CN108315430A - Purposes of the PDL1 SNP genotype as prediction neoadjuvant chemotherapy in breast curative effect marker - Google Patents

Abstract

The invention provides the use of the PDL1 SNP genotype in preparing markers for diagnosis and prediction of neoadjuvant chemotherapy efficacy of breast cancer. Also provided is the use of the PDL1 SNP genotype as a marker in the preparation of reagents for diagnosing and predicting the curative effect of neoadjuvant chemotherapy for breast cancer. Also provided is a kit for detecting the PDL1 SNP genotype, which contains the primers shown in SEQ ID NO.1-18. The present invention finds that in all breast cancer populations and HR-positive populations, after the SNP rs4143815 C to G change in the non-coding region of PDL1 gene, patients with GC genotype are more likely to achieve pCR than patients with GG and CC genotypes. Through the detection of the SNP in the non-coding region of the PDL1 gene in the patient's blood, patients with the GC genotype can be detected as early as possible, and neoadjuvant chemotherapy can be performed in order to achieve complete remission of the pathology and improve the survival time.

Description

PDL1 SNP genotype as a marker for predicting the efficacy of neoadjuvant chemotherapy in breast cancer use

technical field

The invention belongs to the field of medical detection, and relates to a marker for predicting the curative effect of neoadjuvant chemotherapy for breast cancer, specifically the use of PDL1 SNP genotype as a marker for predicting the curative effect of neoadjuvant chemotherapy for breast cancer.

Background technique

Breast cancer is one of the most common malignancies in women worldwide. According to the latest global cancer epidemiological statistics (GLOBOCAN) report, in 2012 alone, there were 1.67 million new cases of breast cancer worldwide, and 522,000 people died of breast cancer. In the same year, the age-standardized incidence rate of breast cancer in China reached 21.6 per 100,000 people. Although the incidence of breast cancer in China is still lower than that of developed countries (as high as 100 per 100,000 people in North America and Northern Europe), it has been on the rise since the 1980s, and the growth rate is more than twice the global average. . In economically developed areas such as the southeast coast of my country, breast cancer has become the malignant tumor with the highest incidence rate in women.

The high incidence has attracted the attention of medical workers around the world to breast cancer. Over the years, the treatment mode of breast cancer has gradually changed from a single surgical treatment in the 19th century to a comprehensive treatment mode combining surgery, chemotherapy, radiotherapy and targeted therapy. Among them, breast cancer neoadjuvant chemotherapy (Neoadjuvant chemotherapy, NAT) has also become a routine item in the treatment of breast cancer. Neoadjuvant chemotherapy can not only reduce the tumor burden for locally advanced patients, but also provide opportunities for surgery and breast conservation. On the other hand, it can also evaluate the response of patients to treatment early, so as to adjust subsequent treatment and predict the long-term prognosis of patients. The pathological complete response (pCR) of neoadjuvant chemotherapy refers to the primary tumor in the surgical specimen after treatment, or the primary tumor plus axillary lymph nodes, with no residual tumor cells or only residual intraductal carcinoma. Several large clinical trials have confirmed that the prognosis of patients who achieved pCR in NAT is much better than that of patients who did not achieve pCR. However, not all patients can achieve pCR results in the same NAT treatment. Therefore, how to early predict the patient's response to NAT treatment and discover the predictive indicators of pCR has become the focus and hotspot of current research.

Programmed death-ligand 1 (Programmed death-ligand-1, PDL1) is a cell membrane transmembrane glycoprotein. The protein is mainly expressed on the cell membrane surface of T cells, NK cells, dendritic cells and tumor cells, and its function is mainly related to immunosuppression. Programmed death-1 (PD1) is mainly expressed on the surface of T cell membrane, and after binding with its ligand PDL1, it can cause T cell apoptosis and induce T cell differentiation to inhibit T cell. Congenital loss of PDL1 expression is often associated with autoimmune diseases such as systemic lupus erythematosus. The acquired PDL1 expression changes are related to the immune escape of tumors.

Basic experiments suggest that PDL1 is regulated and influenced by various cellular pathways, such as MAPK and PI3K/AKT pathways. In terms of epigenetics, PDL1 is also regulated by many miRNAs (Micro-Ribonucleic acid, miRNA), thus participating in the development of tumors such as epithelial-mesenchymal transition (Epithelial-Mesenchymal Transition, EMT). In addition, it is also reported in the research of gastric cancer and lung cancer that the single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) in the non-coding region of PDL1 is associated with both the risk of tumor incidence and prognosis.

SNP refers to the polymorphic change of a specific base in the genome. Unlike gene mutations, a mutation base at a site needs to reach a certain frequency (greater than 1/1000) in the population to be called a SNP. SNP changes in the population are often associated with disease onset, drug metabolism and efficacy. According to the distribution of SNP sites on the genome, it can be divided into SNPs in coding regions and SNPs in non-coding regions. SNPs in the coding region can be divided into synonymous substitutions and non-synonymous substitutions. The two can directly change the protein product or affect the translation process, respectively, and ultimately affect the protein function. Although SNPs in non-coding regions do not participate in protein synthesis, they can affect cell function by affecting various biological processes such as gene splicing, transcription factor binding, and mRNA degradation. SNP mutations in non-coding regions are often reported to be associated with tumorigenesis. The clinical value of PDL1SNP in gastric cancer and lung cancer mentioned above is reflected by the SNP in the non-coding region.

The applicant's neoadjuvant clinical trials (SHPD001 and SHPD002) of paclitaxel combined with cisplatin weekly therapy have obtained preliminary results, showing a high pCR rate. Among them, the pCR rate in the general population is 34.4%, and it is as high as 64.7% and 52.4% in human epidermal growth factor receptor 2 (Human epidermal growth factor 2, HER2) positive breast cancer and triple negative breast cancer. This curative effect is generally believed to be related to metronomic chemotherapy and immune regulation. However, in hormone receptor (Hormone receptor, HR)-positive patients, the pCR rate is not ideal, so we hope to find predictors of pCR in these patients, so as to improve the treatment effect. It has been suggested in the literature that cisplatin can increase the expression level of PDL1 in tumor cells, and this drug is also one of the chemotherapy drugs used by patients in NAT clinical trials in our department.

Contents of the invention

The purpose of the present invention is to provide the use of PDL1 SNP genotype as a marker for predicting the curative effect of neoadjuvant chemotherapy for breast cancer, which will solve the problem that there is no suitable biomarker in the prior art to predict the curative effect of neoadjuvant chemotherapy for breast cancer patients technical problem.

The invention provides the use of the PDL1 SNP genotype in the preparation of markers for diagnosis and prediction of the curative effect of neoadjuvant chemotherapy for breast cancer.

Further, in the PDL1 SNP genotype, rs4143815C>G.

Further, the breast cancer is hormone receptor positive.

The present invention also provides the use of the PDL1 SNP genotype as a marker in the preparation of a reagent for diagnosis and prediction of the curative effect of neoadjuvant chemotherapy for breast cancer.

The present invention also provides the use of the PDL1 SNP gene as a marker in the preparation of reagents for diagnosing and predicting the curative effect of neoadjuvant chemotherapy for breast cancer. The sequence of the PDL1 SNP gene is shown in SEQ ID NO.20.

The present invention also provides a kit for detecting PDL1 SNP genotype, which contains the primers shown in SEQ ID NO.2-19.

The present invention detects the SNP genotype in the whole blood sample of the patient before neoadjuvant chemotherapy, and if the change of SNP rs4143815C to G in the non-coding region of the PDL1 gene is found, it means that the patient is more likely to achieve pCR; thereby predicting that the patient has the potential for treatment. Sensitivity and tolerance. Patients with GC genotype were more likely to achieve pCR than patients with GG and CC genotypes; while patients with GG genotype were more likely to have DFS events and had poorer survival than patients with GC and CC genotypes.

Compared with the prior art, the technical progress of the present invention is remarkable. The present invention found that in all populations and HR-positive populations, after the change of SNP rs4143815C to G in the non-coding region of PDL1 gene, patients with GC genotype were more likely to achieve pCR than patients with GG and CC genotypes; patients with GG genotype were more likely to achieve pCR than patients with GC and CC Patients with the genotype were more prone to DFS events and had poorer survival. Through the detection of SNP in the non-coding region of the PDL1 gene in the patient's blood, patients with GC genotype can be found as early as possible, so that neoadjuvant chemotherapy can be carried out, so as to achieve complete remission of pathology and improve survival time. If it is found that the effect of neoadjuvant chemotherapy is not good, other means such as surgery are used to avoid overtreatment and delay the timing of surgery.

Description of drawings

Figure 1 shows the disease-free survival time of patients with different PDL1 SNP genotypes; Log-rank P value=0.01.

Figure 2 shows the disease-free survival time of patients with different SNP genotypes in the HR positive subgroup, Log-rank p=0.003.

Detailed ways

Example 1

In order to clarify the predictive effect of the SNP genotype in the non-coding region of the PDL1 gene (as shown in SEQ ID NO.1) on the sensitivity of breast cancer neoadjuvant chemotherapy, especially in order to clarify its predictive effect in HR-positive breast cancer; at the same time, observe Tolerance and safety of treatment in patients with different genotypes. We hypothesized that in all populations as well as in HR-positive breast cancer, the PDL1 SNP genotype could predict patient sensitivity and tolerance to treatment, and performed sensitivity analysis by detecting the SNP genotype in whole blood samples of patients before neoadjuvant chemotherapy .

1 Materials and methods

1.1 Main Instruments

1.2 Main kit consumables

1.3 Enrolled patients and sample collection

All the selected cases come from the patients enrolled in two clinical trials of the Breast Surgery Department of Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine. Two clinical trials are registered on the ClinicalTrials.gov website as SHPD001 (NCT02199418) and SHPD002 (NCT02221999), respectively. Both groups received a weekly neoadjuvant chemotherapy regimen of paclitaxel combined with cisplatin, and SHPD002 patients were randomized to receive endocrine therapy at the same time.

Whole blood samples were collected from patients before treatment for subsequent SNP detection. The basic information and tumor data of the patients were collected and counted, including patient age, menstrual status, family history, primary tumor size before operation, biopsy specimen estrogen and progesterone receptors, biopsy specimen HER-2 receptors, adverse reactions during chemotherapy, and whether or not PCR. Both clinical trials have been approved by the Ethics Committee of Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine. All patients signed informed consent, voluntarily enrolled and provided specimens and clinical data for relevant medical research.

1.4 SNP site selection

Through the public database (NCBI/TargentScan), the detected SNP sites were selected according to the following criteria: A. rare allele frequency (Minor Alleles Frequency, MAF)>0.05 and B. located in the 3'UTR region of the PDL1 gene, or located in Other non-coding regions of the PDL1 gene have however been reported to have predictive or prognostic value in other tumors. A total of 9 SNP sites were selected. The DNA extraction and SNP genotype detection of the specimens were all completed by Shanghai Yuzhi Biotechnology Co., Ltd.

