaattatggg 420
gagaagtgct gatgaattta ccgggaataa caaggctctg cagttgcgat tacaggagag 480
ggaaggatcc atgagggggc aaggttgaga gtgaccaggc tcttctagtt ttccccagtt 540
gtggtacgga atgggagggg ttggaatgaa tatattcgtc cttatattca aaagccacag 600
g 601
<210> 39
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gccgggttcg ggaagccaga ggggaggtgc cctgggccgc agccgccgcg cctcatccgc 60
cgtcccgcgc ggtctcctgg ctccccagca gccgcctgag cccggccata gataccctgg 120
cgattattaa acaaaccttg acgtggacgg gttggctgcc agggacgcgc gcggcccgcg 180
tcgtggcaac gcccgcgccg ccccgccctg ggtgacagcc gcgcgcccca ggggtggact 240
gagacgcgca gccgcgcggg ctggagggca gcagggcgtc tacagcattc ctccccattc 300
ggggttctgg gtgcccacgc gtgtccagaa aagcagaagt taagtctcta tagaagtttg 360
gggctaggct ctcaggagat cacctctttg gcccctttat tctttcgcct ttcattcctt 420
taattcttca tttgtagcgc cttgggttta cctgtatgaa acttgtaact aaaatttcag 480
tacttcttac ttctagcttt tactttgttc cttgacccac tgggtgggta cccaagttaa 540
tagagtttag ctccaggatg ttcacaaatt aaagttaatc cctcaggttt ggataagatt 600
t 601
<210> 40
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
ctggctgcag gtttttgggt gagtgtggag tttctttttg attgttgcat gtggggaagg 60
gcagagtcga cgggaacagt cactgcgggc accctagccc tggtgcgtct actgtcctct 120
gctcggctcc ccccatcggt gagtgcgccc gcccgcccga ctgtgcgggg ctgcggttgg 180
ggggaggggg gagcgggatc atctgaggcc agagccactg ccgtgtgtgc ggggaggggg 240
agcggcggga gagagagggg agggacaggc taggtgtctg ctgctccacg ccactgctgc 300
cggcgccccg acctcatccc cagcagcccc ctctgcagct aagggttacc accgcaccac 360
ctctcttctt tgcctgcctc agcggccaag gctctgcggt aggagacaac ccagccgggt 420
ggcgggtggg cctgtctagg tttgggtttg ggtcttgctg aggcccgcat gagagggggt 480
ggccgtgact cggtgtcccc tctttgcagg gggctctgct gcgccgcgca ggcccctcct 540
cctacatcct ctttgggggg tcactggaaa gcagagctta ggcccactct ctgtgcttag 600
t 601
<210> 41
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
aaagagttac caggtgttaa ctcaccagcc ccgcaagctg agaggcgtgc agccccggcg 60
cgcacttttg cgcggaccgc gcacccctcc aagctcgcag ggcgccgggg gagacaggcg 120
ggagtggcgg ctgaccgtgc ctccatcgtc atcatcatct ccgtggcaag ctactgtggg 180
gaggtaatgc agagaccact accacacatt ggccctccct tcaccctccc gcggctaaaa 240
ataaaaataa agaagagaag aaatgaaagc acaaggagtt ctttaaaagc cccaggagac 300
gacagagcag tgagcagatc agaatccacg gagcccaccg ccaggcagag cgaaccgcct 360
gtttacatcc ggctgcctgg gagcggatac caggcaacca agagagatgg ggaatcttac 420
caaatcttaa tacatgtgtg gttccttgag aatatccaat ccacagtaaa cagagcagga 480
gtttaacggt gcgcctgaag actggattac ttagaatcgg gaaaataatc ttcatggtgt 540
ttctgtgccc acacgcacgg cacccacttc cccgatctag cgttcggctc ctgggttcgg 600
g 601
<210> 42
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
ttatctattt atatatttat aattacatat tgcacttgga ccagcaaggc ttgcagagtc 60
attcacggta gaagttaata aagttaaata gatgggaatc tttgtaagta caattgatct 120
cctctggttt ggaaacgaat ctcctcgtcg ttgtaaagtg ttctcgcggg gtgggacaga 180
gagaggagca ttgcgagggg gaagcagaga cagagagcac tgagggcagg ggtcgccttc 240
ccggggcccg ctccccccgg gagcgcgcct ttcccagact cgcacctcca aggtcaggac 300
gcggtggttc cacataagcg gctcgcggtc accacttctt tcaggtcact ctcgggtttc 360
ccggccacca taaagggcca cgtctgcggg aaagaagagg gtggaggagg taagtggccg 420
tggcggaggg ggtggggtgg actcggggcc tgagatgaaa ggaagggggg cggtgggggc 480
aggcggtgct ctggagagac cggttatctc tgcaatttac tcccggtcct agcgcaccct 540
gcggctgcat aaagcaaata gctgctttgg cgccaagtca cagctcgagg tggtggtggt 600
g 601
<210> 43
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
cgcggctgga ctgggaccga gcgcatccct gtgtctcagg gggtttgctt tgtgcaggat 60
gggaccttat tatttttaga cagtggggca aacgtgtctt ttaaagagat ccgaagggtg 120
aaactaatga aaaaggaaat gaattactag aaaagccgtg aagggataaa accgggagca 180
gcaggaagtt gaaggaagga cgaccgaagc taggtgtatc tgcacctgcc tcttgacacg 240
ccaaaaacaa aggggggtta aggcaagaat gtagactaaa tgctgtgagg attagggtaa 300
cgcgagccct caagacgtct cggagaccgc ctacagggct gcaaatacca gggtccattt 360
