CN113249469A - Clonal eosinophilia hypereosinophilia fusion gene detection probe composition, kit and application thereof - Google Patents

Abstract

The invention provides a clonal eosinophilia hypereosinophilia fusion gene detection probe composition, a kit and application thereof. The invention firstly classifies the related fusion genes, wherein the clonal eosinophilia-mediated polycythemia fusion detection core genes FGFR1, PDGFRA and PDGFRB are I-class genes, so the full length of the gene transcript is taken as a target region; the other genes are class II genes, and only the fragment near the fusion breakpoint is used as the target region. According to the base complementary pairing principle, a complementary sequence of a target region is designed to serve as a probe to form a liquid phase chip, the probe relates to 39 target genes, the eosinophilia fusion gene is detected by capturing the corresponding target sequence in a transcriptome library and combining high-throughput sequencing, multiple fusions can be detected at one time, the capability of finding new fusions is realized, the detection cost is reduced, and the accuracy of the detection result is improved.

Description

Clonal eosinophilia hypereosinophilia fusion gene detection probe composition, kit and application thereof

Technical Field

The invention belongs to the technical field of liquid phase capture and the technical field of transcriptome sequencing analysis, and relates to a clonal eosinophilia-mediated granulocytopenia fusion gene detection probe composition, a kit and application thereof.

Background

Eosinophilia (Eosinophilia) refers to a group of diseases with Eosinophilia or tissue damage in peripheral blood, usually higher than 0.5 × 10 in absolute counts⁹The term,/L, is used. If the peripheral blood eosinophil count is higher than 1.5X 10⁹the/L, i.e. whether or not there is organ damage, is called hypereosinophia syndrome (HE). Wherein the number of eosinophils is 1.5 to 5X 10⁹the/L, referred to as moderate boost,>5×10⁹the/L is called the heaviness increase. Eosinophilia syndrome can be classified into four categories according to the etiology: reactive hypereosinophilia, secondary hypereosinophilia, idiopathic hypereosinophilia, and clonal hypereosinophilia.

Wherein, the clonal eosinophilia disease refers to eosinophil originated from blood tumor clone, which mainly comprises: the marrow-line and gonorrhoea-line tumors are accompanied by PDGFRA, PDGFRB, FGFR1 rearrangement or PCM1-JAK2, ETV6-JAK2 or BCR-JAK2 fusion genes; ② chronic eosinophilic leukemia-nonspecific (CEL-NOS), including chaperone ETV6-ABL1, ETV6-FLT3 or other kinase fusion genes; ③ atypical chronic myelogenous leukemia with eosinophilia (aCML-Eo); chronic myelomonocytic leukemia with eosinophilia (CMML-Eo); chronic myelogenous leukemia in accelerated or acute phase (occasionally); sixthly, other myeloproliferative tumors in the acute stage (occasionally); seventhly, acute myeloid leukemia with eosinophilia (AML-Eo), especially with t (8; 21) (q 22; q 22) or inv (16) (p13.1q22) (only occasionally); acute lymphoblastic leukemia, if it is proved that the eosinophilic granulocyte comes from malignant clone; ninthly systemic mastocytosis (eosinophils proved to be clonal).

For patients with clonal eosinophilia, accurate detection of the relevant fusion is of great significance for assisting in identifying the disease type of the patients and guiding clinical treatment. As positive for the FIP1L1-PDGFRA gene (including patients presenting with acute leukemia), a small dose of imatinib is recommended; positive for the presence of PDGFRB gene rearrangement or ETV6-ABL1 fusion gene, standard dose imatinib is recommended; the ETV6-FLT3 fusion gene exists, and sunitinib or sorafenib is recommended; presence of JAK2 gene rearrangement: luccotinib treatment is one of the options.

In addition, with regard to clonal HE caused by the myeloid and lymphoid tumors with PDGFRA, PDGFRB, FGFR1 rearrangement or PCM1-JAK2, ETV6-JAK2 or BCR-JAK2 fusion genes, the potential fusions caused by PDGFRA, PDGFRB and FGFR1 rearrangement are very diverse, and only by way of example with PDGFRB, the PDGFRB fusions related to eosinophilia which have been found so far have approached 20, and there may be new fusion types which have not been found so far. Therefore, the fusion screening method which is comprehensive and accurate and simultaneously needs to have new fusion discovery capability plays a crucial role in the auxiliary diagnosis and medication guidance of the clonal eosinophilia.

At present, the mainstream clinical fusion gene screening methods mainly comprise a second-generation transcriptome double-end sequencing method and a multiple PCR method. Double-end sequencing (pair-end sequencing), namely, fixed-length reads (reads) are respectively and initially detected at two ends of a sequencing library, the sequencing reads at the two ends form a pair, the reads are returned to a genome, and the returned positions of the paired reads on the genome are used for judging whether fusion is supported. The method can screen all fusion events in the transcriptome at one time, but because the expressed genes in the transcriptome are more than ten thousand, and the expression level of some housekeeping genes (house-eating genes) is extremely high, the fusion transcripts with lower expression level are difficult to detect under the condition of limited sequencing amount, thus easily causing false negative and misleading diagnosis or treatment. On the other hand, the transcriptome itself is very large, and the data volume based on the next generation sequencing is very large, which means that the cost of fusion analysis is very high, and the popularization and application of the fusion analysis in clinic are prevented.

The fusion screening by the multiplex PCR method is easy to be clinically applied at present, and the working principle is that a plurality of pairs of primers are added into the same reaction system, and a plurality of fused DNA fragments are amplified simultaneously. The method is economic, simple and convenient and high in sensitivity, but for different fusions, primers need to be independently debugged to enable the fusions to work in the same system, new fusions cannot be flexibly added, and the expandability is poor. In addition, the method mainly aims at detecting the known fusion, and does not have the capability of discovering new fusion, thereby possibly leading to the omission of some new fusions. And at present, a device for detecting clonal eosinophilia by a multiplex PCR method has not been used temporarily.

Therefore, a method for detecting a clonal hypereosinophilia fusion gene, which is comprehensive, has higher sensitivity, strong expandability and certain new fusion discovery capability, is needed.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides a clonal eosinophilia-associated hypereosinophilia fusion gene detection probe composition, a kit and application thereof, wherein the related fusion gene is detected by combining liquid phase capture and second-generation transcriptome sequencing, the detection cost of the fusion gene is reduced, the result accuracy is improved, and the clonal eosinophilia-associated hypereosinophilia fusion gene detection probe composition has certain new fusion discovery capability.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a detection probe composition for eosinophilia fusion gene, wherein a target gene of the detection probe composition comprises a class I gene and a class II gene;

the target region of the detection probe composition is the full length of the transcript of the class I gene and the region near the fusion breakpoint of the class II gene;

the class I genes include FGFR1, PDGFRA and PDGFRB;

the class II genes include ABL1, BCR, CCDC88C, CDK5RAP2, CEP110, CEP85L, CPSF6, CUX1, DTD1, ETV6, FGFR1OP1, FGFR1OP2, FIP1L1, FLT3, GIT2, GOLGA4, HIP1, JAK2, KANK1, KIF5B, LRRFIP1, MYO18A, NDE1, NIN, PCM1, PDE4DIP, PRKG2, RABEP1, cc1, STRN, TPM3, TPR, TP53BP1, TRIM24, WDR48, and ZNF 198.

In the invention, firstly, the related fusion genes are divided into two types according to the clinical diagnosis and treatment background of the clonal eosinophilia. Wherein, the cloned eosinophilia polycythemia lipolytica fusion detection core genes FGFR1, PDGFRA and PDGFRB are I-class genes, so the full length of the gene transcript is used as a target region; the other genes are class II genes, and only the vicinity of the fusion break point is used as a target region.

The fusion breakpoint vicinity region in the invention can be a region within 100-300 bp of the fusion breakpoint vicinity, and preferably a region around 200 bp of the fusion breakpoint.

The target genes of the detection probe composition are shown in the following table 1:

the fusion genes detected by the detection probe composition are shown in the following table 2:

preferably, the detection probe composition comprises a nucleic acid sequence shown in SEQ ID number 1-310.