1.5 DNA extraction: TIANamp Genomic DNA Kit kit was used.

A. Add 20 μl Proteinase K solution to the sample blood (200 μl) and mix well.

B. Add 200 μl buffer GB to the mixture, 70°C x 10min.

C. Add 200 μl of 100% ethanol to the mixture obtained in the previous step, and shake for 15 seconds.

D. Add the above solution into an adsorption column CB3, centrifuge at 12000rpm for 30s, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

E. Add 500 μl of buffer solution to the adsorption column CB3, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and put the adsorption column CB3 into a collection tube.

F. Add 600 μl of rinse solution PW to the adsorption column CB3, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

G. Repeat operation step F.

H. Put the adsorption column CB3 back into the collection tube, centrifuge at 12000rpm for 2min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to dry.

I. Put the adsorption column CB3 in another clean centrifuge tube, add 100 μl of elution buffer TE dropwise to the middle of the adsorption membrane, leave it at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 2 minutes, and collect the solution in the centrifuge tube.

1.6 PCR reaction

The following primers were designed online by Assay Design Suite V2.0 Online.

Primer sequence:

A. Taking a 384-well plate as an example, add 1 μl DNA sample to each well, centrifuge and set aside.

B. Carry out PCR with kit of Qiagen company, reaction system is as follows:

C. Add 4 μl reaction system to each well of DNA sample, put the 384-well plate into the PCR instrument, and the reaction procedure is as follows:

D. After PCR, the 384-well plate is ready for use.

1.7SAP processing

A. Use the SEQUENOM kit to configure the SAP reaction solution, the system is as follows:

B. Add 2 μl of SAP reaction system to each well of the 384-well plate PCR reaction system.

C. Perform the SAP procedure as follows:

D. After the SAP is finished, the 384-well plate is ready for use.

1.8 Extension reaction

A. Prepare the iPlex reaction system with the SEQUENOM company kit, the reaction system is as follows:

B. Add 2 μl extension reaction system to each well of the 384-well plate SAP reaction system.

C. Carry out the extension procedure as follows:

D. After the extension, the 384-well plate is ready for use.

1.9 Product purification

A. Put a little resin on the 384-hole resin scraper, scrape off the excess resin to cover it evenly, and place it at room temperature for 20 minutes.

B. Centrifuge the extended 384-well plate at 1000 rpm for 1 min, add 25 μl of deionized water to each well, and place it upside down on the resin plate. Buckle the resin plate backwards, let the resin fall into the 384-well plate, and seal the film. C. Take the long axis of the 384-orifice plate as the axis, turn the 384-orifice plate over for 20 minutes, and centrifuge at 3500 rpm for 5 minutes for later use.

1.10 SNP genotype detection

A. Nanodispenser SpectroCHIP chip sampling;

The detection samples were transferred from the 384-well reaction plate to the MassARRAY SpectroCHIP chip covered with matrix.

B.MassARRAY Analyzer Compac mass spectrometry detection;

After the sample is transferred to the SpectroCHIP chip, it can be put into the mass spectrometer for detection. It only takes 3-5 seconds for each detection point, and the analysis is fully automatic.

C.TYPER software analyzes the experimental results and obtains typing data.

1.11 Statistical analysis

Hardy-Weinberg equilibrium of SNP genotypes was done by chi-square test. Linkage disequilibrium analysis among different SNPs was done by HaploView software (http://broad.mit.edu/mpg/haploview). When analyzing the SNP genotype, a total of four models were established: Assuming that at a certain SNP site, "T" is a common allele and "A" is a rare/mutant allele, then the dominant model compares TT vs TA+AA difference; recessive model compares TT+TA vs AA difference; co-dominant model compares TT+AA vs TA difference; additive model compares TT vs TA vs AA difference.

The correlation between PDL1 SNP position and side effects was calculated by Spearman correlation analysis; the correlation between different genotypes of PDL1 SNP and pCR was analyzed by logistic regression model for univariate and multivariate analysis. The present invention uses disease-free survival (DFS) to evaluate the survival status, which is defined as the occurrence of any of the following events from surgical resection of the lesion: 1. distant metastasis; 2. local recurrence in the same side region; 3. contralateral breast cancer; 4. die. The comparison of survival curves between different groups was drawn by the Kaplan-Meier method, and the differences in survival rates were compared by the log-rank test. The COX risk model was adjusted for multiple factors, and HR and 95% CI were calculated.

The present invention analyzes the data through STATA 12.0 and SPSS 23.0, and the bilateral p<0.05 means that there is a statistical difference.

2 Experimental results

2.1 The predictive value of PDL1 SNP expression

The present invention detects a total of 9 SNP sites in the non-coding region of the PDL1 gene, and the position and genotype frequency of each site are shown in Table 1. Among them, rs7864231 and rs10217310 were not detected as mutants, so subsequent analysis was not carried out in this paper. The remaining 7 loci were tested and all conformed to the Hardy-Weinberg equilibrium, so they were included in the follow-up analysis. Through multivariate logistic regression analysis, we found a SNP site in the non-coding region of PDL1, the cumulative model of the change from rs4143815C to G (rs4143815C>G) (OR1=9.284, p1=0.049; OR2=13.501, p2=0.023) and total A dominant model (OR=2.800, p=0.042) was associated with pCR in patients treated with neoadjuvant therapy (Table 1). By further calculations, this altered dominance model was also correlated with PR expression (p=0.043).

Table 1 Relationship between different SNP genotypes and pCR

a: p<0.05, there is a statistical difference.

b: Multivariate analysis adjusted for age, tumor size, ER, PR, HER2, and ki67.

2.2 Prognostic value of PDL1 SNP expression

At a median follow-up of 18 months, patients with rs4143815C>G (shown in SEQ ID NO. 20) exhibited different DFS. Among them, the Kaplan-Meier plot showed that the survival of patients with GG genotype was worse than that of patients with GC+CC type (Log-rank p=0.01; Figure 1). The COX multivariate risk model also suggested that patients with the rs4143815GG genotype had a higher risk of recurrence (HR=20.098, 95% CI 2.774-145.596; p=0.003; see Table 2 for details), which was an independent predictor of DFS. In addition, the patient's age (HR=0.125, 95%CI 0.024-0.656; p=0.014), ki67 status (HR=1.050, 95%CI 1.015-1.088; p=0.005), postoperative lymph node status (HR=9.750, 95% 1.894-50.194; p=0.006) was also an independent predictor of DFS.

Table 2 Relationship between PDL1 SNP genotype and survival (COX multivariate risk model)

a: p<0.05, there is a statistical difference.

2.3 Side reaction analysis

The adverse reaction analysis of patients with different rs4143815 genotypes found that CC+CG patients were more prone to neutropenia (rho=-0.183, p=0.032) than GG patients, but less prone to edema (rho=0.179 , p=0.036) adverse reactions. Compared with CG patients, CC+GG patients were more likely to have elevated aspartate aminotransferase (rho=-0.186, p=0.030). The specific adverse reaction spectrum is shown in Table 3.

Table 3 Side effects of patients with different rs4143815 genotypes

Table 3Adverse events according to different genotype of rs4143815

a: p<0.05, there is a statistical difference.

2.4 Subgroup analysis

In subgroup analysis, we observed similar conclusions in HR-positive breast cancer as in the overall population.

In terms of predicting pCR, multivariate logistic regression analysis in HR-positive breast cancer showed that rs4143815C>G patients with GC genotype were more prone to pCR than GG+CC genotype (OR=2.362, p=0.022; Table 4).

In terms of survival, the Kaplan-Meier plot of HR-positive breast cancer showed that the survival of GG genotype was worse than that of GC+CC group (Log-rank p=0.003; Figure 2). The COX multivariate risk model indicated that rs4143815C>G patients with GG genotype had a higher recurrence risk than GC+CC type (HR=10.781, p=0.008; Table 4). The predictive value of PDL1 was not observed in HER2-positive BC, and a valid subgroup analysis could not be performed due to the small number of TNBC breast cancer patients.

Table 4 Relationship between PDL1SNP genotype and pCR and DFS in HR positive subgroup

a: p<0.05, there is a statistical difference.

3 Conclusion

In the present invention, we discuss the predictive value of pCR of neoadjuvant chemotherapy and the long-term prognostic value of different genotypes of PDL1 SNP in patients. This is the first report on the predictive and prognostic value of PDL1 SNP genotype for neoadjuvant chemotherapy in breast cancer patients and the first report of PDL1 SNP genotype in HR-positive breast cancer. It is also the first time that a relationship between PDL1 SNP genotype and side effects of neoadjuvant chemotherapy has been observed.

We found that in all populations and HR-positive populations, after SNP rs4143815C to G in the non-coding region of PDL1 gene was changed, patients with GC genotype were more likely to achieve pCR than patients with GG and CC genotypes; patients with GG genotype were more likely to achieve pCR than patients with GC and CC Patients with the genotype were more prone to DFS events and had poorer survival.

Rs4143815 is located in the non-coding 3'UTR region of the PDL1 gene. Although SNPs in non-coding regions do not participate in protein synthesis, they can affect cell function by affecting various biological processes such as gene splicing, transcription factor binding, and mRNA degradation. The present invention discovers the clinical predictive value of rs4143815. On the one hand, the gene polymorphism changes at this site cause changes in the expression of PDL1 protein, thereby affecting cell functions. Under the induction of IFNγ, the expression of PDL1 protein on the surface of macrophages/monocytes in rs4143815CC genotype carriers was much higher than that in GG genotype individuals.

sequence listing

<110> Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine

<120> Use of PDL1 SNP genotype as a marker for predicting the efficacy of neoadjuvant chemotherapy in breast cancer