ttaaaaatcg agtgctccta gaggtggggt gggggtggga gagccgggaa accagcaaag 420
caatttcttt ccattgagta gacaaaaact tgggccacag gggcatcttg tattttgaac 480
agtgacccga tgaccccgct ggccaggtgt agacagcctc aagtcctaaa tatttgcatt 540
gtcctaactt cccagttgcc tcggatttta atgccctttc actctgcgtt acagtcaatt 600
a 601
<210> 44
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
cagctgagcc ggggttagtg cagcggtggg ggtggggtgt gggggcgggc ggggaggagc 60
tcggcggctc cccgctgctg caggtaatcc gcagcccgct cctccgggcc gccaaacccg 120
aggcccagcg ccacttactg gctgcgctgg gaatcggggc cggagcgcac ccggcgagcg 180
cacagcctcc tacctgcgcc ccgctggggt ccctgggctc attcccctgc cccttccgag 240
cttaaaaccg ccggcacgac gccccttcct cctggactgg gctagggcac taacttccac 300
ggcacttcgg atcgcacgga agaatgatat ctttcacctg ggcgcacggg gtggctaatc 360
tccaagtaga gattacgcaa cacctggtat tttaaacgtg gattacctct attcatgatc 420
aaatgggaat tcttcaagat ctagttttag tgtaccatgc ttttcgcaga taatcgatgc 480
aaagtgcacg gttttggtgc acagaacttt aacattcaac aaatatacag tgagcgccta 540
tgccagacaa tgtggtacaa gacagaccca gaagtgtgta gtctgtattc gaagtgcaag 600
c 601
<210> 45
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
agtcacctcc cctctcagtg tccttctctg taaaatggga attacaatag aatttatctc 60
cgagttgttt tagggaacaa aggaaatata atccatgcaa agtgcctggt ccacaggaag 120
cgctttcgca ttgtcagtgt tggaggtgat gtttgctttg cagggctgtt caatcccctg 180
cccacttcta aacacattta actttgggga ggggcctctt ggggaggaca aaagaaggag 240
gctggggctt ccgctgccct ctgcagacct ggtgtgcgat gacgctgcag cgggggaccg 300
cggttttaaa ggcatcatcg gtagaaacgg cctcacgcat ttccataatt cactctgcat 360
caattaatct ttcactgtcg ataggaacct gcggctgaat tccctccccg ggaggactcg 420
ccattctcaa gctctgaggg gttttcttct cggctggcat catgctaatt tccctcgagt 480
acccctggaa agaggtttta gaacaagacg tggtgcttgg caggaatttg acatctgaaa 540
caactgtcgc tcagggagga gggggttgtg acatttgcta attgccaatc gagcactaat 600
g 601
<210> 46
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
acgctgcttc aaacccctgc cggtgcactt gagctacctt caaaacaccg acttcctaca 60
ctgtcttaag atgtaaattt caaacccgga cgcagaactc ctaaaaaaaa gtaaagagta 120
acaaaaatta agcagataga aaactacttc ttgaacatac aagaattgtc tacaagaaag 180
gcggtaataa taactcctaa taataataac tcctttcact gtttccagtt gatttgagta 240
ggagacggca gttttgaagt aaaactggaa acctgatccc caccccttca cttctcgagc 300
cgggcctcgt cccttaattc tagaaatagc gatcgcgaaa gctaagtgcc gagaagtgcg 360
agagagtgcg agggcgtttt tggatagcag aaatgttagc tcaaaatcga caacttacag 420
ttattcctaa aaatgtttaa atggcattca tgtaaaagac acggattcac ttactccgat 480
gacagttttc ctcccctagg cgaagcacgc agtgcgccgg aacggattat ttttagggtg 540
gtgggagacc agctgctgcc tcctatcgag tttctaaatt gcacagactg ctctgggccg 600
g 601
<210> 47
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
caagggacgg ctagatgatc catctgccaa catcaatatt tggagggaaa gcttaaaata 60
tgctgttttt ccaagggtta aactttaaag gggtgaaaac acaactgatg aaatgttagc 120
atcaaatcgg ttaaccacta actcctttcc atattcgtcg tcgaggctct tttaagtttc 180
catgagaagc gaaacttcat tctctttgct cagaatagag actcctccac ctatgcgcgt 240
ttaaccacag agttgttctt gattgtaagg gacttcgccc acttggttga agtggagagc 300
cggtcctcat tccagacgtc ccgcacggca gtcgctcatg gctccctcca ggccgggagc 360
caggaaggtg gtccttcacc gaggtgcgca ccaagcggcg gtccacccag gcaatggggt 420
gcaccaattt gcctccaaaa atttgctgcg cacacaacct gttcacagtt cctgatcaga 480
aagggctaac gagatgtctg tctacagctg tctctgggcg agggcgcagg ctttgggaag 540
tggttcagcg cgatcgggga caccgcttgc agcccttggt cctgcacccc taggaggggt 600
g 601
<210> 48
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
cctggctcct cttaaagcat cctctccttc gaacggccgg gagtggcagc tttattggga 60
aacgcgcgct ctgggggccc aggtggctgg ggggaccgga gaggcgcgcg ctttgcccac 120
ttctcttggt ggcatcttgt gcgacccagc ccgtcctctc ccgacttcga tcattaccgc 180
tcccacccca acacgcatac ttcccccacc ccacccccaa gagccagcgc ggctttaaca 240
ttaaacaaac gcagcgagcg cctccgagct gcgctctcgg ccgggtctgc gcggcggcgg 300