According to the base complementary pairing principle, a complementary sequence of a target region is designed to be used as a probe, and all the probes are dissolved in an IDTE solution to form a liquid phase chip.

Preferably, the probes in the detection probe composition are modified with a labeling molecule I, which may be biotin.

In the invention, the probe exists in a liquid phase, and by carrying a biotin label, the streptavidin magnetic bead can be used for adsorbing the probe and a target region combined with the probe, then denaturation treatment is carried out to separate the probe from the target region, and the magnetic bead is used for absorbing and discarding the probe, thereby realizing capture and enrichment of the target region.

In a second aspect, the present invention provides a kit for detecting a clonal eosinophilia-mediated fusion gene, wherein the kit comprises the detection probe composition of the first aspect.

Preferably, the detection kit further comprises any one or a combination of at least two of a reverse transcription reagent, a sequencing library construction reagent, a PCR reagent or a probe hybridization reagent.

Preferably, the reverse transcription reagent comprises a single-stranded cDNA synthetase and/or a double-stranded cDNA synthetase.

Preferably, the sequencing library building reagent comprises any one of or a combination of at least two of an RNA fragmentation enzyme, a terminal-plus-a tailase, a ligase, or a linker.

Preferably, the PCR reagents comprise a DNA polymerase and/or PCR primers.

Preferably, the probe hybridization reagent comprises a hybridization buffer and/or an rnase inhibitor.

Preferably, the detection kit further comprises a nucleic acid purification reagent.

Preferably, the nucleic acid purification reagent comprises magnetic beads and/or a washing solution.

Preferably, the magnetic beads are modified with a labeling molecule II, and the labeling molecule I can be combined with the labeling molecule II, for example, the magnetic beads can be streptavidin magnetic beads;

it should be noted that, the specific types of the labeling molecules I and II are not limited in the present invention, and only the labeling molecules I and II can be combined with each other, i.e., the biotin and streptavidin magnetic bead system is used in the present invention.

In a third aspect, the present invention provides a method for detecting a clonal hypereosinophilia-associated fusion gene for the purpose of non-disease diagnosis, comprising the following steps:

extracting total RNA in a sample, carrying out mRNA enrichment by using magnetic beads, carrying out thermal interruption on the enriched RNA, carrying out reverse transcription on the RNA into cDNA, carrying out terminal repair, adding A tail, connecting a joint and PCR amplification, and constructing a pre-library; hybridizing a capture target fragment from the pre-library by using the detection probe composition of the first aspect, performing PCR amplification, and constructing a probe capture transcriptome library; and sequencing and data processing the probe-captured transcriptome library, and analyzing the cloned eosinophilia-related fusion gene.

In the present invention, the method for detecting clonal eosinophilia-mediated fusion gene based on targeted capture of transcriptome library is shown in FIG. 1:

(1) designing a probe: designing a detection probe composition aiming at the class I gene and the class II gene; (2) constructing an expression transcript library; (3) enriching target genes: capturing a target gene by using the detection probe composition, eluting non-target cDNA, and purifying to obtain target cDNA; (4) sequencing; (5) and (6) analyzing.

Preferably, the reverse transcription comprises one-strand cDNA synthesis and two-strand cDNA synthesis.

Preferably, the conditions for the single-stranded cDNA synthesis are: taking the fragmented RNA as a template, adding components required by one-strand synthesis, and carrying out total reaction of 25 μ L (hot cover 105 ℃, 25 ℃ for 10min, 42 ℃ for 15min, 70 ℃ for 15min, 4 ℃ hold);

preferably, the conditions for the two-strand cDNA synthesis are: using the single-stranded cDNA as a template, 65. mu.L of the total reaction system (hot lid 105 ℃, 16 ℃ for 30 min, 65 ℃ for 15min, 4 ℃ hold) was prepared.

Preferably, the sequencing of the capture transcriptome library is performed in a paired-end sequencing mode, and the data processing comprises:

(1) aligning the read obtained by sequencing to a reference genome, and screening the read which cannot be completely matched with a single gene as a candidate fusion gene sequence, wherein the candidate fusion gene sequence comprises joint reads (Junction reads) and cross-breakpoint reads (bridging reads) as shown in FIG. 2;

(2) comparing the candidate fusion gene sequence with a reference genome annotation file, and predicting the fusion gene according to the overlapping condition of the reading sequence;

(3) and performing correction analysis according to the minimum reading support criterion and a checking tool, filtering a prediction result, and removing false positive fusion.

In the present invention, the conditions for determining the final positive fusion gene are: the sum of the numbers of the supported binding reading sequences and cross breakpoint reading sequences of the candidate fusion genes after the data processing is not less than 4.

In a fourth aspect, the present invention provides a detecting apparatus for a clonal hypereosinophilia-associated fusion gene, comprising:

a nucleic acid extraction module: for extracting RNA from a clonal eosinophilia sample;

a reverse transcription module: for reverse transcription of the extracted RNA into cDNA;

pre-library construction module: the method is used for carrying out terminal repair, A tail addition, linker connection and PCR amplification on the reverse transcription cDNA to construct a pre-library;

a hybrid capture module: capturing a target fragment from a pre-library by using the detection probe composition of the first aspect, and performing PCR amplification to construct a probe capture library;

a sequencing module: for sequencing and data processing of the probe capture library.

In a fifth aspect, the invention provides a use of the detection probe composition of the first aspect, the detection kit of the second aspect or the detection apparatus of the fourth aspect in the preparation of a diagnostic product for clonal hypereosinophilia.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a detecting probe composition for eosinophilia fusion genes, which takes clonal eosinophilia fusion detection core genes FGFR1, PDGFRA and PDGFRB as class I genes, takes the full length of a transcript of the class I gene and the area near the fusion end point of the class II gene as target areas respectively, and takes the gene as a gene carrier of the class II genes; the cloned eosinophilia fusion gene is detected by combining liquid phase capture and second-generation transcriptome sequencing, so that the sequencing cost required by the fusion gene detection is lower, the sensitivity is higher, meanwhile, the method disclosed by the invention also has the capability of new fusion discovery, and the result is more reliable.

Drawings

FIG. 1 is a schematic flow chart of the detection of clonal eosinophilia fusion genes based on the sequencing of a targeted capture transcriptome library.

FIG. 2 is a view showing the reading pattern of the fusion of the gene for clonal hypereosinophilia in the present invention.

FIG. 3 is a general technical roadmap for the detection method of the present invention.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1

This example performed a fusion assay on bone marrow samples from patients with clonal eosinophilia.

(1) Nucleic acid extraction

The sample and EL buffer were added at a ratio of 1:5 and incubated on ice, vortexed briefly for 2 times during incubation to lyse red blood cells, followed by 400 ℃ at 4 ℃×gCentrifuging for 10min, and discarding the supernatant; adding EL buffer solution (2 times of sample volume) at a ratio of 1:2, mixing by brief vortex, and stirring at 400 deg.C×gCentrifuging for 10min, and discarding the supernatant;

adding a proper RLT buffer solution according to the volume of the sample to dissolve the white blood cells, uniformly mixing by vortex or blowing, transferring to a lysis column, centrifuging at the maximum rotation speed for 2 min, and collecting filtrate; adding 1 volume time of 70% ethanol into the filtrate, blowing, mixing, transferring to a filter column, 8000×gCentrifuging for 15s, and discarding the collection pipe; transfer the filter column to a fresh collection tube and add 350. mu.L RW1 buffer, 8000×gCentrifuging for 15s, and discarding the filtrate;

slightly and uniformly mixing 10 mu L of DNase I and 70 mu L of RDD buffer solution, adding the mixture into a filter column, and incubating for 15min at 30 ℃; 350. mu.L of RW1 buffer, 8000×gCentrifuging for 15s, and discarding the collection pipe; transfer the filter column to a fresh collection tube and add 500. mu.L of RPE buffer, 8000×gCentrifuging for 15s, and discarding the filtrate;

add 500. mu.L RPE buffer, 20000×gCentrifuging for 3 min, discarding the filtrate, transferring the filter column to a new collecting tube, centrifuging at full speed for 1min, and discarding the centrifuge tube; the filter column was transferred to a 1.5 mL centrifuge tube and 50. mu.L of nucleic-free H was added₂O, standing at room temperature for 5min at 8000×gCentrifuging for 1min to obtain filtrate as the extracted RNA;

(2) mRNA enrichment

The RNA extracted above was quantified using a Qubit, 1000 ng of RNA was put into a 1.5 mL centrifuge tube, the volume was made up to 50. mu.L with ribozyme-free water, and the RNA was pipetted and mixed on ice for further use.