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cactaagagg gaaaacttac ctctttctta tcaaagtttg taaatatatg tataacagaa 4680

gagggagaaa atattaataa atgcacagat tggctgaaat agagtataaa tcttttactc 4740

ccctacttca acataaactg caaaaggaga gtgacttttc tttcactctg acttccgtat 4800

tcctcatgct taaaatagtg cctagcacag aagaggtgct caatcagtgt ttgctaaacg 4860

aaataattag tcacatttca agcaggatga ctaaatgaag aatagaatct aggcagatac 4920

tctggaagag tggctgtgag tcattcatat cttagtatga attagtcaaa tccaactctc 4980

tccccttccc actccccact gttagtagaa gaatctgttt attgagagaa tagatttata 5040

atttagaata agtgagaggg gcagaagagg agatttgaa ggatggcacc tgaaggagga 5100

ctagcatggc tgagacagtg aagtggaagc cttgaatagc taaagggtaa gatgaaagta 5160

tttagctgta gggggaaaaa gcattgacag gttggaaaag taaaagtcag attctccttg 5220

ctctgaaatt ttgtacaggg caggttctac taggtatgtt acaatgcaga aaaaacatga 5280

aataattgag aggaatttgg tgcaatatta tcttcttggc ttcttttgag tgggcagatt 5340

tttttcacgg cctgtaacta taataaattt gaaacttctc atcttttagt aacttttttc 5400

acttaagttt atgtggctgt gggcaatgga atgaagatat tgaacttcca attccctgtt 5460

gggtttccac aattacaagt caatcatgac tggttattag aagactattt cagttagaac 5520

caccaagtcc catattgtca tattgtatgt ttaattatta agtgaagcag tcttcttttc 5580

gtgttttcca taattagggc attccagaaa gatgaggata tttgctgtct ttatattcat 5640

gacctactgg catttgctga acggtaagac accaaatcct tccattaggt tctatatttt 5700

aaatatttta accatgagtt taaaactaaa atgatcattt aaaatgcatg caattttctt 5760

atagagagaa cattctattc tttcttctac tttacacaat ggcaaagtct tctttctact 5820

ttacgcaatg ataaagttac ctgtgtcatt ttgtaaaaat atagagaata tagacaaatt 5880

gaaagacaca aaataatcta ttacccattt cccagggtta actactgaaa atatctgggg 5940

aaatggcctg tatgtataca tttatttgtt tgctttcaac aaggccaaga tcctttgatc 6000

tttcagtctt ggttgctctg tgacatgcct ttcctgatga ggatacttta aggaagaatt 6060

gtaagataca tggaaaatgt caggctaaca cagtactggc atcaccctgt gctctttcct 6120

gaactccata ccaatgtact tcttgccaga aaactgatca aaagtttagg gaagtaaaaa 6180

gagatgactg ttagaatcta ccattccctc tatgtaggaa gcaaataggt gtcctgtcaa 6240

aggacattct ggggatgtct acatgaaacc aagtctccct ggttgtaagg actccatctc 6300

catataatat ttatacagta atatatgttt ataaattgtg ggggcaactt gtttagctaa 6360

ttttattatt ctgctattgg gacactgtgt ctcagcatga gatatagtgt cccaaaacat 6420

atttcaagcc cattggataa aatatgtgtt tagcaagttc ttaaatataa tgataacata 6480

accgaccaga taaagtgatt tataaacgct gtgccaattt tgtaaatgtt tcgaggaatt 6540

ttcccttttc tgaagattgt ccttctttct ttttagcatt tactgtcacg gttcccaagg 6600

acctatatgt ggtagagagtat ggtagcaata tgacaattga atgcaaattc ccagtagaaa 6660

aacaattaga cctggctgca ctaattgtct attgggaaat ggaggataag aacattattc 6720

aatttgtgca tggagaggaa gacctgaagg ttcagcatag tagctacaga cagagggccc 6780

ggctgttgaa ggaccagctc tccctgggaa atgctgcact tcagatcaca gatgtgaaat 6840

tgcaggatgc aggggtgtac cgctgcatga tcagctatgg tggtgccgac tacaagcgaa 6900

ttactgtgaa agtcaatggt aagaattatt atagatgaga ggcctgatct ttatgaaaa 6960

catattccaa gtgttgaaga cttttcattc ttgtaagtcc atacttattt tcaaacagaa 7020

cagcatagtc tgttcattca ttcattcaat tcatgaattc attcacataa ttatccaatt 7080

tcttgagcac ctatttgata gtcactggaa atccagagac aaacaacaca gagccatgtt 7140

ctacagtatg tacagttttc caaaaagaat ttctagtctt tactttttta ttacaaatgg 7200

aatacgtata cttgcaaata attcagatac tgtggaagag atcaaatgaa ttgcaaaagt 7260

gtccctcctc ccttcaccac tatctcccat ggcatgcaga gagagtaacc attatttgtg 7320

tgtccctcca gaaatttttt tattcaacta ctattttttt attttattag gtccgtcagt 7380

tttccttttt tgagcctctc tatatcaaat gcaaataaat atattcagaa caaacccccac 7440

tgtaaggttc acattaaaaa agacttgaag tcaccctatg aagacaaaaa ataatcacat 7500

taagtgtgaa agaacctatt cttccagtac aggataagcc atacttactg ggcatatatt 7560

catcttgaaa atctatactg atgttgtctt ggggaattga aaaggaacta ggagtgttag 7620

ttcctcggta ttgacccaca gttatgttat caggtcactt gagttcaaag ttttgtgttg 7680

gcactagcta agtaaaggaa aacacctctg ctttcattgt tgagtttcac agaattgaga 7740

gctgaaagga tcccaggcag gagcagctaa tccaaactcc cacaaagaac aaaaatcccc 7800

cagaggatct tctgttctta tatttcctgc aatggcgtcc ctgtcatatc ccacaatggc 7860

ctccctgcca tttggatatc ccttccatat cctgttgaaa ttactcccta atagtaagct 7920

gaaatctgcc cctctagttg tagtcttggg attatttcat ttacatgatg accttttaat 7980

atttgactag aattaaatca tctcccccttg gtctttccat tcctgggcta actaccatca 8040

atctgagggc taacaataca agtagaaaaa gtatacattt gtcactgatc actgatcaat 8100

tattaatcaa tgatcactga taactataaa ctcaaaaaca aaatcatgtg gggattaaga 8160

gaaatgtatc agttttatgt tgtatttctg gtccctgata ctggctcagg taatgccact 8220

attgtcaaga agataccact tgtaaagtag atttaatttt catttatattt taccatatgc 8280

ttctccattc atgacatctc ttgagatgtt gtggtttata ctttcagttt ttctccagtc 8340

catccgcaaa tatcaggcat ctactgtgtt ccaagatatt aaagaaatca tcatgactta 8400

gcctcatcaa cagcattgct agatctggga tggaaaggaa gagtataatc ctggcagtca 8460

ggaagaaggc agcataaagt ataagtttct gcttccaaaa aaggtctctc atcagcctgt 8520

agggagtgtg tagggaaggg acagctgtcc ttgtagtagg gaagggtttt attcaggtcg 8580

tctgggctcc ataatatccc ttgtgtatct gcagtctcct ttgccatgga tcaacacaat 8640

aggaaatctt ccggcactga tggtttttcc aagggggagt tcttcctgga gcaaagcaaa 8700

tgaccaacca ggtttgagga cctgatttgt ttgacaattc cattttgtat tgtaaattac 8760

ttaattggca ttctactccc aatccatctt gtcatttgca tacagtggtt ttgggattga 8820

gttcagctat accaaaagtc tgaaccttct gcacttagaa caaggcaacc accaagcttc 8880

acttgcactg aggccgtgtc tccaatggaa atgaggcagc tggcttgcag gagcttccca 8940

actcagggaa gtagaactcc tgagtcacct ccatatgcaa atgatttcac agtaatgctg 9000

ttgaacttca cttcccatca cagcaaatgt gtggtaacat agcttcccca caggagttta 9060

ctcaccatgg tattttaaag gtgaaacatt tcaaaactga aatttgaaag aatttagttt 9120

tggattcact caattatcac tatcacttcg ggtgttattg cacctttctt gtttgtgagt 9180

ttaaatgcca gactctcagg ccactaactt tcaattaaaa gtgtttttct ttaatcgctg 9240

aacctaacag cagggaaaac gaaatgttca ttcagacttt cagaaccttc aatgagatta 9300

ggcagctgaa agatcaaagt gttgcatagt tgtcccgata aagctatttg gatcatatgg 9360

accaaatcga ctgctgtcat tccccaccaa ccccatctct ccccaaaatt cccagccctg 9420

tttaagtgtt ctctgtagca tttatctcta tctagtatat tgtgtagcat atcatatcat 9480

acttttctgt tttgtttatt gtctctctcc tcctagaata taaactccac aagcacaaag 9540

atttgggcct gttttataat attgttgcat ccccagggcc tgatatacag cagagtggtg 9600

gtacgaaaag agcacacaaa aaaatatttg ttgagtcaat gaatgaatga tttcctcaaa 9660

taggattagc ctaaaatttt ggaaacatga acagatttgg atatgtgaaa atttatttcc 9720

agactgttca tcaggaactg ttagcagctt ctaaagggta cactggagca gcagtagtaa 9780

aaggaggaag aggagcagct ctgctactgc tactatcgag tactactaca attagcactt 9840

gcttattctg tgtgttaggc cctgtactga acactctgtc taaattagtt catttcctcc 9900

tggaaatgac tctagggggt aagtgcttca tcatgtaaga tgagtatttt tcacattttg 9960

ttgtgtctga aatctgagtg tgtctttcaa tgatggaatc tttgattcca tgataagtgg 10020

tattattccc attttaagga tgaggaaact gaggtccaaa gaaattaagt aatttgccca 10080

aattcaccca gcctagaaaa tgataaagct agttctaaac ccaagcagat tagctctgaa 10140

gtctgggccc ttaataacca ctttttatg cctatatttg tacctctggt gtacgtatca 10200

agttatatgt tgacttcaaa actatcatga ccttttcttg gttttgattg tccaacatta 10260

gtatagtgtt ctgggtctgc aaaaattttg attackcatc tcatctgtaa aacattttga 10320