cggcgttaca aattgtaaat ttaaattgct cgccggattc attacctccc ctctttgatt 360
tcagccagcc gtgaaaaatt atactggtgt accactgaga aactgttttg ccgcaaagag 420
cctacgccta atcactggct ttctccctcc gacaaaagtg taattatttt gtttggggtg 480
taaatatagg ccgcgcctcg cacacacact cagcgtcccg cagcgccgca gagcaccgcc 540
cgccgccgga aggaatcggg cccccgagta ggagcgggcg tgaaggctga gccccgctgt 600
c 601
<210> 49
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
gaggagaggg gcctgggaag caggatctcc tgcctggcgc agattggggt gccaggatgg 60
cttctcagag ggatcagcgc tgcaactcgg tcgccgcggg gccctggtga caggcgcacg 120
ccccgccctt tctcctccca agacctgcgg gcttttgtta tgcagatggc ggcaggaggc 180
tgagcctgag gaggaggggg ttggtgggga ggaggagaga cggagagggg ggaggaaagt 240
gcagctcgcg cgaccagcct cctcttccaa cgtcacattg tgcgagagac aaaacccggg 300
cgcgccggag ctcacacgcg cacgcacaca catccgaccc cgtcgcctct tctctcctgg 360
tgctgcccag aaagccagcc ctcccttccc ttcttggggc gcagaggctc agccagctca 420
gagcgcagcc tggagccgac ccagaagggc gaagaaagcc cagcggacga gcctcctttc 480
tctgctgcct gcccgggctg gggcgtccca tcccccgccc tgaactccga tctctcccac 540
cccacccctc tctgggtttc acccggacag agccgggagc tgggtgtcgc ccccgtttgg 600
a 601
<210> 50
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
ggagaggggc ctgggaagca ggatctcctg cctggcgcag attggggtgc caggatggct 60
tctcagaggg atcagcgctg caactcggtc gccgcggggc cctggtgaca ggcgcacgcc 120
ccgccctttc tcctcccaag acctgcgggc ttttgttatg cagatggcgg caggaggctg 180
agcctgagga ggagggggtt ggtggggagg aggagagacg gagagggggg aggaaagtgc 240
agctcgcgcg accagcctcc tcttccaacg tcacattgtg cgagagacaa aacccgggcg 300
cgccggagct cacacgcgca cgcacacaca tccgaccccg tcgcctcttc tctcctggtg 360
ctgcccagaa agccagccct cccttccctt cttggggcgc agaggctcag ccagctcaga 420
gcgcagcctg gagccgaccc agaagggcga agaaagccca gcggacgagc ctcctttctc 480
tgctgcctgc ccgggctggg gcgtcccatc ccccgccctg aactccgatc tctcccaccc 540
cacccctctc tgggtttcac ccggacagag ccgggagctg ggtgtcgccc ccgtttggaa 600
t 601
<210> 51
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
atacacagaa ttattgtttc agtgatgacg ggggtgggaa agggaaggat gtggggggat 60
gggtcccagg ctcttaaaat cgtgtgccac caacgtcaca accgccatca tgatgtgtac 120
gcatgtttac aagaaatgtt cagcatccat ccgcccgagc tctgcatccg actggagact 180
ttacacctgc ttcctacgaa gagggagctc ttgccagcga gcccatgctt acccataatc 240
tacgaagaga tcttttcatc tcctctccgt gaacacttgg ggaggggctg ggactccagg 300
cgacacggaa acgccaccac acgcacactc gacggagcac aacactttct gagctgccat 360
ccatttttaa tttattggat ccaagagaaa ataacaaccg tagggagggg ggaggagaag 420
ggtggagggg gagagagcag gaatcaattc ttggtggaga aaataatcta tgactagatg 480
acgcttaaag gcacgacaag agattttatg aagttcccct aacggaggga ataatgaagt 540
gcctggcaga gaaggaaatg gagataagag ggaggctggt gtcaggagga tgtccacagg 600
g 601
<210> 52
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
agagagcaca ttttgaataa gcaaagtgga gatctcactg acaaaacaca aaggctaccc 60
cagcatcctc aggcaggggg cgcatacctg ggccccctat gcccaaggtc tcctcctgga 120
atcccctgct gtcctcgggc acccagagcg ggaacgccag gttttcccca tcacgcaagt 180
gcaggcggcg cgggggttcc ggaggctgtg agtgtcaatc acctaccctt atcagttctc 240
cgtccaacac ttacagtcac aaagctgggc tggaaaagtg ataaggggac gtgactggcc 300
ggcaaagaag ttttgatgaa gctttgcagg acaagatgta aatggacgtt tgatttcccg 360
gcgagccctc cccgccccct ctctgcagtt cctcctctgc ctcaaattca aagcccctct 420
gtgcccttct actttgctct ggcctctcgc cccagtctct ccctctgaca cctggagagt 480
cacttatgtt tctgcacaca gaaattcaga catgcagctc tggacagcta tggaaagtaa 540
taggttcttt gcattaaaaa aaatcaatta aaaatagaaa ttaatttaaa ttttaaaatt 600
a 601
<210> 53
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
acccagtcgc agagagcaca ttttgaataa gcaaagtgga gatctcactg acaaaacaca 60
aaggctaccc cagcatcctc aggcaggggg cgcatacctg ggccccctat gcccaaggtc 120
tcctcctgga atcccctgct gtcctcgggc acccagagcg ggaacgccag gttttcccca 180
tcacgcaagt gcaggcggcg cgggggttcc ggaggctgtg agtgtcaatc acctaccctt 240