Preparing a mixed solution in advance: the mixture was prepared on ice according to table 3 below, vortexed, and centrifuged instantaneously.

Aspirate 20. mu.L of Oligo dT Beads d (T) and mix well with vortex, equilibrate at room temperature for more than 30 min₂₅And washing twice with 100 μ L of RNA 2 × Binding Buffer;

adding 50 mu L of 2 XRNA Binding Buffer into each reaction, and uniformly mixing by blowing for 10 times; transferring 50 mu L of magnetic bead solution into a 50 mu L of Total RNA reaction tube, and blowing, beating and uniformly mixing;

carrying out reaction in a PCR instrument (thermal cover is more than or equal to 75 ℃, incubation is carried out at 65 ℃ for 5min, Hold is carried out at 4 ℃, and the reaction system is 100 muL);

after the reaction is finished, uniformly blowing and stirring the product, and standing for 5min at room temperature; transferring the centrifuge tube to a magnetic rack until the liquid is completely clarified (about 2 min), removing the supernatant, and retaining the magnetic beads;

taking the centrifugal tube off the magnetic frame, washing twice by 200 mu L of Wash Buffer, adding 50 mu L of Tris Buffer into the reaction tube, and blowing and uniformly mixing; carrying out reaction in a PCR instrument (thermal cover is more than or equal to 90 ℃, incubation is carried out for 2 min at 80 ℃, Hold is carried out at 25 ℃, and the reaction system is 50 muL);

after the reaction is finished, taking down the product from the PCR instrument, adding 50 mu L of 2 xRNA Binding Buffer into a reaction tube, blowing and mixing uniformly, and incubating for 5min at room temperature;

transferring the centrifuge tube to a magnetic rack until the liquid is completely clarified (about 2 min), removing the supernatant, and retaining the magnetic beads; the reaction tube was washed once with 200. mu.L of Wash Buffer.

(3) mRNA Pre-library construction

Taking down the centrifugal tube from the magnetic frame, adding 11.5 mu L of the mixed solution prepared in advance into the reaction tube, and blowing, beating and uniformly mixing;

carrying out reaction in a PCR instrument (thermal cover is more than or equal to 105 ℃, incubation is carried out at 94 ℃ for 10min, Hold is carried out at 4 ℃, and the reaction system is 12 muL); when the temperature of the sample is reduced to 65 ℃, the sample is quickly taken out and placed on ice to stand for 1 min.

The centrifuge tube was transferred to a magnetic rack until the liquid was completely clear (about 2 min); carefully pipette 10. mu.L of the supernatant into a new 200. mu.L PCR tube, discard the beads, and place on ice;

preparing a chain of synthetic mixed solution on ice according to table 4, blowing, beating, mixing uniformly, and performing instantaneous centrifugation:

carrying out reaction in a PCR instrument (the thermal cover is more than or equal to 80 ℃, the incubation is carried out for 10min at 25 ℃, the incubation is carried out for 15min at 42 ℃, the incubation is carried out for 15min at 70 ℃, the Hold is carried out at 4 ℃, and the reaction system is 20 muL);

preparing a two-chain synthesis mixed solution on ice according to the following table 5, blowing, beating and mixing uniformly, and performing instantaneous centrifugation:

the reaction was carried out in a PCR instrument (hot lid. ltoreq.40 ℃, 16 ℃ incubation for 60 min, 4 ℃ Hold, reaction system: 80 μ L)

Adding an End Prep Reaction Buffer and an End Prep Enzyme Mix into ds cDNA to prepare a connection system with the total volume of 60 mu L, carrying out Reaction in a PCR instrument (a hot cover is more than or equal to 75 ℃, incubation is carried out at 20 ℃ for 30 min, incubation is carried out at 65 ℃ for 30 min, Hold is carried out at 4 ℃, the Reaction system is 60 mu L), and carrying out ice operation in the whole process;

adding the Ligation Master Mix, Ligation Enhancer, Adapter and nucleic-free H₂O preparing a connecting system with the total volume of 93.5 mu L, performing connecting reaction in a PCR instrument (hot cover 105 ℃, incubation at 20 ℃ for 15min, Hold at 4 ℃), and performing ice operation in the whole process;

purifying the ligation product, adding a PCR Primer Mix and an Ultra II Q5 Master Mix into the purified adaptor ligation product to prepare a PCR reaction system with the total volume of 50 mu L, and performing library amplification reaction in a PCR instrument (hot cover 105 ℃, pre-denaturation at 98 ℃ for 30 s, denaturation at 98 ℃ for 10 s, annealing and extension at 65 ℃ for 75 s, amplification for 10 cycles, complete extension at 65 ℃ for 5min, and Hold at 4 ℃);

and purifying the PCR product to obtain a purified product, namely the pre-library.

Example 2 fusion Gene-related fragment Capture of interest

(1) Capturing target fragments

Putting the pre-library with the total amount of 2 mu g into a new 1.5 mL centrifuge tube, adding 5 mu L of Human Cot-1 DNA and 2 mu L of Universal bottles, covering a tube cover, pricking the tube cover with a disposable syringe, putting the tube cover into a vacuum concentrator, and drying at the constant temperature of 60 ℃ until no liquid exists;

adding a total volume of 17 μ L of Hybridization solution (including 2 × Hybridization Buffer, Hybridization Buffer Enhancer, probe composition and nucleic-Free Water, wherein the concentration of the probe composition in the reaction system is 400 amol/probe/rxn), mixing, standing at room temperature for 5min, transferring to a PCR tube, and running HYB program (hot lid 100 ℃, 95 ℃ for 30 s, 65 ℃ for 4 h, 65 ℃ Hold);

after the HYB program is operated, transferring the cleaned 17 mu L M-270 magnetic bead solution into a hybridization reaction tube, blowing and uniformly mixing, and incubating for 45 min at 65 ℃ in a PCR instrument;

after incubation at 65 ℃, adding 100 mu L of 1 × Wash Buffer1 preheated at 65 ℃ into a hybridization reaction tube, blowing and uniformly mixing, transferring the centrifugal tube to a magnetic frame until the liquid is completely clarified, sucking and removing the supernatant, and keeping magnetic beads;

taking down the centrifuge tube, adding 150 μ L of 1 × Stringent Wash Buffer preheated at 65 deg.C, blowing, mixing, incubating at 65 deg.C for 5min, transferring the centrifuge tube to magnetic frame until the liquid is completely clarified, removing supernatant, and retaining magnetic beads; repeating the steps once;

taking down the centrifuge tube, adding 150 mu L of room temperature 1 × Wash Buffer1, washing, removing supernatant and keeping magnetic beads; then adding 150 mu L of room temperature 1 XWash Buffer 2, washing, removing supernatant by suction, and keeping magnetic beads; 150 μ L of room temperature 1 × Wash Buffer3 was added, washed, the supernatant was aspirated off, the beads were retained, and 20 μ L of nucleic-free H was added₂And O, blowing, beating and mixing uniformly for later use.

(2) Capture library amplification and sequencing

Adding 30 mu L of PCR Master Mix into the magnetic bead resuspension solution, blowing, uniformly mixing, and carrying out library amplification reaction in a PCR instrument (hot cover 105 ℃, pre-denaturation at 98 ℃ for 45 s, denaturation at 98 ℃ for 15s, annealing at 60 ℃ for 30 s, extension at 72 ℃ for 30 s, amplification for 7-9 cycles, complete extension at 72 ℃ for 1min, and Hold at 4 ℃);

purifying the PCR product to obtain a purified product, namely a probe capture library;

performing double-ended sequencing by adopting a well-known Illumina NGS sequencing technology, and analyzing and processing the obtained double-ended sequencing data by the following steps:

1) comparing the read sequences obtained by sequencing with a reference genome, and screening out the read sequences which can not be completely matched with a single gene as candidate fusion gene sequences;

2) and comparing the candidate fusion gene sequence with the reference genome annotation file, and predicting the fusion gene according to the overlapping condition of the reading sequence. Performing further correction analysis according to the minimum reading support criterion and by using a checking tool;

3) the prediction results were filtered to remove false positive fusions.