actcgtgtgtttgtgcatgc aatttgtgtgt gtaattataa aaatttact ttctgttaat 10380

atataagttg tatcataaga aactgccgtt tttgaagagc aaaaaaaggt tgaatgttac 10440

cagttacatc tggttcaacc taatagacat ttgtacaaaa acagacattt taagaggttg 10500

aaataaaaat ttaataaaca atattttcag tttttactaa ttgtgatgct tcactatcat 10560

tagctaatat gtcaaggcat aatatacctt agggtgaact ttatcattaa caaaggtgga 10620

tggtgtcaat aatcttgagg tttgtgtttt tttatataac actgcgaggt ctaattaagt 10680

acttactgtt taccacctca tacagtggcc gataaaaagt gtcacttctg ctgtttcctc 10740

tgggttgtgc ttgaattatt agtattatct tcagtcctca gtttctttgt gggaaacttt 10800

ttaattagtt gtttaatttt gtaagatggt tagtttagtc aaaattagat aagagaattt 10860

gaaaatccgt agctacccca aagcaaccta cacataagaa ctattatttttgtgttttga 10920

aatcataatt ttattgattt ccagtgtttc cactggtagt ggtttcattg atataggagt 10980

atcaaaacat cactcattat ttatttcagt ttcatttgat cctagccgtt ttgtattaac 11040

tctctgtgaa gaaattacct cacaaatcta ttgctgtcct tggtaaagga atggagaatt 11100

aaggctctag atcattagtg gttacactat agtattagaa gtaaaaaaaa gattatacca 11160

acaaaataag aacatgttaa tgtacttgta atgaataaac atgaataaag ctcttatgct 11220

atataggtgc actaaacaat ctactagaat tgtcagcaaa ctacgtatct taatcctgaa 11280

agggtcccaa accaatgatc taaaattgaa tcaaactttc ttccttgagc ataattactt 11340

aaatgattta ttaaaatagc cagcatttaa aagcttaaaa tgtaaatatc ataatgtggt 11400

atcctagata gcatcccaga acagaaaaag gatattaggg aaaaactgga ggaatggaat 11460

aaattatgca gtttagttat taataatgta ctaacgtcct tagttatgac gattgtacca 11520

tggtaatgta agatactaac aatagaggaa accgggtaag gagtatacag taactctata 11580

ctatctttgc aacttttttg taaatttaaa acttctaaaa taaagaacaa atttaaacat 11640

taaaaagtat caccaggaac atatatcact gtttacagat gaaatactat gtattttcat 11700

atctaatttc tgatcattga cttcaaatca gaaaagtgaa tgacacctca aaatcaggtt 11760

ttctgtttac tgaagtctaa gaaaagaaag cataccagct ggagagattc atgtttataa 11820

agacagattt ataacaacaa aaataaaata tccaagaata aatttaagaa gaagcacttt 11880

actgagaaac atatgaaaac ctgaacaaat ggagagggat attttgtatt tgaatagaaa 11940

gacttctggt ttaaagataa ttctctttaa attatttttt gtagaaattt aaggggtaca 12000

agagcagtgttgtcacatgg atatattaca tagtggtgaa gtctggggtt ttagtgtaaa 12060

ttaatcttta cattttgttt gagcccaata aatgtaccaa catgattttt atagaaagat 12120

agtcattcct attaatccaa acttgtccca actttgaatt gaattgaggc agagctagca 12180

ggtgttcccc acggctgagg catctgaaca ttaagcatat ccctctgaga accagcctgc 12240

attgatactc tttctaatgt ggacagcatc aagctatgta cgtagttctg tgctcagcaa 12300

aagccctgac ttctttttgt ttatgtccta gccccataca acaaaatcaa ccaaagaatt 12360

ttggttgtgg atccagtcac ctctgaacat gaactgacat gtcaggctga gggctacccc 12420

aaggccgaag tcatctggac aagcagtgac catcaagtcc tgagtggtaa gaccaccacc 12480

accaattcca agagagagga gaagcttttc aatgtgacca gcacactgag aatcaacaca 12540

acaactaatg agattttcta ctgcactttt aggagattag atcctgagga aaaccataca 12600

gctgaattgg tcatcccagg taatattctg aatgtgtcca ttaaaatatg tctaacactg 12660

tcccctagca cctagcatga tgtctgccta tcatagtcat tcagtgattg ttgaataaat 12720

gaatgaatga ataacactat gtttacaaaa tatatcctaa ttcctcacct ccattcatcc 12780

aaaccatatt gttacttaat aaacattcag cagatattta tggaatatac cttttgttcc 12840

atgcattgta gtactcattg gatacacata gaataataag actcagttca cactcttcag 12900

gaaacagata aaaaactaag aaacaaacaa aaaacaggca atccaacacc atgtgggaaa 12960

tgctttcata gccgggaaac ctggggaata cctgagagga atactcaatt caggccttgt 13020

ttcaggaatc caaatcctgg cacatcagag ctgcttccct ctttccaggg tggcaggaaa 13080

taaatggaac atatttttct atcttatgcc aaacatgagg gaccctttct ccccggtgcc 13140

tctcccaagg tagtctacaa tatttcaact ctagcagtct gcttagtgca tagaacatga 13200

ggctgtgtgt ccctgggcaa attacktagac ttctgtgtgc ttcactttcc ctgtaggatt 13260

ataatctact gagcaagctt attgtaaggg tcagattagc aacagtgtat gaaaatgatt 13320

tgagaccatt gcctgcacaa attcaactat ttttttttat ctcactactc tacagaagta 13380

ggtagggtgg gagacagagt ctgatgagag gctcagaatg tgaaagaaag tgaggcgagt 13440

gagcatgata tttaatataa acacaaagat attctgagaa gagctgctca ctgccccctc 13500

ccccaataca tgttgatagg aaaatgccac gtacttcagc aaaaacaact gaaaaattag 13560

atagaaaagt caatcaatag gaaaagataa tccaggacgg tgttgtgaac agaaagaggg 13620

ggaaaaaact ttagaaaatg atggggatgc tcttactggg gtacgagtcc tcaggtattg 13680

aactggcttt cagtaaaagc tagattagtg ggttcctgcc atttacaagc tgttttatga 13740

caacttactt gttgggtggc ctacagtaac tcacctaact gcactgagtc tgtttcctca 13800

tctgtaaatt ggggattttt ttttaaatac ctggcatgcc taactcataa agttgttctg 13860

aaactgaaat aaaacatacg tgaacaggca ttgtaaactg taagttacgg aaaaagctgg 13920

ctgttgttgt gtctttaaag tttcacctgg gtagtcaaag atggatcatg ggtctcagtg 13980

gagagctgag ccaggcagga gctgactaag ggtgagaggt gggagttagc agcctctgaa 14040

catctgtgta ccatgggacc ccctttcctc ctgcatggta ccccagacaa ggagcctagt 14100

aagagatact aatggcttgt tgtccagaga tgttcaaact gcagagaaag ataagacaac 14160

aagcattggc ctccaatcat gatgacagat aggaggaggt gggagctcct tagcagtgct 14220

ggttggcctt ccatgttcta ctgtgggcca tctctgccat gtactgtagg ctactagctt 14280

ctatattaaa gaatgcaaga ggggccagga gcggaggctc atgcctgtaa tctcagcact 14340

ttgggaggcc aaggtggggca gatcacttga ggtcaggagt ttgtgaccag cctggccaac 14400

atggtgaaac tctgccttta ctaaaaatat aaaaattagc tgggtgtggt ggtgtgcacc 14460

tgtaatccca gctactcggg agactgaggc acaagaattg cttgaacctg ggaggcggaa 14520

gttgcagtga gcccagattg cgccactgca ctccaccctg ggcaacagag aaagactctg 14580

cctcaaaaaa aaaaaaaaaa agcaagagga agtgaaataa tcaaggccgc catttaatag 14640

tgagcagcca ctccatgtgg tactgtgcaa gcacattta aatattagcc tcacaagaaa 14700

tgtattagca tttgtatttt gtacactggt taagtatctt gcccaagacc tcaaaactgg 14760

ttaagggcag cagaatttag ccccagcacc accttttcaa agcctgggct tctcacactt 14820

ctccatgctg ttcccatttt aacacaggta tctcgccatt ccagccactc aaactttggc 14880

atttaagaaa attatcctaa agctaaacta aacttcaagg atgaccattc tcctgacccc 14940

ttcccatcaa aattttatct ttagtcagtt tgttttcgtt ttgttttgtt tttcagaact 15000

acctctggca catcctccaa atgaaaggac tcacttggta attctggggag ccatcttatt 15060

atgccttggt gtagcactga cattcatctt ccgtttaaga aaaggtagta tttccttaat 15120

tgcagtggtc tccactgggg gtgaggaagg ggtgagaatt ggatcatggc tgcaaggaaa 15180

cccgacttaa cctctgcaag gtggtgcaaa ggcattccac tgttcaacag caattatatt 15240

gaagctgagt gggatcactg ggtgaagatg aagcgtaagg ggtgaggggc aggagaatgg 15300

gtatggatgg aggtagaaga tgcagtgtca tacagtttttttctatcatg aaaataacca 15360

cagacttaca gaagagaaag agctaaaatg cccgtcattt tcagttgcat tttagtcttg 15420

cattagttgc aaccagctgg tttctgggta ccctaagtaa taaaaatagt tcctctgtag 15480

aactgtagta tgtttaccat agagtatttt gcaaaatttt tggtagagga tgttacataa 15540

tttgcatgtg ttcatttctc catttacctg tgggaacaat taaaatccag gaaaatgagt 15600

atattcaaat aatttcctcc catttaagat gagtcagagt aaataattcc tccaatactt 15660

agagaagtat accaagagat ccagtgatgg tatagagttg tctgatgtta aatagggaag 15720

tagaatatgg aaggggattc caatagtcgt tgaaaaattc cccataaccc cttacatggg 15780

ggaaagtagt gttaactgag agagtagaga taagctgttt ccaaaaatta tattcttaac 15840

aggactgaga tagccagaat ataaggatca agtttcaatg acagtaagat cctgagatgg 15900

agttgatttg cacaaagaaa taattgttgc cagcatgcat tttgaatatt tctctggaaa 15960

aaaagattag ttggcagtag aaatggatag aaatcaatag atattaaaat acctcagaat 16020