atcagttctc cgtccaacac ttacagtcac aaagctgggc tggaaaagtg ataaggggac 300
gtgactggcc ggcaaagaag ttttgatgaa gctttgcagg acaagatgta aatggacgtt 360
tgatttcccg gcgagccctc cccgccccct ctctgcagtt cctcctctgc ctcaaattca 420
aagcccctct gtgcccttct actttgctct ggcctctcgc cccagtctct ccctctgaca 480
cctggagagt cacttatgtt tctgcacaca gaaattcaga catgcagctc tggacagcta 540
tggaaagtaa taggttcttt gcattaaaaa aaatcaatta aaaatagaaa ttaatttaaa 600
t 601
<210> 54
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
cgttttttaa gcccaacttc aataaatctt gacgaggcct ggaattcaga gagtatccca 60
ggcggctgag ggagagggtt gtccaggccg gggctgggga tggagctgga cgtggcgggg 120
cgatatccca tccgcccgag aagggagcct gggcccggat ccactatctc tgcagaggcc 180
ctcagaagac cctctctagg aggccaggtc tgtcccacgg ccgcccttgc ccactctcaa 240
acctattaga gaaagagggt cctccgctgc agtcgctctc ctgccagggg cacagtccac 300
gcgggctcac acatttctag ggtctgcgac cacccgcctg gaaccggccc tcagcccctg 360
ctagaccctc ccacccccca atcaaccctc cgcccggaaa gcctcctttc ccacaacttg 420
ggcgtgcaaa agcagcaagt tctgcccaag tccagcgggg ttcgcattcc gccctcccca 480
ccgcgcccca gctccgctcg ggcccgcgca aaccccggcg cagcccctgc attaatgagc 540
gcgagaactg accgaggtct gtaaagaccg tcctcttccc atccccctat accattcaaa 600
c 601
<210> 55
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
actgcgtgtg ctggaaacgc tggtttaggt gctgcagctg caggctggag tagatggtcc 60
tcggcttgcg gagctttttg gcgggggcct gagggcgccg ctcggagggt tccggggaca 120
gccgcggctt ctccgagtct ggtgggcagc acaacacggt gtagggggcg aggagggagc 180
gagggggtag gacggacagt ttcataagct tatttgcatc tcgtctgcat gccgtcggca 240
gcaaatagca aaaagcgcag ccaccaggat cgcccccttg gcccctgccc tctaccttcc 300
gcgtggagcg tttcaacccc cacgttcacc gcgccaggtg atacaagacg cagttctgct 360
cccagggagc tcccactcgt atgggtcctg tgaccctagg aagaaagcgc cgagggccac 420
agccagggta ccgcggcgcc tgcggtggga ggtcggagtt cagccacggg cggtggcgcc 480
agcttggggt agggcggggc agaaaggcta ggtccagggc tgagccgagg cctagagatg 540
acagaggaag gacattgcag gcagaggctc agagcaatgc tcagaggctt gagacggtgt 600
c 601
<210> 56
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
ccgccgcctg ctgaagacgg tgctgatgat cctgctggcc ttcctggtgt gctggggccc 60
actcttcggg ctgctgctgg ccgacgtctt tggctccaac ctctgggccc aggagtacct 120
gcggggcatg gactggatcc tggccctggc cgtcctcaac tcggcggtca accccatcat 180
ctactccttc cgcagcaggg aggtgtgcag agccgtgctc agcttcctct gctgcgggtg 240
tctccggctg ggcatgcgag ggcccgggga ctgcctggcc cgggccgtcg aggctcactc 300
cggagcttcc accaccgaca gctctctgag gccaagggac agctttcgcg gctcccgctc 360
gctcagcttt cggatgcggg agcccctgtc cagcatctcc agcgtgcgga gcatctgaag 420
ttgcagtctt gcgtgtggat ggtgcagcca ccgggtgcgt gccaggcagg ccctcctggg 480
gtacaggaag ctgtgtgcac gcagcctcgc ctgtatgggg agcagggaac gggacaggcc 540
cccatggtct tcccggtggc ctctcggggc ttctgacgcc aaatgggctt cccatggtca 600
c 601
<210> 57
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
cctggcaggc ggtgtcggcg ccggcggggg actcggcgcc cagggcccct tccaggccct 60
ccctcgccgg gctccccacc agctccggag gtggcggcga tgcggaccga gtgagcggcg 120
gtggggccag gccgggcgcc gggcgggagc cgaggccgcg gcggactccg caatcccgca 180
gccggtgact ggagcccacc tctgcagaga caaaggttag aaaaagaggg ggtgaggctc 240
tgggaaagca gaatgcgggg ggaaattcgc cggggaaatc aggaaaaagg ccgtgaaggc 300
gagcggcgcc tgcacatgac cagccgcagc caatgacgac cgcaaatagg tcataaaaag 360
gtctattacc aagttgtaaa ttttcttcaa acaaaaagga atttactgca attccttgcg 420
gattttgcac atacagctcg caagcgccat gttttttcct cttctgcttt ctctctcccc 480
cgcctcctct cttccttctc ctcctcctct ctctctccat tctctctctc cttcctccct 540
tctctttctc tgtccctctt cacctttccc tttttcttcc cttcgtggtt cttccctccc 600
t 601
<210> 58
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
cgggggcgat gagcatctct gctggggcgc cctaacctgg aaaaccattg aaaacggctc 60
aggcttgcaa gacttcctgc ctccccggga ttcccagaat cttcaagtgg acggaaaggc 120
gattcctaaa caagttatag aaacttctag atgctatttg agatacatag aattctaatt 180