The final positive fusion gene determination conditions were: the sum of the numbers of the supported binding reading sequences and cross breakpoint reading sequences of the candidate fusion genes after the data processing is not less than 4.

Example 3

This example uses bone marrow samples from 6 clinically suspected eosinophilia patients.

The detection method comprises the steps as shown in fig. 3: firstly, extracting RNA in a sample, then carrying out mRNA enrichment, and then carrying out pre-library construction, wherein the pre-library construction comprises the following steps: performing RNA thermal interruption, performing reverse transcription on fragmented RNA to convert the fragmented RNA into cDNA, and performing terminal repair, A tail addition, linker addition and PCR amplification; then, the probe composition of the present invention is used for hybrid capture, which comprises: closing library DNA, washing with magnetic beads, hybridizing, eluting to obtain a capture library, amplifying, and purifying to obtain an on-machine detection;

the specific implementation steps are shown in example 1 and example 2.

The results are shown in Table 6, and in 6 clinically suspected eosinophilia patients, MYO18A-PDGFRB fusion was detected in one case, ETV6-ABL1 fusion was detected in two cases, CUX1-FGFR1 fusion was detected in one case, and related genes were not detected in the other two cases.

Meanwhile, in the present embodiment, the second-generation high-throughput transcriptome sequencing method is adopted for synchronous detection, and it is found that the detection results of 5 samples in total of TR02-TR06 are the same as that in the present embodiment, while no relevant fusion is detected in the TR01 sample by adopting the transcriptome sequencing method.

The above results show that the method for detecting the clonal eosinophilia-polycythemia fusion gene by targeted capturing RNA-seq has higher sensitivity, stronger detection capability on low-expression fusion transcripts and lower cost compared with the RNA-seq.

In conclusion, the clonal eosinophilia-associated hypereosinophilia fusion gene detection probe composition, the kit constructed by applying the same and the detection method provided by the invention enable the sequencing cost required for detecting a sample to be detected to be lower, the sensitivity to be higher and the result to be accurate and reliable.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Mejie transformation medical research (Suzhou) Co., Ltd

<120> clonal eosinophilia hypereosinophilia fusion gene detection probe composition, kit and application thereof