ttggttcatc tctgggaaaa gatgaaaaat aaaagtgtat actcctcaag aacatctagg 16080

atcaaaagca tgtgccctac actattgaat taattaacct cataagttgg gacctgtgga 16140

ataaggatgt ccaccagact tcctagggat tacaaatgtt tcacagaact tgaaatttaa 16200

acttgggtca ctgtatggga tgtagagctg tgctatatgg aaataaaaat gatttctttt 16260

tctcaaggga gaatgatgga tgtgaaaaaa tgtggcatcc aagatacaaa ctcaaagaag 16320

caaagtggta agaatatcag aaggaattgg gaagtaaaag tcaaaggaaa caaaaagcta 16380

aagcaataac aaagagaaat ccatcagtca taatctcctc tccttttaaa gaatgctggt 16440

tcccctttgc ctcacagcta acacaagaac tcctccaccg tctgaggagg tttaggagca 16500

gggaagggga aggagtcagc ttcatttgct aatcttctgt tgccctgcac cctagcagct 16560

ccttgcagca ggggacaagg atgacttagg tggatggata attaattgat tctaaaatat 16620

tgtgtgtcag tattgtaata ctatgttaat tgcaccatgc acggtatctc atttaatccc 16680

ccacccccttg ccattaccaa agagagagag agagagagag agagaaatac tagaatttat 16740

cctcatttta cagtagagaa aacagagggt caagaagata atgtaaagtg cccaagaaca 16800

cacagctgat cacaaaaatc aagcttgggg gccattagcc taaccacaga ccctactct 16860

taaccccatct gcttcaatcc attttgctac aaatgtttaac atttataagc agggcagaaa 16920

aacctcatcc aggttatga actaagaaga aagttatatt aaggtttcta atttttttaa 16980

tgtagttaga aaccaaactt aacaatgagc ccaagtttaa agcagtctaa ttaacctgga 17040

caagctcagg caagtttcat tctgtggccc atagcatcat ctgtgttgta aagctaagta 17100

gcaaatgttg tttgggtcat gctgggggac aagccatccc aatttgctca ggactgaggg 17160

gttttccagg atatcatgta aggataattg ggtacaaata taacctgctg ctttctctca 17220

tttcaaattt atcatttatc atatcagcaa ctatgagtta tgttttttat tagatttctt 17280

gttacttttt ccccagacca cttcccatga aattaatata ctattatcac tctccagata 17340

cacatttgga ggagacgtaa tccagcattg gaacttctga tcttcaagca gggattctca 17400

acctgtggtt taggggttca tcggggctga gcgtgacaag aggaaggaat gggcccgtgg 17460

gatgcaggca atgtgggact taaaaggccc aagcactgaa aatggaacct ggcgaaagca 17520

gaggaggaga atgaagaaag atggagtcaa acaggggagcc tggagggaga ccttgatact 17580

ttcaaatgcc tgaggggctc atcgacgcct gtgacaggga gaaaggatac ttctgaacaa 17640

ggagcctcca agcaaatcat ccattgctca tcctaggaag acgggttgag aatccctaat 17700

ttgagggtca gttcctgcag aagtgccctt tgcctccact caatgcctca atttcttttc 17760

tgcatgactg agagtctcag tgttggaacg ggacagtatt tatgtatgag tttttcctat 17820

ttattttgag tctgtgaggt cttcttgtca tgtgagtgtg gttgtgaatg atttcttttg 17880

aagatatatt gtagtagatg ttacaatttt gtcgccaaac taaacttgct gcttaatgat 17940

ttgctcacat ctagtaaaac atggagtatt tgtaaggtgc ttggtctcct ctataactac 18000

aagtatacat tggaagcata aagatcaaac cgttggttgc ataggatgtc acctttattt 18060

aacccattaa tactctggtt gacctaatct tattctcaga cctcaagtgt ctgtgcagta 18120

tctgttccat ttaaatatca gctttacaat tatgtggtag cttacacaca taatctcatt 18180

tcatcgctgt aaccaccctg ttgtgataac cactattatt ttacccatcg tacagctgag 18240

gaagcaaaca gattaagtaa cttgcccaaa ccagtaaata gcagacctca gactgccacc 18300

cactgtcctt ttataataca atttacagct atattttact ttaagcaatt cttttattca 18360

aaaaccattt attaagtgcc cttgcaatat caatcgctgt gccaggcatt gaatctacag 18420

atgtgagcaa gacaaagtac ctgtcctcaa ggagctcata gtataatgag gagattaaca 18480

agaaaatgta ttattacaat ttagtccagt gtcatagcat aaggatgatg cgaggggaaa 18540

acccgagcag tgttgccaag aggaggaaat aggccaatgt ggtctgggac ggttggatat 18600

acttaaacat cttaataatc agagtaattt tcatttacaa agagaggtcg gtacttaaaa 18660

taaccctgaa aaataacact ggaattcctt ttctagcatt atatttattc ctgatttgcc 18720

tttgccatat aatctaatgc ttgtttatat agtgtctggt attgtttaac agttctgtct 18780

tttctattta aatgccacta aattttaaat tcataccttt ccatgattca aaattcaaaa 18840

gatcccatgg gagatggttg gaaaatctcc acttcatcct ccaagccatt caagtttcct 18900

ttccagaagc aactgctact gcctttcatt catatgttct tctaaagata gtctacattt 18960

ggaaatgtat gttaaaagca cgtattttta aaattttttt cctaaatagt aacacattgt 19020

atgtctgctg tgtactttgc tatttttatt tattttagtg tttcttatat agcagatgga 19080

atgaatttga agttcccagg gctgaggatc catgccttct ttgtttctaa gttatctttc 19140

ccatagcttt tcattatctt tcatatgatc cagtatatgt taaatatgtc ctacatatac 19200

atttagacaa ccaccatttg ttaagtattt gctctaggac agagtttgga tttgtttatg 19260

tttgctcaaa aggagaccca tggggctctcc agggtgcact gagtcaatct agtcctaaaa 19320

agcaatctta ttattaactc tgtatgacag aatcatgtct ggaacttttg ttttctgctt 19380

tctgtcaagt ataaacttca ctttgatgct gtacttgcaa aatcacattt tctttctgga 19440

aattccggca gtgtaccttg actgctagct accctgtgcc agaaaagcct cattcgttgt 19500

gcttgaaccc ttgaatgcca ccagctgtca tcactacaca gccctcctaa gaggcttcct 19560

ggaggtttcg agattcagat gccctgggag atcccagagt ttcctttccc tcttggccat 19620

attctggtgt caatgacaag gagtaccttg gctttgccac atgtcaaggc tgaagaaaca 19680

gtgtctccaa cagagctcct gtgttatct gtttgtacat gtgcatttgt acagtaattg 19740

gtgtgacagt gttctttgtg tgaattacag gcaagaattg tggctgagca aggcacatatag 19800

tctactcagt ctattcctaa gtcctaactc ctccttgtgg tgttggattt gtaaggcact 19860

ttatcccttt tgtctcatgt ttcatcgtaa atggcatagg cagagatgat acctaattct 19920

gcatttgatt gtcacttttt gtacctgcat taatttaata aaatattctt atttattttg 19980

ttacttggta caccagcatg tccatttct tgtttatttt gtgtttaata aaatgttcag 20040

tttaacatcc cagtggagaa agtta 20065

<210> 2

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 2

acgttggatg atttgagggt cagttcctgc 30

<210> 3

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 3

acgttggatg actgagactc tcagtcatgc 30

<210> 4

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 4

acgttggatg tccactggga tgttaaactg 30

<210> 5

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 5

acgttggatg ttgtcacttt ttgtacctgc 30

<210> 6

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 6

acgttggatg acagagctcc ttgtgttatc 30

<210> 7

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 7

acgttggatg tgctcagcca caattcttgc 30

<210> 8

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 8

acgttggatg gaggatccat gccttctttg 30

<210> 9

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 9

acgttggatg cttaacaaat ggtggttgtc 30

<210> 10

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 10

acgttggatg ttaagtccca cattgcctgc 30

<210> 11

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 11

acgttggatg tctcaacctg tggtttaggg 30

<210> 12

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 12

acgttggatg atacctagta gaacctgccc 30

<210> 13

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 13

acgttggatg gcattgacag gttggaaaag 30

<210> 14

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 14

acgttggatg atttgctgcc ttgggcagag 30

<210> 15

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 15

acgttggatg tcgggaagct gcgcagaact 30

<210> 16

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 16

acgttggatg aacaggtggg aaagatgaac 30

<210> 17

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 17

acgttggatg gttgctgatg ggaattgagg 30

<210> 18

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 18

acgttggatg actttggtga ctgtgacctc 30

<210> 19

<211> 30

<212>DNA

<213> Artificial Sequence

<400> 19

acgttggatg catgtgtgtg tcatacacag 30

<210> 20

<211> 20065

<212>DNA

<213> PDL1 gene (Homo sapiens)