tatttaatta ttctaaaaat tccaatcaca atggcgcggc gttagccacc acaactttgc 240
agggcaaaaa aaaaaaaata cttcctgaac taacatatgt ttcacaagtg tgggcgcagc 300
cgggacaatt tcgagacaac ttcgagacaa tttcgaatgg acaaattgcg gagaagttgc 360
ttctgccgct cagaagccgg ttcacctcct tctccaccgc ggcatttcca aaacaacagg 420
gacaagtctc cccggctcgc cgcaggcctg accgcccagc tccgccagga tttgcagaga 480
gcagcgcgct ccatttgcag aaaggaaatc gagtaggtcc tcgcccccga ctggtgcttc 540
ttggggtgtg gggtgcccag ggaatgggct tcctggaagc accaaaggag cctgcggagc 600
c 601
<210> 59
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
tggcgggcgc ggcggcggcg gcggcggctc ccgcgggcag tgcctgggcg ggcggcgggt 60
gggagccagt gtgcgagcgg gactgcgagc gcggggggcg ggccgggggc ggtgcccaga 120
ggagcgcccc gcccccgccc ccgccccgcc cgcgcgcgct attgttccgg cctcgggcgc 180
tgcctagggc cgggggcccg ggggcccggg ccggggcgtg gaggtccttc ctccgggctg 240
ggccgccgcc gggtcccccg cccggccctg catccctgag gggggcgcgc ctgtccagac 300
ggcgcttttc cgtcccgcct ttcgccaccc acggtgagga gagaagcccg aacggcaccc 360
ctccccagaa gtgggacact cccttgccct aaatatttga ggcggcgcta gtccccgccc 420
cgcggcgtgg gtgcgggggg aagaggaagg tggtgacagg accactttgg agatgggtgc 480
tgccttacct ggggggacgc aggccagcgc cggaggatgc tcttagcatg cccgccccct 540
cctcgtctca ctgatctgag tcagcttgac agccccccct ggagattgcc gcgcttaaac 600
t 601
<210> 60
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
gtgccagtgg tccccttgag ggtacttgct acccggtcgc tggaggtaca gttctcatct 60
gtctgccccc tggactcacg cacttacccc actccacctg ttgccaggat cgcctcattt 120
ctgcacatca tttcggggtg gctgagaggg gaccacaaac ccctccaagc ctctcttttg 180
tggttccagc gaggcggtca tccttcactt ctccttttcc agctttcagg aagcagttag 240
ggaatgtatg atgaagcaga aatgagccgt caggatgata ttttaatggc ttatatgtca 300
cgttggtgca tgtggctttg aactggagcc gttcgcgttt cctgcagaga gcgccgcaaa 360
tcccacttga taacttctac aacccacaac tttttttttt ctcacattca ctcttaccct 420
gtatatttgg atgtgtttcc taaaaatacc ctcttgtcaa cgtccccgtt ggattttcca 480
cggactggag aggagaagac tgacttttct tttttgaaga attattttcc ctctccccct 540
cccaacagct acccttcccc ccttaggtcc cctcccttgt tgtgtgtgtg tgtgtgcgcc 600
c 601
<210> 61
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
ttggaggttt gctgcccagg gagaaggaga agcagctctc aggccctttc ggcaacttct 60
cggcttccct tgcatctctg aacgttagtg atccggacct cagatccggt ttccgagggc 120
gccagggtcc tagggctgag gtccattcaa atgcttaggc gactctgccg caggcctccg 180
gaagaggccc ggtcgttaag aaagggggcg gggtaaggag gaacggctag cacctagagc 240
tggggcctgg cctctgcccc caccgccctg cctttggcaa ccagcgaatc aggactgaaa 300
cgtagggaca cagagggaca gagactaagt ccaggatagc gacgatcagt caggcagaca 360
tggagacaga gagatagtga catggcctta ggatcggcag ccacgaaaag cgctcttaca 420
aacatctgat attccagtga ccttccttca ctaactccct aaataatact gcgcctcaca 480
ggcgctaatc cgcagcatat tttatttttc ttttggcact gccatttctt gtctcccact 540
agactgtaaa ctccaccaag cgggcaatct ttcttctctg catccctggc acagagtagg 600
c 601
<210> 62
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
gcaaaacaaa ggagaagtgt ggggcgcctc cgaggtggca aagccagcgg agatgagggt 60
ggcgcaggca tgagctctcc atgcctaggg cgccagggag gcacctaggg gactcatttt 120
atttttagta ttttttaaaa tcgaacaaga acacgccgca gttaaaaaaa atggagataa 180
acagcgatat taaaaggaaa aaatggggga aggaaggtag gtttatttaa aaaagaaagg 240
gaaaaaaatt agcttggtgg ttcccggcaa aggcgagccg gggctggggg tccccagcac 300
gaggccaccg cctcccaccc ggcccccgcc gcactggctc cgccgtccct tcgtttcctt 360
gggtcattcc cagcccgcag gcacaggttc ggccaaatca gaatagctcc cgtcctttca 420
ttttgtttag atttcacctt aagtcgacgc cacggtgtgt atctaaatac ctgtattttt 480
aaaggacgcg cggaagccgc aggtttcacc gctgactcgg aaacatcacg cggtcccgcg 540
cgggagtagc accgtcttcc ccgcagcgcc cgcccctcgc atcctccggg aagcattccg 600
a 601
<210> 63
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
agcaggctgg gcatgtgaag cccaaagaac tccgtttgga aactgcactg ctgtctctgc 60
agtgtggcca cagtgactgc ccgcccacca agcatctaga gcccagaggc aggggtgttg 120
ctgcatggat gtgtccacct ggatacgcac gttttcattt catttaaatg gcaaacaaag 180