<130> 20210527

<160> 310

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<400> 12

tgaagtcaaa cattttgttg tagaggtgcg ggcctaccca cctcccagga tatcctggct 60

gaaaaacaat ctgactctga ttgaaaatct cactgagatc accactgatg tggaaaagat 120

<210> 13

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 13

tcaggaaata aggtatcgaa gcaaattaaa gctgatccgt gctaaggaag aagacagtgg 60

ccattatact attgtagctc aaaatgaaga tgctgtgaag agctatactt ttgaactgtt 120

<210> 14

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 14

aactcaagtt ccttcatcca ttctggactt ggtcgatgat caccatggct caactggggg 60

acagacggtg aggtgcacag ctgaaggcac gccgcttcct gatattgagt ggatgatatg 120

<210> 15

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 15

caaagatatt aagaaatgta ataatgaaac ttcctggact attttggcca acaatgtctc 60

aaacatcatc acggagatcc actcccgaga caggagtacc gtggagggcc gtgtgacttt 120

<210> 16

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 16

cgccaaagtg gaggagacca tcgccgtgcg atgcctggct aagaatctcc ttggagctga 60

gaaccgagag ctgaagctgg tggctcccac cctgcgttct gaactcacgg tggctgctgc 120

<210> 17

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 17

agtcctggtg ctgttggtga ttgtgatcat ctcacttatt gtcctggttg tcatttggaa 60

acagaaaccg aggtatgaaa ttcgctggag ggtcattgaa tcaatcagcc cagatggaca 120

<210> 18

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 18

tgaatatatt tatgtggacc cgatgcagct gccttatgac tcaagatggg agtttccaag 60

agatggacta gtgcttggtc gggtcttggg gtctggagcg tttgggaagg tggttgaagg 120

<210> 19

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 19

aacagcctat ggattaagcc ggtcccaacc tgtcatgaaa gttgcagtga agatgctaaa 60

acccacggcc agatccagtg aaaaacaagc tctcatgtct gaactgaaga taatgactca 120

<210> 20

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 20

cctggggcca catttgaaca ttgtaaactt gctgggagcc tgcaccaagt caggccccat 60

ttacatcatc acagagtatt gcttctatgg agatttggtc aactatttgc ataagaatag 120

<210> 21

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 21

ggatagcttc ctgagccacc acccagagaa gccaaagaaa gagctggata tctttggatt 60

gaaccctgct gatgaaagca cacggagcta tgttatttta tcttttgaaa acaatggtga 120

<210> 22

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 22

ctacatggac atgaagcagg ctgatactac acagtatgtc cccatgctag aaaggaaaga 60

ggtttctaaa tattccgaca tccagagatc actctatgat cgtccagcct catataagaa 120

<210> 23

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 23

gaaatctatg ttagactcag aagtcaaaaa cctcctttca gatgataact cagaaggcct 60

tactttattg gatttgttga gcttcaccta tcaagttgcc cgaggaatgg agtttttggc 120

<210> 24

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 24

ttcaaaaaat tgtgtccacc gtgatctggc tgctcgcaac gtcctcctgg cacaaggaaa 60

aattgtgaag atctgtgact ttggcctggc cagagacatc atgcatgatt cgaactatgt 120

<210> 25

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 25

gtcgaaaggc agtacctttc tgcccgtgaa gtggatggct cctgagagca tctttgacaa 60

cctctacacc acactgagtg atgtctggtc ttatggcatt ctgctctggg agatcttttc 120

<210> 26

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 26

ccttggtggc accccttacc ccggcatgat ggtggattct actttctaca ataagatcaa 60

gagtgggtac cggatggcca agcctgacca cgctaccagt gaagtctacg agatcatggt 120

<210> 27

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 27

gaaatgctgg aacagtgagc cggagaagag accctccttt taccacctga gtgagattgt 60

ggagaatctg ctgcctggac aatataaaaa gagttatgaa aaaattcacc tggacttcct 120

<210> 28

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 28

gaagagtgac catcctgctg tggcacgcat gcgtgtggac tcagacaatg catacattgg 60

tgtcacctac aaaaacgagg aagacaagct gaaggactgg gagggtggtc tggatgagca 120

<210> 29

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 29

gagactgagc gctgacagtg gctacatcat tcctctgcct gacattgacc ctgtccctga 60

ggaggaggac ctgggcaaga ggaacagaca cagctcgcag acctctgaag agagtgccat 120

<210> 30

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 30

tgagacgggt tccagcagtt ccaccttcat caagagagag gacgagacca ttgaagacat 60

cgacatgatg gatgacatcg gcatagactc ttcagacctg gtggaagaca gcttcctgta 120

<210> 31

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 31

actggcggat tcgaggggtt ccttccactt ctggggccac ctctggatcc cgttcagaaa 60

accactttat tgcaatgcag aggttgagag gaggacttgg ttgatgttta aagagaagtt 120

<210> 32

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 32

cccagccaag ggcctcgggg agcgttctaa atatgaatga atgggatatt ttgaaatgaa 60

ctttgtcagt gttgcctctt gcaatgcctc agtagcatct cagtggtgtg tgaagtttgg 120

<210> 33

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 33

agatagatgg ataagggaat aataggccac agaaggtgaa ctttgtgctt caaggacatt 60

ggtgagagtc caacagacac aatttatact gcgacagaac ttcagcattg taattatgta 120

<210> 34

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 34

aataactcta accaaggctg tgtttagatt gtattaacta tcttctttgg acttctgaag 60

agaccactca atccatccat gtacttccct cttgaaacct gatgtcagct gctgttgaac 120

<210> 35

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 35

tttttaaaga agtgcatgaa aaaccatttt tgaaccttaa aaggtactgg tactatagca 60

ttttgctatc ttttttagtg ttaaagagat aaagaataat aattaaccaa ccttgtttaa 120

<210> 36

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 36

tagatttggg tcatttagaa gcctgacaac tcattttcat attgtaatct atgtttataa 60

tactactact gttatcagta atgctaaatg tgtaataatg taacatgatt tccctccaga 120

<210> 37

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 37

gaaagcacaa tttaaaacaa tccttactaa gtaggtgatg agtttgacag tttttgacat 60

ttatattaaa taacatgttt ctctataaag tatggtaata gctttagtga attaaattta 120

<210> 38

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 38

gttgagcata gagaacaaag taaaagtagt gttgtccagg aagtcagaat ttttaactgt 60

actgaatagg ttccccaatc catcgtatta aaaaacaatt aactgccctc tgaaataatg 120

<210> 39

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 39

ggattagaaa caaacaaaac tcttaagtcc taaaagttct caatgtagag gcataaacct 60

gtgctgaaca taacttctca tgtatattac ccaatggaaa atataatgat cagcaaaaag 120

<210> 40

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 40

actggatttg cagaagtttt tttttttttt ttcttcatgc ctgatgaaag ctttggcgac 60

cccaatatat gtattttttg aatctatgaa cctgaaaagg gtcagaagga tgcccagaca 120

<210> 41

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 41

tcagcctcct tctttcaccc cttaccccaa agagaaagag tttgaaactc gagaccataa 60

agatattctt tagtggaggc tggatgtgca ttagcctgga tcctcagttc tcaaatgtgt 120

<210> 42

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 42

gtggcagcca ggatgactag atcctgggtt tccatccttg agattctgaa gtatgaagtc 60

tgagggaaac cagagtctgt atttttctaa actccctggc tgttctgatc ggccagtttt 120

<210> 43

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 43

cggaaacact gacttaggtt tcaggaagtt gccatgggaa acaaataatt tgaactttgg 60

aacagggttg gcattcaacc acgcaggaag cctactattt aaatccttgg cttcaggtta 120

<210> 44

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 44

gtgacattta atgccatcta gctagcaatt gcgaccttaa tttaactttc cagtcttagc 60

tgaggctgag aaagctaaag tttggttttg acaggttttc caaaagtaaa gatgctactt 120

<210> 45

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 45

cccactgtat gggggagatt gaactttccc cgtctcccgt cttctgcctc ccactccata 60

ccccgccaag gaaaggcatg tacaaaaatt atgcaattca gtgttccaag tctctgtgta 120

<210> 46

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 46

accagctcag tgttttggtg gaaaaaacat tttaagtttt actgataatt tgaggttaga 60

tgggaggatg aattgtcaca tctatccaca ctgtcaaaca ggttggtgtg ggttcattgg 120

<210> 47

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 47

cattctttgc aatactgctt aattgctgat accatatgaa tgaaacatgg gctgtgatta 60

ctgcaatcac tgtgctatcg gcagatgatg ctttggaaga tgcagaagca ataataaagt 120

<210> 48

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 48

acttgactac ctactggtgt aatctcaatg caagccccaa ctttcttatc caactttttc 60

atagtaagtg cgaagactga gccagattgg ccaattaaaa acgaaaacct gactaggttc 120

<210> 49

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 49

tgtagagcca attagacttg aaatacgttt gtgtttctag aatcacagct caagcattct 60

gtttatcgct cactctccct tgtacagcct tattttgttg gtgctttgca ttttgatatt 120

<210> 50

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 50

gctgtgagcc ttgcatgaca tcatgaggcc ggatgaaact tctcagtcca gcagtttcca 60

gtcctaacaa atgctcccac ctgaatttgt atatgactgc atttgtgtgt gtgtgtgtgt 120