<400> 20

ggcgcaacgc tgagcagctg gcgcgtcccg cgcggcccca gttctgcgca gcttcccgag 60

gctccgcacc agccgcgctt ctgtccgcct gcaggtaggg agcgttgttc ctccgcgggt 120

gcccacggcc cagtatctct ggctagctcg ctgggcactt taggacggag ggtctctaca 180

ccctttcttt gggatggaga gaggagaagg gaaagggaac gcgatggtct aggggggcagt 240

agagccaatt acctgttggg gttaataaga acaggcaatg catctggcct tcctccaggc 300

gcgattcagt tttgctctaa aaataattta tacctctaaa aataaataag ataggtagta 360

taggataggt agtcattctt atgcgactgt gtgttcagaa tatagctctg atgctaggct 420

ggaggtctgg acacgggtcc aagtccaccg ccagctgctt gctagtaaca tgacttgtgt 480

aagttatccc agctgcagca tctaagtaag tctcttcctg cgctaagcag gtccaggatc 540

cctgaacgga atttatttgc tctgtccatt ctgagaaccc aaaggagtcc taaaagagga 600

atggaggagc ctaagaataa aaatagtata ataaaacatt tcttagacac attgaccttg 660

gcctatgtca aagttcagtc tgggtttgtc ttataacaca aggagtaaaa gtaccattgt 720

tctacctctt tttttaatac ttgaaaaaaa tttactgtgg atgcttttct atgaattaaa 780

taaccttcta aaaaatgttt tcattgctgc attcgattag attgggtaac taaatgaaat 840

taattcctca ctgttgggta taaaggttat ttacagtggt tctgtcttag ccattcactg 900

aactcattgc atatatatct ctggaatatt gctgattgtt tccttcaagt aaacttagaa 960

gtgtaactac ttagtcaaag agcctgaata ttttaaaggc cttttgaaga aaactgaaaa 1020

tgctttccag aaaggatgta tcagttgaca atgacagtcg tcaacagtat ttaaggagaa 1080

ctatgatact ctgaagaaaa acttagcctt tctcagtaaa agtaggtagg cagaggccac 1140

atgacagcag ttagagtgtg gtcttcaagg aagtcacaga aatactgtgg ggaattgaaa 1200

ccccatgtgg aaaatgtaca agagtgtctc agtgtgactg agaaggaggt tgggcatggg 1260

gtttcatgga gtttaataaa gtttggtcac ttagtagagg tttaataaat caactgtctt 1320

aatctttgat cctacttaag aattttttttttgtttttgt agagatgggg ctcttgttat 1380

gttgcccagg ctgttctcga actcctagcc tcaggcgatc ctccctcctc aggctccaga 1440

agtcctggga ttactggcgg gagccaccat gcaggcctct tgctcctact tttgagaaag 1500

gaagtttaac cggttttttttgtctttttttttttttttttgagacagag tctcactctg 1560

ttgcccatgc tggagtgcag tggtgcaatc tcagctcact gcctcccggg ttcaagtgat 1620

tctcctgcct cagcctcccg agtagctggg actacaggca cctgccacca cgcccagcta 1680

atttttgtat ttttagtaga aatggggttt caccatattg gccaggctga tctcgaactc 1740

ctgacctcag gtgatccgcc tgcctcggcc tcccaaagtg ctgggattac aggcatgagc 1800

cactgctcct ggctgcttaa ctttttctct atctcatcct ctacccatc ctacccttgg 1860

aagatagaga agtagtatta gttccatagt gttatactgg gcttccccca gggacaaacc 1920

cacttcccca acctgaatga gccatcactt cttccccagt ttacatttca ttgctcttta 1980

aatgtctcca ttcggatatg ggaattcaca tatggtcata attcttacct gaagaagatg 2040

tcagtcttct tctcttagac caactgccct gatatgaggt ttagaggtta aagaacatgt 2100

gtgtatttac atgatctttg tattctgcct tttcgtccct cactaatgac agctgcaccc 2160

caaggaaatg gagctgtgga agagagggtt tgataagaaa ttaagtaaat attggatcta 2220

atccatcacc ctccaggaag cctttattac tcctaaaaat ttcaaccaaa ttcattaaag 2280

gacaagaact ccaccagagt aggccataaa cattggcaaa attagttgta atccatgact 2340

agattaatg tccctttgtt ttattcccat atggttataa tgctttgctt ggcattaggg 2400

gtattttaag ttttcttctg cctagtaagt gaatttgtgt ttataataca ataatcataa 2460

aatatcacat taatatttta taactgtaca gttataaaat attttataag taatatttat 2520

attttataag taatatttta taactgtaca gttaactctg gcccaaggaa aagatagtct 2580

gtagatgct gcagccccat tttagcaaat gtgacctcac aggcctgaat gccatcgcta 2640

ttccacatct acaggataga cggaaaggaa agaaataaaaaaataggtac ctaacactgg 2700

caagaggatg atgactcatg ttatttcact taaccttttt atcttttaac atgaaggact 2760

catacaggtt gataagaaac cagtgacata aacagaccaa aaaatgatca gatctttcaa 2820

attagcaaaa aaataatatt ttttaaacaa tgggtgaaaa tacagtgtaa cagtaccaat 2880

tatcaacatg tgttgagaac cagaaaaatg ttctttttct ttgatcagca acactatttg 2940

ggaaaatcta tcctcagggc ctagcctggg gccctggcac acagtaggca ctcaacgaat 3000

atttgctgaa cacacaaata cttatgatat tttaaaaaat tggcaacaat ctgataccta 3060

acaatagagg gattaaatat tatggaactg ttaaataaga tgcttatgaa taccatgcag 3120

taagatgggc aatatttatg ccataagctt taatgaaaca aatgggtatt aaatgtatga 3180

taaggttata aattactttt taaaagatta cagggaaaaa aattgaaaga tatacactga 3240

aatgtttttt gctcacagtg gtgacaaggt ttctcagcac tggcactgtt gacgttttag 3300

gctgtatgtc tttgctgtgg gaggctggcc tgtgcactgc agggtgtttg gcagcactct 3360

tggcctctgc ccctagatag caatagcagt cctccctcaa ccagcccaat tttgacaacc 3420

aaaaatgttt ccaggcatca ccagatgctc cctgggtgag agtgatgaaa tagtagggga 3480

ttttcccctt cttttcttat tttctgtaat tccattatat tactttaata ataaagaaaa 3540

aaacataaaa aataaacgaa tgttattatt ctacgtcagt ttggatgttt ggactccatt 3600

ttggggttct ttccattata tcacttggtc tgctaaacat tctacggttt ggtaaggtga 3660

agtgattcat gaaattttgg ttttattttt ttcctgatac taaaaataaa aattctttc 3720

acttggaaat ttggacacag aacaccaaaa aaaatccata atctcatctc tctttttctg 3780

tcttttcctt ccttttttcc ctttaaaaac aataaagagt gaaacctacc tgttctccct 3840

ctaatttaat tcctaaatat aatcactgtc aatatcttgg aatttcctg tgtctaaaca 3900

cacacacaca cttttttttt tcagcaaaag tggatttctg ctacatgtag tgttctgcaa 3960

cttactttct atgtgtttac aaaatcagta catgtacata tgctgaattc agtccttaat 4020

ggtattatat tttgtgaata taccaaaatt tgtttaacca cttagacaat ctaggatatt 4080

ctcagtttgc tgttatgagc aatgctcttc ctttacatat acagacatat atatatatat 4140

gtgtgtgtgtgtgtgtttttgt tttagtagga tagatttcta ggagagggtg aaaggtctta 4200

tgacatccgc atttacgatt gtaataggaa gtatcaaagt gccccctaaa gaaaaaaatc 4260

ctcccattag tgggtaagaa agcctatttg ttcatatctt cacaaacact aaatattaga 4320

aatatttaca attgtggtca agctcataag tgaaaatggt atttcatatc ttatattttt 4380

tattgtgaga ttgaacatct ttcatatgtt tacatgtcac ctgtatttct tattctctga 4440

actatatgtt atgacctttc actttttttc ctcatgggtt atgtgtagtt tgtatagttg 4500

tcttattgat tgttaggagc tatttatata ttaggaacat taatctcctg tcttatatat 4560

acgtggcatc gattagttga tcatttgtga gttcatgtct gtatacaaag attggagagg 4620

cactaagagg gaaaacttac ctctttctta tcaaagtttg taaatatatg tataacagaa 4680

gagggagaaa atattaataa atgcacagat tggctgaaat agagtataaa tcttttactc 4740

ccctacttca acataaactg caaaaggaga gtgacttttc tttcactctg acttccgtat 4800

tcctcatgct taaaatagtg cctagcacag aagaggtgct caatcagtgt ttgctaaacg 4860

aaataattag tcacatttca agcaggatga ctaaatgaag aatagaatct aggcagatac 4920

tctggaagag tggctgtgag tcattcatat cttagtatga attagtcaaa tccaactctc 4980

tccccttccc actccccact gttagtagaa gaatctgttt attgagagaa tagatttata 5040

atttagaata agtgagaggg gcagaagagg agatttgaa ggatggcacc tgaaggagga 5100

ctagcatggc tgagacagtg aagtggaagc cttgaatagc taaagggtaa gatgaaagta 5160

tttagctgta gggggaaaaa gcattgacag gttggaaaag taaaagtcag attctccttg 5220

ctctgaaatt ttgtacaggg caggttctac taggtatgtt acaatgcaga aaaaacatga 5280

aataattgag aggaatttgg tgcaatatta tcttcttggc ttcttttgag tgggcagatt 5340

tttttcacgg cctgtaacta taataaattt gaaacttctc atcttttagt aacttttttc 5400

acttaagttt atgtggctgt gggcaatgga atgaagatat tgaacttcca attccctgtt 5460

gggtttccac aattacaagt caatcatgac tggttattag aagactattt cagttagaac 5520

caccaagtcc catattgtca tattgtatgt ttaattatta agtgaagcag tcttcttttc 5580

gtgttttcca taattagggc attccagaaa gatgaggata tttgctgtct ttatattcat 5640

gacctactgg catttgctga acggtaagac accaaatcct tccattaggt tctatatttt 5700

aaatatttta accatgagtt taaaactaaa atgatcattt aaaatgcatg caattttctt 5760

atagagagaa cattctattc tttcttctac tttacacaat ggcaaagtct tctttctact 5820

ttacgcaatg ataaagttac ctgtgtcatt ttgtaaaaat atagagaata tagacaaatt 5880

gaaagacaca aaataatcta ttacccattt cccagggtta actactgaaa atatctgggg 5940

aaatggcctg tatgtataca tttatttgtt tgctttcaac aaggccaaga tcctttgatc 6000

tttcagtctt ggttgctctg tgacatgcct ttcctgatga ggatacttta aggaagaatt 6060

gtaagataca tggaaaatgt caggctaaca cagtactggc atcaccctgt gctctttcct 6120

gaactccata ccaatgtact tcttgccaga aaactgatca aaagtttagg gaagtaaaaa 6180

gagatgactg ttagaatcta ccattccctc tatgtaggaa gcaaataggt gtcctgtcaa 6240

aggacattct ggggatgtct acatgaaacc aagtctccct ggttgtaagg actccatctc 6300

catataatat ttatacagta atatatgttt ataaattgtg ggggcaactt gtttagctaa 6360

ttttattatt ctgctattgg gacactgtgt ctcagcatga gatatagtgt cccaaaacat 6420

atttcaagcc cattggataa aatatgtgtt tagcaagttc ttaaatataa tgataacata 6480

accgaccaga taaagtgatt tataaacgct gtgccaattt tgtaaatgtt tcgaggaatt 6540

ttcccttttc tgaagattgt ccttctttct ttttagcatt tactgtcacg gttcccaagg 6600

acctatatgt ggtagagagtat ggtagcaata tgacaattga atgcaaattc ccagtagaaa 6660

aacaattaga cctggctgca ctaattgtct attgggaaat ggaggataag aacattattc 6720

aatttgtgca tggagaggaa gacctgaagg ttcagcatag tagctacaga cagagggccc 6780

ggctgttgaa ggaccagctc tccctgggaa atgctgcact tcagatcaca gatgtgaaat 6840

tgcaggatgc aggggtgtac cgctgcatga tcagctatgg tggtgccgac tacaagcgaa 6900

ttactgtgaa agtcaatggt aagaattatt atagatgaga ggcctgatct ttatgaaaa 6960