gtcttgggca gatttcgaaa agcactggtg agctgcggag acatttccga gcaggagctg 240
atgtggaggg ttttcagcgc cgggtgaggg tcccgcagcc acagcgatgt tctagcctcc 300
gccgcggaac tcacagaggt tcccccgcca tagcctcccg cgtgcgctcc tgcgcttcca 360
ccagacctta tttataagtc tgttgatttc gcaggccgca ccttgcaagg ggcacacggt 420
ccgtcttatt atttcataat cacttcttct ctttttgttt tccacccggc ctggctctgt 480
gtctaggact cagcgtgcat tccctgggct cggcctgccc cgcctgccta agaatcccac 540
aaatcattga cagcgtgggg gaggggagat tgcctgtagc agggaaatgg cgccctagaa 600
a 601
<210> 64
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
agcacccttg ccccagagtg agcatcaaca ccgtgatcac ggttattacc tgggaggtct 60
cctgcccagg atccaggcgg tgaggctgcc ctggaggcag gctcgctccg ccacaggcgc 120
gcgggcagca gcggccaagc cgaggatggc agggcctgcg ggtgaccagg cctccggcca 180
agggcacgcg acccccagcg cttcccaggt gctcccctga agcccgggac gcgtgggcac 240
cggcgcgggg cggtgccggg gaaacgcagc catcgcctca gggcacgcca gccaatgggc 300
ggcctcaccg atgcggtaat gccccaacgc ggcgtttcat tggccagctc tctttcaggc 360
tccaactatt tattagattt tttttttttt ttaagaaatt gcaaataggc ctaaacaaaa 420
tccagaggag agggaaggga tgcctgagca cagatgggtg ggagaacacg ccccgaggtg 480
cagcgttggc acgagaagcc ctgtctgcga gccccgagga ggagatgcct ccccaagccc 540
tgtgcagaca gaagagtccg cccctccctc tcccgcacct gctgaacttt ggggtcagca 600
g 601
<210> 65
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
gctgcctgcg cggaaggagc cgcctccctc ctttcagcga acgcattcgg ggttcccccc 60
tcccgccaag tcctttgtct ggaaggggac tggcaggctc tgtcgtcgcc gtgtgccccc 120
ctcctttccc cagcctctac cccctccaac taactttcct ttcttccctt ctcttttctt 180
ccctctctgg atgggtaatc acaggctccg ctgccaagcg cgctgcgagg agccgctgcc 240
cgttcccgtc ggtgcccctc cccgcggcag cctggccgag tccgcccagc ctgcctagga 300
cgtccctgac ccgcgcgctc agtttcattc agtggccgtg ggatgttgca tctgagtttt 360
tcctttaggc caaaactttg tcccgaggtt tttctgagag ggttttattc tccaacagca 420
acaacaacat gcgaattctt aaaactagta tttatttcga ttcaggcagt cagggaaatg 480
gcagaatcac cagccgtttg cagaattcta cgctcgccaa ggcaagtact attagtatat 540
tttctcccaa cgttttcgtt gtaaaataca catgacaaaa tttgccctct taaccatttt 600
a 601
<210> 66
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
tagccctgtt taaaaaaatg gctgattaag ttgagtgcgc atgtctttgt gtgtctattc 60
ccgatatgca ctctgggaat gagagaggag agcgagggag agggagaggg agagggggga 120
gagagaggtg taattagtga ggtgatcaac attccccaga ctgcaaatga cgcgcagccc 180
ggactcagag cagctccgga gcctcacggc ttccaaagct cccagcgctg cggctggagc 240
cccggatgcg gcgaccgcgg agaggacagg ggatggggac ctccgggtct cgcaccctac 300
gcgcccctgc gcgcgactcc caaacttcat tagacacaca cgcgctctta cacacaaaca 360
cagacacaca cagagcaaga aggggaaaga aataaaacat agataccgcc aaagcatggc 420
cctttaacca ttcgagattt tttttttatt ttaatgaggc aaagtaatta ttatcggacc 480
agagatttgt ttactaggga gctttaagca gaaatatgag ttagggtaat gaagagcttt 540
tctctgccta gtttattctg ctgtgcacat gaatgcactt taatggcgaa atgcgggggc 600
c 601
Claims (14)
1. A methylated biomarker or combination thereof for breast cancer detection, selected from any at least one of the following methylated biomarkers: cg23035715, cg26371731, cg04541368, cg13973436, cg16304215, cg20072171, cg08402365, cg21501525, cg22778178, cg08599259, cg25566568, cg15634980, cg07458308, cg01348584, cg14140881, cg25756435, cg00594560, cg08279008, cg09760908, cg18087672, cg14868703, cg17632299, cg18786873, cg20631750, cg 25926, cg 24556128, cg 22978855, cg23524195, cg 208607, cg 3522435300, cg 254443, cg 143528, cg 8245528, cg 254543, cg 25067946, cg 227946, cg 797746, cg 7746, cg 2357946, cg 2087946, cg 20835, cg 2087746, cg 7246, cg 640426, cg 7746, cg 640426, cg 7246, cg 640426, cg 7746, cg 640426, cg 6446, cg 640426, cg 7279, cg 6427, cg 640426, cg 6427, cg 7279, cg 6427, cg 640435, cg 640426, cg 7279, cg 6427, cg 640426, cg 640435, cg 7279, cg 640435, cg 640426, cg 7279, cg 640435, cg 727968, cg 64.