<210> 51

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 51

tttcagcaaa ttccagattt gtttcctttt ggcctcctgc aaagtctcca gaagaaaatt 60

tgccaatctt tcctactttc tatttttatg atgacaatca aagccggcct gagaaacact 120

<210> 52

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 52

atttgtgact ttttaaacga ttagtgatgt ccttaaaatg tggtctgcca atctgtacaa 60

aatggtccta tttttgtgaa gagggacata agataaaatg atgttataca tcaatatgta 120

<210> 53

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 53

tatatgtatt tctatataga cttggagaat actgccaaaa catttatgac aagctgtatc 60

actgccttcg tttatatttt tttaactgtg ataatcccca caggcacatt aactgttgca 120

<210> 54

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 54

cttttgaatg tccaaaattt atattttaga aataataaaa agaaagatac ttacatgttc 60

ccaaaacaat ggtgtggtga atgtgtgaga aaaactaact tgatagggtc taccaataca 120

<210> 55

<211> 94

<212> DNA

<213> Artificial Synthesis

<400> 55

aaatgtatta cgaatgcccc tgttcatgtt tttgttttaa aacgtgtaaa tgaagatctt 60

tatatttcaa taaatgatat ataatttaaa gtta 94

<210> 56

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 56

agatgcaggg gcgcaaacgc caaaggagac caggctgtag gaagagaagg gcagagcgcc 60

ggacagctcg gcccgctccc cgtcctttgg ggccgcggct ggggaactac aaggcccagc 120

<210> 57

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 57

gcaggcagct gcagggggcg gaggcggagg agggaccagc gcgggtggga gtgagagagc 60

gagccctcgc gccccgccgg cgcatagcgc tcggagcgct cttgcggcca caggcgcggc 120

<210> 58

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 58

gcgtcctcgg cggcgggcgg cagctagcgg gagccgggac gccggtgcag ccgcagcgcg 60

cggaggaacc cgggtgtgcc gggagctggg cggccacgtc cggacgggac cgagacccct 120

<210> 59

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 59

ctcgtagcgc attgcggcga cctcgccttc cccggccgcg agcgcgccgc tgcttgaaaa 60

gccgcggaac ccaaggactt ttctccggtc cgagctcggg gcgccccgca gggcgcacgg 120

<210> 60

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 60

ggtacccgtg ctgcagtcgg gcacgccgcg gcgccggggc ctccgcaggg cgatggagcc 60

cggtctgcaa ggaaagtgag gcgccgccgc tgcgttctgg aggagggggg cacaaggtct 120

<210> 61

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 61

ctggagaccc cgggtggcgg acgggagccc tccccccgcc ccgcctccgg ggcaccagct 60

ccggctccat tgttcccgcc cgggctggag gcgccgagca ccgagcgccg ccgggagtcg 120

<210> 62

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 62

cgagcgccgg ccgcggagct cttgcgaccc cgccaggacc cgaacagagc ccgggggcgg 60

cgggccggag ccggggacgc gggcacacgc ccgctcgcac aagccacggc ggactctccc 120

<210> 63

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 63

ccgaggcgga acctccacgc cgagcgaggg tcagtttgaa aaggaggatc gagctcactg 60

tggagtatcc atggagatgt ggagccttgt caccaacctc taactgcaga actgggatgt 120

<210> 64

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 64

gtggagctgg aagtgcctcc tcttctgggc tgtgctggtc acagccacac tctgcaccgc 60

taggccgtcc ccgaccttgc ctgaacaagc ccagccctgg ggagcccctg tggaagtgga 120

<210> 65

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 65

gagtccttcc tggtccaccc cggtgacctg ctgcagcttc gctgtcggct gcgggacgat 60

gtgcagagca tcaactggct gcgggacggg gtgcagctgg cggaaagcaa ccgcacccgc 120

<210> 66

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 66

gcatcacagg ggaggaggtg gaggtgcagg actccgtgcc cgcagactcc ggcctctatg 60

cttgcgtaac cagcagcccc tcgggcagtg acaccaccta cttctccgtc aatgtttcag 120

<210> 67

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 67

agatgctctc ccctcctcgg aggatgatga tgatgatgat gactcctctt cagaggagaa 60

agaaacagat aacaccaaac caaaccgtat gcccgtagct ccatattgga catccccaga 120

<210> 68

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 68

gaaaagatgg aaaagaaatt gcatgcagtg ccggctgcca agacagtgaa gttcaaatgc 60

ccttccagtg ggaccccaaa ccccacactg cgctggttga aaaatggcaa agaattcaaa 120

<210> 69

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 69

aacctgacca cagaattgga ggctacaagg tccgttatgc cacctggagc atcataatgg 60

actctgtggt gccctctgac aagggcaact acacctgcat tgtggagaat gagtacggca 120

<210> 70

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 70

cagcatcaac cacacatacc agctggatgt cgtggagcgg tcccctcacc ggcccatcct 60

gcaagcaggg ttgcccgcca acaaaacagt ggccctgggt agcaacgtgg agttcatgtg 120

<210> 71

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 71

tgtaaggtgt acagtgaccc gcagccgcac atccagtggc taaagcacat cgaggtgaat 60

gggagcaaga ttggcccaga caacctgcct tatgtccaga tcttgaagac tgctggagtt 120

<210> 72

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 72

ttaataccac cgacaaagag atggaggtgc ttcacttaag aaatgtctcc tttgaggacg 60

caggggagta tacgtgcttg gcgggtaact ctatcggact ctcccatcac tctgcatggt 120

<210> 73

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 73

gttgaccgtt ctggaagccc tggaagagag gccggcagtg atgacctcgc ccctgtacct 60

ggagatcatc atctattgca caggggcctt cctcatctcc tgcatggtgg ggtcggtcat 120

<210> 74

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 74

atcgtctaca agatgaagag tggtaccaag aagagtgact tccacagcca gatggctgtg 60

cacaagctgg ccaagagcat ccctctgcgc agacaggtaa cagtgtctgc tgactccagt 120

<210> 75

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 75

gtgcatccat gaactctggg gttcttctgg ttcggccatc acggctctcc tccagtggga 60

ctcccatgct agcaggggtc tctgagtatg agcttcccga agaccctcgc tgggagctgc 120

<210> 76

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 76

gcctcgggac agactggtct taggcaaacc cctgggagag ggctgctttg ggcaggtggt 60

gttggcagag gctatcgggc tggacaagga caaacccaac cgtgtgacca aagtggctgt 120

<210> 77

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 77

gtgaagatgt tgaagtcgga cgcaacagag aaagacttgt cagacctgat ctcagaaatg 60

gagatgatga agatgatcgg gaagcataag aatatcatca acctgctggg ggcctgcacg 120

<210> 78

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 78

cgcaggatgg tcccttgtat gtcatcgtgg agtatgcctc caagggcaac ctgcgggagt 60

acctgcaggc ccggaggccc ccagggctgg aatactgcta caaccccagc cacaacccag 120

<210> 79

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 79

agaggagcag ctctcctcca aggacctggt gtcctgcgcc taccaggtgg cccgaggcat 60

ggagtatctg gcctccaaga agtgcataca ccgagacctg gcagccagga atgtcctggt 120

<210> 80

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 80

gtgacagagg acaatgtgat gaagatagca gactttggcc tcgcacggga cattcaccac 60

atcgactact ataaaaagac aaccaacggc cgactgcctg tgaagtggat ggcacccgag 120

<210> 81

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 81

aggcattatt tgaccggatc tacacccacc agagtgatgt gtggtctttc ggggtgctcc 60

tgtgggagat cttcactctg ggcggctccc cataccccgg tgtgcctgtg gaggaacttt 120

<210> 82

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 82

tttcaagctg ctgaaggagg gtcaccgcat ggacaagccc agtaactgca ccaacgagct 60

gtacatgatg atgcgggact gctggcatgc agtgccctca cagagaccca ccttcaagca 120

<210> 83

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 83

cagctggtgg aagacctgga ccgcatcgtg gccttgacct ccaaccagga gtacctggac 60

ctgtccatgc ccctggacca gtactccccc agctttcccg acacccggag ctctacgtgc 120

<210> 84

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 84

gctcctcagg ggaggattcc gtcttctctc atgagccgct gcccgaggag ccctgcctgc 60

cccgacaccc agcccagctt gccaatggcg gactcaaacg ccgctgactg ccacccacac 120

<210> 85

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 85

acgccctccc cagactccac cgtcagctgt aaccctcacc cacagcccct gctgggccca 60

ccacctgtcc gtccctgtcc cctttcctgc tggcaggagc cggctgccta ccaggggcct 120

<210> 86

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 86

cttcctgtgt ggcctgcctt caccccactc agctcacctc tccctccacc tcctctccac 60

ctgctggtga gaggtgcaaa gaggcagatc tttgctgcca gccacttcat cccctcccag 120

<210> 87

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 87

agatgttgga ccaacacccc tccctgccac caggcactgc ctggagggca gggagtggga 60

gccaatgaac aggcatgcaa gtgagagctt cctgagcttt ctcctgtcgg tttggtctgt 120

<210> 88

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 88

gttttgcctt cacccataag cccctcgcac tctggtggca ggtgccttgt cctcagggct 60

acagcagtag ggaggtcagt gcttcgtgcc tcgattgaag gtgacctctg ccccagatag 120

<210> 89

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 89

aggtggtgcc agtggcttat taattccgat actagtttgc tttgctgacc aaatgcctgg 60

taccagagga tggtgaggcg aaggccaggt tgggggcagt gttgtggccc tggggcccag 120

<210> 90