catattccaa gtgttgaaga cttttcattc ttgtaagtcc atacttattt tcaaacagaa 7020

cagcatagtc tgttcattca ttcattcaat tcatgaattc attcacataa ttatccaatt 7080

tcttgagcac ctatttgata gtcactggaa atccagagac aaacaacaca gagccatgtt 7140

ctacagtatg tacagttttc caaaaagaat ttctagtctt tactttttta ttacaaatgg 7200

aatacgtata cttgcaaata attcagatac tgtggaagag atcaaatgaa ttgcaaaagt 7260

gtccctcctc ccttcaccac tatctcccat ggcatgcaga gagagtaacc attatttgtg 7320

tgtccctcca gaaatttttt tattcaacta ctattttttt attttattag gtccgtcagt 7380

tttccttttt tgagcctctc tatatcaaat gcaaataaat atattcagaa caaacccccac 7440

tgtaaggttc acattaaaaa agacttgaag tcaccctatg aagacaaaaa ataatcacat 7500

taagtgtgaa agaacctatt cttccagtac aggataagcc atacttactg ggcatatatt 7560

catcttgaaa atctatactg atgttgtctt ggggaattga aaaggaacta ggagtgttag 7620

ttcctcggta ttgacccaca gttatgttat caggtcactt gagttcaaag ttttgtgttg 7680

gcactagcta agtaaaggaa aacacctctg ctttcattgt tgagtttcac agaattgaga 7740

gctgaaagga tcccaggcag gagcagctaa tccaaactcc cacaaagaac aaaaatcccc 7800

cagaggatct tctgttctta tatttcctgc aatggcgtcc ctgtcatatc ccacaatggc 7860

ctccctgcca tttggatatc ccttccatat cctgttgaaa ttactcccta atagtaagct 7920

gaaatctgcc cctctagttg tagtcttggg attatttcat ttacatgatg accttttaat 7980

atttgactag aattaaatca tctcccccttg gtctttccat tcctgggcta actaccatca 8040

atctgagggc taacaataca agtagaaaaa gtatacattt gtcactgatc actgatcaat 8100

tattaatcaa tgatcactga taactataaa ctcaaaaaca aaatcatgtg gggattaaga 8160

gaaatgtatc agttttatgt tgtatttctg gtccctgata ctggctcagg taatgccact 8220

attgtcaaga agataccact tgtaaagtag atttaatttt catttatattt taccatatgc 8280

ttctccattc atgacatctc ttgagatgtt gtggtttata ctttcagttt ttctccagtc 8340

catccgcaaa tatcaggcat ctactgtgtt ccaagatatt aaagaaatca tcatgactta 8400

gcctcatcaa cagcattgct agatctggga tggaaaggaa gagtataatc ctggcagtca 8460

ggaagaaggc agcataaagt ataagtttct gcttccaaaa aaggtctctc atcagcctgt 8520

agggagtgtg tagggaaggg acagctgtcc ttgtagtagg gaagggtttt attcaggtcg 8580

tctgggctcc aataatccc ttgtgtatct gcagtctcct ttgccatgga tcaacacaat 8640

aggaaatctt ccggcactga tggtttttcc aagggggagt tcttcctgga gcaaagcaaa 8700

tgaccaacca ggtttgagga cctgatttgt ttgacaattc cattttgtat tgtaaattac 8760

ttaattggca ttctactccc aatccatctt gtcatttgca tacagtggtt ttgggattga 8820

gttcagctat accaaaagtc tgaaccttct gcacttagaa caaggcaacc accaagcttc 8880

acttgcactg aggccgtgtc tccaatggaa atgaggcagc tggcttgcag gagcttccca 8940

actcagggaa gtagaactcc tgagtcacct ccatatgcaa atgatttcac agtaatgctg 9000

ttgaacttca cttcccatca cagcaaatgt gtggtaacat agcttcccca caggagttta 9060

ctcaccatgg tattttaaag gtgaaacatt tcaaaactga aatttgaaag aatttagttt 9120

tggattcact caattatcac tatcacttcg ggtgttattg cacctttctt gtttgtgagt 9180

ttaaatgcca gactctcagg ccactaactt tcaattaaaa gtgtttttct ttaatcgctg 9240

aacctaacag cagggaaaac gaaatgttca ttcagacttt cagaaccttc aatgagatta 9300

ggcagctgaa agatcaaagt gttgcatagt tgtcccgata aagctatttg gatcatatgg 9360

accaaatcga ctgctgtcat tccccaccaa ccccatctct ccccaaaatt cccagccctg 9420

tttaagtgtt ctctgtagca tttatctcta tctagtatat tgtgtagcat atcatatcat 9480

acttttctgt tttgtttatt gtctctctcc tcctagaata taaactccac aagcacaaag 9540

atttgggcct gttttataat attgttgcat ccccagggcc tgatatacag cagagtggtg 9600

gtacgaaaag agcacacaaa aaaatatttg ttgagtcaat gaatgaatga tttcctcaaa 9660

taggattagc ctaaaatttt ggaaacatga acagatttgg atatgtgaaa atttatttcc 9720

agactgttca tcaggaactg ttagcagctt ctaaagggta cactggagca gcagtagtaa 9780

aaggaggaag aggagcagct ctgctactgc tactatcgag tactactaca attagcactt 9840

gcttattctg tgtgttaggc cctgtactga acactctgtc taaattagtt catttcctcc 9900

tggaaatgac tctagggggt aagtgcttca tcatgtaaga tgagtatttt tcacattttg 9960

ttgtgtctga aatctgagtg tgtctttcaa tgatggaatc tttgattcca tgataagtgg 10020

tattattccc attttaagga tgaggaaact gaggtccaaa gaaattaagt aatttgccca 10080

aattcaccca gcctagaaaa tgataaagct agttctaaac ccaagcagat tagctctgaa 10140

gtctgggccc ttaataacca ctttttatg cctatatttg tacctctggt gtacgtatca 10200

agttatatgt tgacttcaaa actatcatga ccttttcttg gttttgattg tccaacatta 10260

gtatagtgtt ctgggtctgc aaaaattttg attackcatc tcatctgtaa aacattttga 10320

actcgtgtgtttgtgcatgc aatttgtgtgt gtaattataa aaatttact ttctgttaat 10380

atataagttg tatcataaga aactgccgtt tttgaagagc aaaaaaaggt tgaatgttac 10440

cagttacatc tggttcaacc taatagacat ttgtacaaaa acagacattt taagaggttg 10500

aaataaaaat ttaataaaca atattttcag tttttactaa ttgtgatgct tcactatcat 10560

tagctaatat gtcaaggcat aatatacctt agggtgaact ttatcattaa caaaggtgga 10620

tggtgtcaat aatcttgagg tttgtgtttt tttatataac actgcgaggt ctaattaagt 10680

acttactgtt taccacctca tacagtggcc gataaaaagt gtcacttctg ctgtttcctc 10740

tgggttgtgc ttgaattatt agtattatct tcagtcctca gtttctttgt gggaaacttt 10800

ttaattagtt gtttaatttt gtaagatggt tagtttagtc aaaattagat aagagaattt 10860

gaaaatccgt agctacccca aagcaaccta cacataagaa ctattatttttgtgttttga 10920

aatcataatt ttattgattt ccagtgtttc cactggtagt ggtttcattg atataggagt 10980

atcaaaacat cactcattat ttatttcagt ttcatttgat cctagccgtt ttgtattaac 11040

tctctgtgaa gaaattacct cacaaatcta ttgctgtcct tggtaaagga atggagaatt 11100

aaggctctag atcattagtg gttacactat agtattagaa gtaaaaaaaa gattatacca 11160

acaaaataag aacatgttaa tgtacttgta atgaataaac atgaataaag ctcttatgct 11220

atataggtgc actaaacaat ctactagaat tgtcagcaaa ctacgtatct taatcctgaa 11280

agggtcccaa accaatgatc taaaattgaa tcaaactttc ttccttgagc ataattactt 11340

aaatgattta ttaaaatagc cagcatttaa aagcttaaaa tgtaaatatc ataatgtggt 11400

atcctagata gcatcccaga acagaaaaag gatattaggg aaaaactgga ggaatggaat 11460

aaattatgca gtttagttat taataatgta ctaacgtcct tagttatgac gattgtacca 11520

tggtaatgta agatactaac aatagaggaa accgggtaag gagtatacag taactctata 11580

ctatctttgc aacttttttg taaatttaaa acttctaaaa taaagaacaa atttaaacat 11640

taaaaagtat caccaggaac atatatcact gtttacagat gaaatactat gtattttcat 11700

atctaatttc tgatcattga cttcaaatca gaaaagtgaa tgacacctca aaatcaggtt 11760

ttctgtttac tgaagtctaa gaaaagaaag cataccagct ggagagattc atgtttataa 11820

agacagattt ataacaacaa aaataaaata tccaagaata aatttaagaa gaagcacttt 11880

actgagaaac atatgaaaac ctgaacaaat ggagagggat attttgtatt tgaatagaaa 11940

gacttctggt ttaaagataa ttctctttaa attatttttt gtagaaattt aaggggtaca 12000

agagcagtgttgtcacatgg atatattaca tagtggtgaa gtctggggtt ttagtgtaaa 12060

ttaatcttta cattttgttt gagcccaata aatgtaccaa catgattttt atagaaagat 12120

agtcattcct attaatccaa acttgtccca actttgaatt gaattgaggc agagctagca 12180

ggtgttcccc acggctgagg catctgaaca ttaagcatat ccctctgaga accagcctgc 12240

attgatactc tttctaatgt ggacagcatc aagctatgta cgtagttctg tgctcagcaa 12300

aagccctgac ttctttttgt ttatgtccta gccccataca acaaaatcaa ccaaagaatt 12360

ttggttgtgg atccagtcac ctctgaacat gaactgacat gtcaggctga gggctacccc 12420

aaggccgaag tcatctggac aagcagtgac catcaagtcc tgagtggtaa gaccaccacc 12480

accaattcca agagagagga gaagcttttc aatgtgacca gcacactgag aatcaacaca 12540

acaactaatg agattttcta ctgcactttt aggagattag atcctgagga aaaccataca 12600

gctgaattgg tcatcccagg taatattctg aatgtgtcca ttaaaatatg tctaacactg 12660

tcccctagca cctagcatga tgtctgccta tcatagtcat tcagtgattg ttgaataaat 12720

gaatgaatga ataacactat gtttacaaaa tatatcctaa ttcctcacct ccattcatcc 12780

aaaccatatt gttacttaat aaacattcag cagatattta tggaatatac cttttgttcc 12840

atgcattgta gtactcattg gatacacata gaataataag actcagttca cactcttcag 12900

gaaacagata aaaaactaag aaacaaacaa aaaacaggca atccaacacc atgtgggaaa 12960

tgctttcata gccgggaaac ctggggaata cctgagagga atactcaatt caggccttgt 13020

ttcaggaatc caaatcctgg cacatcagag ctgcttccct ctttccaggg tggcaggaaa 13080

taaatggaac atatttttct atcttatgcc aaacatgagg gaccctttct ccccggtgcc 13140

tctcccaagg tagtctacaa tatttcaact ctagcagtct gcttagtgca tagaacatga 13200

ggctgtgtgt ccctgggcaa attacktagac ttctgtgtgc ttcactttcc ctgtaggatt 13260

ataatctact gagcaagctt attgtaaggg tcagattagc aacagtgtat gaaaatgatt 13320

tgagaccatt gcctgcacaa attcaactat ttttttttat ctcactactc tacagaagta 13380

ggtagggtgg gagacagagt ctgatgagag gctcagaatg tgaaagaaag tgaggcgagt 13440

gagcatgata tttaatataa acacaaagat attctgagaa gagctgctca ctgccccctc 13500

ccccaataca tgttgatagg aaaatgccac gtacttcagc aaaaacaact gaaaaattag 13560

atagaaaagt caatcaatag gaaaagataa tccaggacgg tgttgtgaac agaaagaggg 13620

ggaaaaaact ttagaaaatg atggggatgc tcttactggg gtacgagtcc tcaggtattg 13680

aactggcttt cagtaaaagc tagattagtg ggttcctgcc atttacaagc tgttttatga 13740

caacttactt gttgggtggc ctacagtaac tcacctaact gcactgagtc tgtttcctca 13800

tctgtaaatt ggggattttt ttttaaatac ctggcatgcc taactcataa agttgttctg 13860

aaactgaaat aaaacatacg tgaacaggca ttgtaaactg taagttacgg aaaaagctgg 13920