2. The methylated biomarker or combination thereof for breast cancer detection according to claim 1, which comprises at least one of any of the following methylated biomarkers: cg23035715, cg26371731, cg04541368 and cg 13973436.
3. The methylation biomarker or the combination thereof for breast cancer detection according to claim 2, wherein the methylation biomarker or the combination thereof comprises a marker cg23035715, and at least one marker selected from cg26371731, cg04541368 and cg 13973436.
4. The methylated biomarker or combination thereof for breast cancer diagnosis according to claim 3, which comprises the biomarker cg23035715, and cg26371731, cg04541368 and cg13973436 of the following biomarkers.
5. The methylation biomarker or the combination thereof for breast cancer diagnosis according to claim 3 or 4, wherein the biomarker further comprises at least one biomarker selected from the following biomarkers: cg16304215, cg20072171, cg08402365, cg21501525, cg22778178, cg08599259, cg25566568, cg15634980, cg07458308, cg01348584, cg14140881, cg25756435, cg00594560, cg08279008, cg09760908, cg18087672, cg14868703, cg17632299, cg 86818773, cg20631750, cg25924096, cg 24615528.
6. The methylation biomarker or the combination thereof for breast cancer diagnosis according to claim 3, 4 or 5, wherein the biomarker further comprises at least one biomarker selected from the following biomarkers: cg22889755, cg23524195, cg20861607, cg22435300, cg25504443, cg14351528, cg25824543, cg23413809, cg24797187, cg26877715, cg01167274, cg12361223, cg04430835, cg27111970, cg22851944, cg 23228540.
7. The methylation biomarker or the combination thereof for breast cancer diagnosis according to claim 3, 4, 5 or 6, wherein the biomarker further comprises at least one biomarker selected from the group consisting of: cg26225694, cg07790615, cg14825633, cg11901043, cg 2404927, cg21962423, cg14135814, cg01070209, cg14841828, cg04947764, cg 013978141, cg00436496, cg01832036, cg27125093, cg21319323, cg06916239, cg11667451, cg03355998, cg19019849, cg 220488, cg15012484, cg20817483, cg21254450 and cg 23134869.
8. The methylated biomarker or a combination thereof for breast cancer diagnosis according to claim 7, which comprises the markers cg23035715, cg26371731, cg04541368, cg13973436, cg16304215, cg20072171, cg08402365, cg21501525, cg22778178, cg08599259, cg25566568, cg15634980, cg07458308, cg01348584, cg 141881, cg 256435, cg00594560, cg08279008, cg09760908, cg18087672, cg14868703, cg 32217617699, cg18786873, cg20631750, cg 254096, cg 245528, cg 889755, cg 24195, cg 208208607, cg 3561300, cg 3506352247743, cg 25927943, cg 6792357946, cg 67927746, cg 6792357943, cg 6792774092357935, cg 6792357946, cg 640435, cg 640477409235798, cg 6404774092357747, cg 640435, cg 640477409247, cg 640447, cg 64047747, cg 640435, cg 640447, cg 6447, cg 64048, cg 640435, cg 640447, cg 6447, cg 64048, cg 6447, cg 640447, cg 6447, cg 64048, cg 6447, cgc, cg 6405, cgc 798, cg 6447, cg 6405, cg 64048, cg 640435, cgc, cg 640435, cg 6405, cg 64048, cg 640435.