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 90

agccccaaac tgggggctct gtatatagct atgaagaaaa cacaaagtgt ataaatctga 60

gtatatattt acatgtcttt ttaaaagggt cgttaccaga gatttaccca tcgggtaaga 120

<210> 91

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 91

gatgctcctg gtggctggga ggcatcagtt gctatatatt aaaaacaaaa aagaaaaaaa 60

aggaaaatgt ttttaaaaag gtcatatatt ttttgctact tttgctgttt tattttttta 120

<210> 92

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 92

taaattatgt tctaaaccta ttttcagttt aggtccctca ataaaaattg ctgctgcttc 60

atttatctat gggctgtatg aaaagggtgg gaatgtccac tggaaagaag ggacacccac 120

<210> 93

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 93

acgggccctg gggctaggtc tgtcccgagg gcaccgcatg ctcccggcgc aggttccttg 60

taacctcttc ttcctaggtc ctgcacccag acctcacgac gcacctcctg cctctccgct 120

<210> 94

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 94

ctgcttttgg aaagtcagaa aaagaagatg tctgcttcga gggcaggaac cccatccatg 60

cagtagaggc gctgggcaga gagtcaaggc ccagcagcca tcgaccatgg atggtttcct 120

<210> 95

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 95

ctccaaggaa accggtgggg ttgggctggg gagggggcac ctacctagga atagccacgg 60

ggtagagcta cagtgattaa gaggaaagca agggcgcggt tgctcacgcc tgtaatccca 120

<210> 96

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 96

cagcactttg ggacaccgag gtgggcagat cacttcaggt caggagtttg agaccagcct 60

ggccaactta gtgaaacccc atctctacta aaaatgcaaa aattatccag gcatggtggc 120

<210> 97

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 97

gcacacgcct gtaatcccag ctccacagga ggctgaggca gaatcccttg aagctgggag 60

gcggaggttg cagtgagccg agattgcgcc attgcactcc agcctgggca acagagaaaa 120

<210> 98

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 98

aacaaaaagg aaaacaaatg atgaaggtct gcagaaactg aaacccagac atgtgtctgc 60

cccctctatg tgggcatggt tttgccagtg cttctaagtg caggagaaca tgtcacctga 120

<210> 99

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 99

gaggctagtt ttgcattcag gtccctggct tcgtttcttg ttggtatgcc tccccagatc 60

gtccttcctg tatccatgtg accagactgt atttgttggg actgtcgcag atcttggctt 120

<210> 100

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 100

ttcttacagt tcttcctgtc caaactccat cctgtccctc aggaacgggg ggaaaattct 60

ccgaatgttt ttggtttttt ggctgcttgg aatttacttc tgccacctgc tggtcatcac 120

<210> 101

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 101

actgtcctca ctaagtggat tctggctccc ccgtacctca tggctcaaac taccactcct 60

cagtcgctat attaaagctt atattttgct ggattactgc taaatacaaa agaaagttca 120

<210> 102

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 102

caatatgttt tcatttctgt agggaaaatg ggattgctgc tttaaatttc tgagctaggg 60

attttttggc agctgcagtg ttggcgacta ttgtaaaatt ctctttgttt ctctctgtaa 120

<210> 103

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 103

aaatagcacc tgctaacatt acaatttgta tttatgttta aagaaggcat catttggtga 60

acagaactag gaaatgaatt tttagctctt aaaagcattt gctttgagac cgcacaggag 120

<210> 104

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 104

agtgtctttc cttgtaaaac agtgatgata atttctgcct tggccctacc ttgaagcaat 60

gttgtgtgaa gggatgaaga atctaaaagt cttcataagt ccttgggaga ggtgctagaa 120

<210> 105

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 105

aaaaatataa ggcactatca taattacagt gatgtccttg ctgttactac tcaaatcacc 60

cacaaatttc cccaaagact gcgctagctg tcaaataaaa gacagtgaaa ttgacctgaa 120

<210> 106

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 106

ctcctgaggc tgccagcagc cagcagtgac tgcccgccct atctgggacc caggatcgct 60

ctgtgagcaa cttggagcca gagaggagat caacaaggag gaggagagag ccggcccctc 120

<210> 107

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 107

cagccctgct gcccagcagc agcctgtgct cgccctgccc aacgcagaca gccagaccca 60

gggcggcccc tctggcggct ctgctcctcc cgaaggatgc ttggggagtg aggcgaagct 120

<210> 108

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 108

tgggccgctc ctctccccta cagcagcccc cttcctccat ccctctgttc tcctgagcct 60

tcaggagcct gcaccagtcc tgcctgtcct tctactcagc tgttacccac tctgggacca 120

<210> 109

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 109

agcagtcttt ctgataactg ggagagggca gtaaggagga cttcctggag ggggtgactg 60

tccagagcct ggaactgtgc ccacaccaga agccatcagc agcaaggaca ccatgcggct 120

<210> 110

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 110

ttccgggtgc gatgccagct ctggccctca aaggcgagct gctgttgctg tctctcctgt 60

tacttctgga accacagatc tctcagggcc tggtcgtcac acccccgggg ccagagcttg 120

<210> 111

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 111

gtcctcaatg tctccagcac cttcgttctg acctgctcgg gttcagctcc ggtggtgtgg 60

gaacggatgt cccaggagcc cccacaggaa atggccaagg cccaggatgg caccttctcc 120

<210> 112

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 112

cagcgtgctc acactgacca acctcactgg gctagacacg ggagaatact tttgcaccca 60

caatgactcc cgtggactgg agaccgatga gcggaaacgg ctctacatct ttgtgccaga 120

<210> 113

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 113

atcccaccgt gggcttcctc cctaatgatg ccgaggaact attcatcttt ctcacggaaa 60

taactgagat caccattcca tgccgagtaa cagacccaca gctggtggtg acactgcacg 120

<210> 114

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 114

gagaagaaag gggacgttgc actgcctgtc ccctatgatc accaacgtgg cttttctggt 60

atctttgagg acagaagcta catctgcaaa accaccattg gggacaggga ggtggattct 120

<210> 115

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 115

tgatgcctac tatgtctaca gactccaggt gtcatccatc aacgtctctg tgaacgcagt 60

gcagactgtg gtccgccagg gtgagaacat caccctcatg tgcattgtga tcgggaatga 120

<210> 116

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 116

aggtggtcaa cttcgagtgg acataccccc gcaaagaaag tgggcggctg gtggagccgg 60

tgactgactt cctcttggat atgccttacc acatccgctc catcctgcac atccccagtg 120

<210> 117

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 117

gccgagttag aagactcggg gacctacacc tgcaatgtga cggagagtgt gaatgaccat 60

caggatgaaa aggccatcaa catcaccgtg gttgagagcg gctacgtgcg gctcctggga 120

<210> 118

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 118

agaggtgggc acactacaat ttgctgagct gcatcggagc cggacactgc aggtagtgtt 60

cgaggcctac ccaccgccca ctgtcctgtg gttcaaagac aaccgcaccc tgggcgactc 120

<210> 119

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 119

ccagcgctgg cgaaatcgcc ctgtccacgc gcaacgtgtc ggagacccgg tatgtgtcag 60

agctgacact ggttcgcgtg aaggtggcag aggctggcca ctacaccatg cgggccttcc 120

<210> 120

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 120

catgaggatg ctgaggtcca gctctccttc cagctacaga tcaatgtccc tgtccgagtg 60

ctggagctaa gtgagagcca ccctgacagt ggggaacaga cagtccgctg tcgtggccgg 120

<210> 121

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 121

gggcatgccc cagccgaaca tcatctggtc tgcctgcaga gacctcaaaa ggtgtccacg 60

tgagctgccg cccacgctgc tggggaacag ttccgaagag gagagccagc tggagactaa 120

<210> 122

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 122

acgtgacgta ctgggaggag gagcaggagt ttgaggtggt gagcacactg cgtctgcagc 60

acgtggatcg gccactgtcg gtgcgctgca cgctgcgcaa cgctgtgggc caggacacgc 120

<210> 123

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 123

caggaggtca tcgtggtgcc acactccttg ccctttaagg tggtggtgat ctcagccatc 60

ctggccctgg tggtgctcac catcatctcc cttatcatcc tcatcatgct ttggcagaag 120

<210> 124

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 124

gaagccacgt tacgagatcc gatggaaggt gattgagtct gtgagctctg acggccatga 60

gtacatctac gtggacccca tgcagctgcc ctatgactcc acgtgggagc tgccgcggga 120

<210> 125

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 125

accagcttgt gctgggacgc accctcggct ctggggcctt tgggcaggtg gtggaggcca 60

cggctcatgg cctgagccat tctcaggcca cgatgaaagt ggccgtcaag atgcttaaat 120

<210> 126

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 126

tccacagccc gcagcagtga gaagcaagcc cttatgtcgg agctgaagat catgagtcac 60

cttgggcccc acctgaacgt ggtcaacctg ttgggggcct gcaccaaagg aggacccatc 120

<210> 127

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 127

ctatatcatc actgagtact gccgctacgg agacctggtg gactacctgc accgcaacaa 60

acacaccttc ctgcagcacc actccgacaa gcgccgcccg cccagcgcgg agctctacag 120

<210> 128

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 128

gcaatgctct gcccgttggg ctccccctgc ccagccatgt gtccttgacc ggggagagcg 60

acggtggcta catggacatg agcaaggacg agtcggtgga ctatgtgccc atgctggaca 120