ctgttgttgt gtctttaaag tttcacctgg gtagtcaaag atggatcatg ggtctcagtg 13980

gagagctgag ccaggcagga gctgactaag ggtgagaggt gggagttagc agcctctgaa 14040

catctgtgta ccatgggacc ccctttcctc ctgcatggta ccccagacaa ggagcctagt 14100

aagagatact aatggcttgt tgtccagaga tgttcaaact gcagagaaag ataagacaac 14160

aagcattggc ctccaatcat gatgacagat aggaggaggt gggagctcct tagcagtgct 14220

ggttggcctt ccatgttcta ctgtgggcca tctctgccat gtactgtagg ctactagctt 14280

ctatattaaa gaatgcaaga ggggccagga gcggaggctc atgcctgtaa tctcagcact 14340

ttgggaggcc aaggtggggca gatcacttga ggtcaggagt ttgtgaccag cctggccaac 14400

atggtgaaac tctgccttta ctaaaaatat aaaaattagc tgggtgtggt ggtgtgcacc 14460

tgtaatccca gctactcggg agactgaggc acaagaattg cttgaacctg ggaggcggaa 14520

gttgcagtga gcccagattg cgccactgca ctccaccctg ggcaacagag aaagactctg 14580

cctcaaaaaa aaaaaaaaaa agcaagagga agtgaaataa tcaaggccgc catttaatag 14640

tgagcagcca ctccatgtgg tactgtgcaa gcacattta aatattagcc tcacaagaaa 14700

tgtattagca tttgtatttt gtacactggt taagtatctt gcccaagacc tcaaaactgg 14760

ttaagggcag cagaatttag ccccagcacc accttttcaa agcctgggct tctcacactt 14820

ctccatgctg ttcccatttt aacacaggta tctcgccatt ccagccactc aaactttggc 14880

atttaagaaa attatcctaa agctaaacta aacttcaagg atgaccattc tcctgacccc 14940

ttcccatcaa aattttatct ttagtcagtt tgttttcgtt ttgttttgtt tttcagaact 15000

acctctggca catcctccaa atgaaaggac tcacttggta attctggggag ccatcttatt 15060

atgccttggt gtagcactga cattcatctt ccgtttaaga aaaggtagta tttccttaat 15120

tgcagtggtc tccactgggg gtgaggaagg ggtgagaatt ggatcatggc tgcaaggaaa 15180

cccgacttaa cctctgcaag gtggtgcaaa ggcattccac tgttcaacag caattatatt 15240

gaagctgagt gggatcactg ggtgaagatg aagcgtaagg ggtgaggggc aggagaatgg 15300

gtatggatgg aggtagaaga tgcagtgtca tacagtttttttctatcatg aaaataacca 15360

cagacttaca gaagagaaag agctaaaatg cccgtcattt tcagttgcat tttagtcttg 15420

cattagttgc aaccagctgg tttctgggta ccctaagtaa taaaaatagt tcctctgtag 15480

aactgtagta tgtttaccat agagtatttt gcaaaatttt tggtagagga tgttacataa 15540

tttgcatgtg ttcatttctc catttacctg tgggaacaat taaaatccag gaaaatgagt 15600

atattcaaat aatttcctcc catttaagat gagtcagagt aaataattcc tccaatactt 15660

agagaagtat accaagagat ccagtgatgg tatagagttg tctgatgtta aatagggaag 15720

tagaatatgg aaggggattc caatagtcgt tgaaaaattc cccataaccc cttacatggg 15780

ggaaagtagt gttaactgag agagtagaga taagctgttt ccaaaaatta tattcttaac 15840

aggactgaga tagccagaat ataaggatca agtttcaatg acagtaagat cctgagatgg 15900

agttgatttg cacaaagaaa taattgttgc cagcatgcat tttgaatatt tctctggaaa 15960

aaaagattag ttggcagtag aaatggatag aaatcaatag atattaaaat acctcagaat 16020

ttggttcatc tctgggaaaa gatgaaaaat aaaagtgtat actcctcaag aacatctagg 16080

atcaaaagca tgtgccctac actattgaat taattaacct cataagttgg gacctgtgga 16140

ataaggatgt ccaccagact tcctagggat tacaaatgtt tcacagaact tgaaatttaa 16200

acttgggtca ctgtatggga tgtagagctg tgctatatgg aaataaaaat gatttctttt 16260

tctcaaggga gaatgatgga tgtgaaaaaa tgtggcatcc aagatacaaa ctcaaagaag 16320

caaagtggta agaatatcag aaggaattgg gaagtaaaag tcaaaggaaa caaaaagcta 16380

aagcaataac aaagagaaat ccatcagtca taatctcctc tccttttaaa gaatgctggt 16440

tcccctttgc ctcacagcta acacaagaac tcctccaccg tctgaggagg tttaggagca 16500

gggaagggga aggagtcagc ttcatttgct aatcttctgt tgccctgcac cctagcagct 16560

ccttgcagca ggggacaagg atgacttagg tggatggata attaattgat tctaaaatat 16620

tgtgtgtcag tattgtaata ctatgttaat tgcaccatgc acggtatctc atttaatccc 16680

ccacccccttg ccattaccaa agagagagag agagagagag agagaaatac tagaatttat 16740

cctcatttta cagtagagaa aacagagggt caagaagata atgtaaagtg cccaagaaca 16800

cacagctgat cacaaaaatc aagcttgggg gccattagcc taaccacaga ccctactct 16860

taaccccatct gcttcaatcc attttgctac aaatgtttaac atttataagc agggcagaaa 16920

aacctcatcc aggttatga actaagaaga aagttatatt aaggtttcta atttttttaa 16980

tgtagttaga aaccaaactt aacaatgagc ccaagtttaa agcagtctaa ttaacctgga 17040

caagctcagg caagtttcat tctgtggccc atagcatcat ctgtgttgta aagctaagta 17100

gcaaatgttg tttgggtcat gctgggggac aagccatccc aatttgctca ggactgaggg 17160

gttttccagg atatcatgta aggataattg ggtacaaata taacctgctg ctttctctca 17220

tttcaaattt atcatttatc atatcagcaa ctatgagtta tgttttttat tagatttctt 17280

gttacttttt ccccagacca cttcccatga aattaatata ctattatcac tctccagata 17340

cacatttgga ggagacgtaa tccagcattg gaacttctga tcttcaagca gggattctca 17400

acctgtggtt taggggttca tcggggctga gcgtgacaag aggaaggaat gggcccgtgg 17460

gatgcaggca atgtgggact taaaaggccc aagcactgaa aatggaacct ggcgaaagca 17520

gaggaggaga atgaagaaag atggagtcaa acaggggagcc tggagggaga ccttgatact 17580

ttcaaatgcc tgaggggctc atcgacgcct gtgacaggga gaaaggatac ttctgaacaa 17640

ggagcctcca agcaaatcat ccattgctca tcctaggaag acgggttgag aatccctaat 17700

ttgagggtca gttcctgcag aagtgccctt tgcctccact caatgcctca atttgttttc 17760

tgcatgactg agagtctcag tgttggaacg ggacagtatt tatgtatgag tttttcctat 17820

ttattttgag tctgtgaggt cttcttgtca tgtgagtgtg gttgtgaatg atttcttttg 17880

aagatatatt gtagtagatg ttacaatttt gtcgccaaac taaacttgct gcttaatgat 17940

ttgctcacat ctagtaaaac atggagtatt tgtaaggtgc ttggtctcct ctataactac 18000

aagtatacat tggaagcata aagatcaaac cgttggttgc ataggatgtc acctttattt 18060

aacccattaa tactctggtt gacctaatct tattctcaga cctcaagtgt ctgtgcagta 18120

tctgttccat ttaaatatca gctttacaat tatgtggtag cttacacaca taatctcatt 18180

tcatcgctgt aaccaccctg ttgtgataac cactattatt ttacccatcg tacagctgag 18240

gaagcaaaca gattaagtaa cttgcccaaa ccagtaaata gcagacctca gactgccacc 18300

cactgtcctt ttataataca atttacagct atattttact ttaagcaatt cttttattca 18360

aaaaccattt attaagtgcc cttgcaatat caatcgctgt gccaggcatt gaatctacag 18420

atgtgagcaa gacaaagtac ctgtcctcaa ggagctcata gtataatgag gagattaaca 18480

agaaaatgta ttattacaat ttagtccagt gtcatagcat aaggatgatg cgaggggaaa 18540

acccgagcag tgttgccaag aggaggaaat aggccaatgt ggtctgggac ggttggatat 18600

acttaaacat cttaataatc agagtaattt tcatttacaa agagaggtcg gtacttaaaa 18660

taaccctgaa aaataacact ggaattcctt ttctagcatt atatttattc ctgatttgcc 18720

tttgccatat aatctaatgc ttgtttatat agtgtctggt attgtttaac agttctgtct 18780

tttctattta aatgccacta aattttaaat tcataccttt ccatgattca aaattcaaaa 18840

gatcccatgg gagatggttg gaaaatctcc acttcatcct ccaagccatt caagtttcct 18900

ttccagaagc aactgctact gcctttcatt catatgttct tctaaagata gtctacattt 18960

ggaaatgtat gttaaaagca cgtattttta aaattttttt cctaaatagt aacacattgt 19020

atgtctgctg tgtactttgc tatttttatt tattttagtg tttcttatat agcagatgga 19080

atgaatttga agttcccagg gctgaggatc catgccttct ttgtttctaa gttatctttc 19140

ccatagcttt tcattatctt tcatatgatc cagtatatgt taaatatgtc ctacatatac 19200

atttagacaa ccaccatttg ttaagtattt gctctaggac agagtttgga tttgtttatg 19260

tttgctcaaa aggagaccca tggggctctcc agggtgcact gagtcaatct agtcctaaaa 19320

agcaatctta ttattaactc tgtatgacag aatcatgtct ggaacttttg ttttctgctt 19380

tctgtcaagt ataaacttca ctttgatgct gtacttgcaa aatcacattt tctttctgga 19440

aattccggca gtgtaccttg actgctagct accctgtgcc agaaaagcct cattcgttgt 19500

gcttgaaccc ttgaatgcca ccagctgtca tcactacaca gccctcctaa gaggcttcct 19560

ggaggtttcg agattcagat gccctgggag atcccagagt ttcctttccc tcttggccat 19620

attctggtgt caatgacaag gagtaccttg gctttgccac atgtcaaggc tgaagaaaca 19680

gtgtctccaa cagagctcct gtgttatct gtttgtacat gtgcatttgt acagtaattg 19740

gtgtgacagt gttctttgtg tgaattacag gcaagaattg tggctgagca aggcacatatag 19800

tctactcagt ctattcctaa gtcctaactc ctccttgtgg tgttggattt gtaaggcact 19860

ttatcccttt tgtctcatgt ttcatcgtaa atggcatagg cagagatgat acctaattct 19920

gcatttgatt gtcacttttt gtacctgcat taatttaata aaatattctt atttattttg 19980

ttacttggta caccagcatg tccatttct tgtttatttt gtgtttaata aaatgttcag 20040

tttaacatcc cagtggagaa agtta 20065

Claims (6)

1. Use of PDL1 SNP genotype in the preparation of markers for diagnosis and prediction of neoadjuvant chemotherapy efficacy in breast cancer. 2. The use according to claim 1, characterized in that: in the PDL1 SNP genotype, rs4143815 C>G. 3. The use according to claim 1, characterized in that: the breast cancer is hormone receptor positive. 4. Use of the PDL1 SNP genotype as a marker in the preparation of reagents for diagnosis and prediction of the curative effect of neoadjuvant chemotherapy for breast cancer. 5. The use of the PDL1 SNP gene as a marker in the preparation of reagents for diagnosis and prediction of the curative effect of neoadjuvant chemotherapy for breast cancer, the sequence of the PDL1 SNP gene is shown in SEQ ID NO.20. 6. A kit for detecting the PDL1 SNP genotype, characterized in that it contains primers shown in SEQ ID NO.2-19.