9. The methylated biomarker or a combination thereof according to any one of claims 1 to 8, wherein the marker cg23035715 has the sequence shown in SEQ ID No.1, cg26371731 has the sequence shown in SEQ ID No.2, cg04541368 has the sequence shown in SEQ ID No.3, cg13973436 has the sequence shown in SEQ ID No.4, cg16304215 has the sequence shown in SEQ ID No.5, cg20072171 has the sequence shown in SEQ ID No.6, cg08402365 has the sequence shown in SEQ ID No.7, cg21501525 has the sequence shown in SEQ ID No.8, cg22778178 has the sequence shown in SEQ ID No.9, cg08599259 has the sequence shown in SEQ ID No.10, cg 255668 has the sequence shown in SEQ ID No.11, cg 15615615634980 has the sequence shown in SEQ ID No.12, cg 07079208 has the sequence shown in SEQ ID No.10, cg 255646 has the sequence shown in SEQ ID No.16, cg 9488116 has the sequence shown in SEQ ID No.16, The sequence of cg08279008 is shown as SEQ ID NO.18, the sequence of cg09760908 is shown as SEQ ID NO.19, the sequence of cg18087672 is shown as SEQ ID NO.20, the sequence of cg14868703 is shown as SEQ ID NO.21, the sequence of cg17632299 is shown as SEQ ID NO.22, the sequence of cg18786873 is shown as SEQ ID NO.23, the sequence of cg20631750 is shown as SEQ ID NO.24, the sequence of cg25924096 is shown as SEQ ID NO.25, the sequence of cg15321298 is shown as SEQ ID NO.26, the sequence of cg22889755 is shown as SEQ ID NO.27, the sequence of cg 24195 is shown as SEQ ID NO.28, the sequence of cg20861607 is shown as SEQ ID NO.29, the sequence of cg22435300 is shown as SEQ ID NO.30, the sequence of cg 50254443 is shown as SEQ ID NO.31, the sequence of cg 5114332 is shown as SEQ ID NO. 2357932, the sequence of cg 266735, the sequence of SEQ ID NO.12 is shown as SEQ ID NO.12, the sequence of cg 267935, the sequence of SEQ ID NO.12, the sequence of cg 266735 is shown as SEQ ID NO.12, the sequence of SEQ ID NO.32, the sequence of cg 266735, the sequence of SEQ ID NO.12 is shown as SEQ ID NO.12, the sequence of cg 1767935, the sequence of SEQ ID NO., The sequence of cg12361223 is shown as SEQ ID NO.38, the sequence of cg04430835 is shown as SEQ ID NO.39, the sequence of cg27111970 is shown as SEQ ID NO.40, the sequence of cg22851944 is shown as SEQ ID NO.41, the sequence of cg 28540 is shown as SEQ ID NO.42, the sequence of cg26225694 is shown as SEQ ID NO.43, the sequence of cg07790615 is shown as SEQ ID NO.44, the sequence of cg14825633 is shown as SEQ ID NO.45, the sequence of cg 01011943 is shown as SEQ ID NO.46, the sequence of cg 2404927 is shown as SEQ ID NO.47, the sequence of cg21962423 is shown as SEQ ID NO.48, the sequence of cg14135814 is shown as SEQ ID NO.49, the sequence of SEQ cg01070209 is shown as SEQ ID NO.50, the sequence of cg 148cg 41828 is shown as SEQ ID NO.46, the sequence of SEQ ID NO. 0040049, the sequence of cg 04496, the sequence of cg 04353535353551 is shown as SEQ ID NO.49, the sequence of SEQ ID NO.4, the sequence of SEQ ID No. 0136397746, the sequence of SEQ ID NO.4, the sequence of SEQ ID No.11, the sequence of cg 0494419453 is shown as SEQ ID NO.11, the sequence of SEQ ID No.11, The sequence of cg06916239 is shown as SEQ ID NO.58, the sequence of cg11667451 is shown as SEQ ID NO.59, the sequence of cg03355998 is shown as SEQ ID NO.60, the sequence of cg19019849 is shown as SEQ ID NO.61, the sequence of cg22009488 is shown as SEQ ID NO.62, the sequence of cg15012484 is shown as SEQ ID NO.63, the sequence of cg 20817417483 is shown as SEQ ID NO.64, the sequence of cg21254450 is shown as SEQ ID NO.65, and/or the sequence of cg23134869 is shown as SEQ ID NO. 66.
10. Use of the methylated biomarkers of any one of claims 1 to 9 or a combination thereof in the preparation of a diagnostic kit for breast cancer.
11. A kit for breast cancer diagnosis, comprising a reagent for detecting the methylation state of the methylated biomarker or combination thereof according to any one of claims 1 to 9 in a sample to be tested.
12. The kit for breast cancer diagnosis according to claim 11, wherein the breast cancer is breast cancer of different stages.
13. The kit for breast cancer diagnosis by detecting cfDNA methylation according to claim 11, wherein the reagents are based on reagents used in pyrosequencing, bisulfite conversion sequencing, methylation chip method, qPCR method, digital PCR method, next generation sequencing method, third generation sequencing method, whole genome methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technique, HPLC method, MassArray, methylation specific PCR, or a combination thereof.
14. The kit for breast cancer diagnosis by detecting cfDNA methylation according to any one of claims 11 to 13, wherein the sample to be tested is blood, serum or plasma.
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| WO2021233329A1 (en) * | 2020-05-19 | 2021-11-25 | 广州市基准医疗有限责任公司 | Methylation biomarker or combination thereof for detecting breast cancer, and application |
| CN114317736A (en) * | 2021-08-19 | 2022-04-12 | 广州市基准医疗有限责任公司 | Methylation marker combination for pan-cancer species detection and application thereof |
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| ROSS等: "breast cancer biomarkers and molecular medcine", 《EXPERT REV MOL DIAGN》 * |
| 符德元: "乳腺癌相关基因异常甲基化的临床实验研究", 《中国博士学位论文全文数据库》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021233329A1 (en) * | 2020-05-19 | 2021-11-25 | 广州市基准医疗有限责任公司 | Methylation biomarker or combination thereof for detecting breast cancer, and application |
| CN114317736A (en) * | 2021-08-19 | 2022-04-12 | 广州市基准医疗有限责任公司 | Methylation marker combination for pan-cancer species detection and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2021233329A1 (en) | 2021-11-25 |
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