<210> 129

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 129

atgaaaggag acgtcaaata tgcagacatc gagtcctcca actacatggc cccttacgat 60

aactacgttc cctctgcccc tgagaggacc tgccgagcaa ctttgatcaa cgagtctcca 120

<210> 130

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 130

agtgctaagc tacatggacc tcgtgggctt cagctaccag gtggccaatg gcatggagtt 60

tctggcctcc aagaactgcg tccacagaga cctggcggct aggaacgtgc tcatctgtga 120

<210> 131

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 131

aaggcaagct ggtcaagatc tgtgactttg gcctggctcg agacatcatg cgggactcga 60

attacatctc caaaggcagc acctttttgc ctttaaagtg gatggctccg gagagcatct 120

<210> 132

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 132

ttcaacagcc tctacaccac cctgagcgac gtgtggtcct tcgggatcct gctctgggag 60

atcttcacct tgggtggcac cccttaccca gagctgccca tgaacgagca gttctacaat 120

<210> 133

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 133

tgccatcaaa cggggttacc gcatggccca gcctgcccat gcctccgacg agatctatga 60

gatcatgcag aagtgctggg aagagaagtt tgagattcgg ccccccttct cccagctggt 120

<210> 134

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 134

tgctgcttct cgagagactg ttgggcgaag gttacaaaaa gaagtaccag caggtggatg 60

aggagtttct gaggagtgac cacccagcca tccttcggtc ccaggcccgc ttgcctgggt 120

<210> 135

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 135

ttccatggcc tccgatctcc cctggacacc agctccgtcc tctatactgc cgtgcagccc 60

aatgagggtg acaacgacta tatcatcccc ctgcctgacc ccaaacccga ggttgctgac 120

<210> 136

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 136

cgagggccca ctggagggtt cccccagcct agccagctcc accctgaatg aagtcaacac 60

ctcctcaacc atctcctgtg acagccccct ggagccccag gacgaaccag agccagagcc 120

<210> 137

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 137

cccagcttga gctccaggtg gagccggagc cagagctgga acagttgccg gattcggggt 60

gccctgcgcc tcgggcggaa gcagaggata gcttcctgta gggggctggc ccctaccctg 120

<210> 138

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 138

gccctgcctg aagctccccc cctgccagca cccagcatct cctggcctgg cctgaccggg 60

cttcctgtca gccaggctgc ccttatcagc tgtccccttc tggaagcttt ctgctcctga 120

<210> 139

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 139

acgtgttgtg ccccaaaccc tggggctggc ttaggaggca agaaaactgc aggggccgtg 60

accagccctc tgcctccagg gaggccaact gactctgagc cagggttccc ccagggaact 120

<210> 140

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 140

tcagttttcc catatgtaag atgggaaagt taggcttgat gacccagaat ctaggattct 60

ctccctggct gacaggtggg gagaccgaat ccctccctgg gaagattctt ggagttactg 120

<210> 141

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 141

gaggtggtaa attaactttt ttctgttcag ccagctaccc ctcaaggaat catagctctc 60

tcctcgcact tttatccacc caggagctag ggaagagacc ctagcctccc tggctgctgg 120

<210> 142

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 142

gctgagctag ggcctagcct tgagcagtgt tgcctcatcc agaagaaagc cagtctcctc 60

cctatgatgc cagtccctgc gttccctggc ccgagctggt ctggggccat taggcagcct 120

<210> 143

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 143

taattaatgc tggaggctga gccaagtaca ggacaccccc agcctgcagc ccttgcccag 60

ggcacttgga gcacacgcag ccatagcaag tgcctgtgtc cctgtccttc aggcccatca 120

<210> 144

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 144

agtcctgggg ctttttcttt atcaccctca gtcttaatcc atccaccaga gtctagaagg 60

ccagacgggc cccgcatctg tgatgagaat gtaaatgtgc cagtgtggag tggccacgtg 120

<210> 145

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 145

gtgtgtgcca gtatatggcc ctggctctgc attggacctg ctatgaggct ttggaggaat 60

ccctcaccct ctctgggcct cagtttcccc ttcaaaaaat gaataagtcg gacttattaa 120

<210> 146

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 146

actctgagtg ccttgccagc actaacattc tagagtattc caggtggttg cacatttgtc 60

cagatgaagc aaggccatat accctaaact tccatcctgg gggtcagctg ggctcctggg 120

<210> 147

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 147

gagattccag atcacacatc acactctggg gactcaggaa ccatgcccct tccccaggcc 60

cccagcaagt ctcaagaaca cagctgcaca ggccttgact tagagtgaca gccggtgtcc 120

<210> 148

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 148

ctggaaagcc cccagcagct gccccaggga catgggaaga ccacgggacc tctttcacta 60

cccacgatga cctccggggg tatcctgggc aaaagggaca aagagggcaa atgagatcac 120

<210> 149

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 149

cctcctgcag cccaccactc cagcacctgt gccgaggtct gcgtcgaaga cagaatggac 60

agtgaggaca gttatgtctt gtaaaagaca agaagcttca gatgggtacc ccaagaagga 120

<210> 150

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 150

atgtgagagg tgggcgcttt ggaggtttgc ccctcaccca ccagctgccc catccctgag 60

gcagcgctcc atgggggtat ggttttgtca ctgcccagac ctagcagtga catctcattg 120

<210> 151

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 151

gtccccagcc cagtgggcat tggaggtgcc aggggagtca gggttgtagc caagacgccc 60

ccgcacgggg agggttggga agggggtgca ggaagctcaa cccctctggg caccaaccct 120

<210> 152

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 152

tgcattgcag gttggcacct tacttccctg ggatccccag agttggtcca aggagggaga 60

gtgggttctc aatacggtac caaagatata atcacctagg tttacaaata tttttaggac 120

<210> 153

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 153

ctcacgttaa ctcacattta tacagcagaa atgctatttt gtatgctgtt aagtttttct 60

atctgtgtac ttttttttaa gggaaagatt ttaatattaa acctggtgct tctcactcac 120

<210> 154

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 154

gatgaacaat acagaaactt cctcagaagt gtatccttgc tgaagaattt acctgaagat 60

aaattaacca agatcattga ctgcttggaa gtggaatact atgacaaagg agattacatc 120

<210> 155

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 155

cattagagag ggcgaggaag gaagtacctt tttcattttg gcaaaaggaa aggtaaaagt 60

aacacagagc acagaaggcc atgatcaacc acagctgata aaaacactgc agaaaggaga 120

<210> 156

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 156

aatactttgg agaaaaagct cttatcagtg atgatgtcag gtcagctaac attattgctg 60

aagaaaatga tgttgcatgc ctggttatag atcgagaaac attcaaccaa actgtcggta 120

<210> 157

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 157

agaagcggga gctggagggc aagctcgcca ccctcagcga ccaggtgaac cggcgggact 60

ttgagtcaga gaagcggctg cggaaggacc tgaagcgcac caaggccctg ctggcagatg 120

<210> 158

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 158

cccagctcat gctggaccac ctgaagaaca gtgctcccag caagcgagag attgcccagc 60

tcaagaacca gctggaggag tcagagttca cctgtgcggc agccgtgaaa gcacggaaag 120

<210> 159

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 159

caatggaggt ggagatcgaa gacctgcacc tgcagattga tgacatcgcc aaagccaaga 60

cagcgctgga ggagcagctg agccgccttc agcgtgagaa gaatgagatc cagaaccggc 120

<210> 160

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 160

tggaggaaga tcaggaagac atgaacgaat tgatgaagaa gcacaaggct gccgtggctc 60

aggcttcccg ggacctggct cagataaatg atctccaagc tcagctagaa gaagccaaca 120

<210> 161

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 161

aagagaagca ggagctgcag gagaagctac aagccctcca gagccaggtg gagttcctgg 60

agcagtccat ggtggacaag tccctggtga gcaggcagga agctaagata cgggagctgg 120

<210> 162

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 162

agacacgcct ggagtttgaa aggacgcaag tgaaacggct ggagagcctg gctagccgtc 60

tcaaggaaaa catggagaag ctgactgagg agcgggatca gcgcattgca gccgagaacc 120

<210> 163

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 163

taaagagttc cagccccacc agctactgga agtcccttgc ccctgatcgg tcagatgatg 60

agcacgaccc tctcgacaac acctccagac cgcgatactc ccacagttat ctgagtgaca 120

<210> 164

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 164

gcgacacaga ggccaagctg acggagacta acgcatagcc caggggagtg gttggcagcc 60

ctctcacccc agggcctgtg gctgcctggg cacctctccc aggaagtggt ggggcaccgg 120

<210> 165

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 165

aagaagacgc cagtttgatg aagaatcact ggaaagcttt agcagtatgc ctgatccagt 60

agatccaaca acagtgacta aaacattcaa gacaagaaaa gcgtctgcac aggccagcct 120

<210> 166

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 166

tggcatctaa agataaaact cccaagtcaa aaagtaagaa gaggaattct actcagctga 60

aaagcagagt taaaaacatc aggtatgaaa gtgccagtat gtctagcaca tgtgaacctt 120

<210> 167

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 167

tgcaaaagta ggaacagaca ttcagcccag actgaagagc ctgttcaagc aaaagtattc 60

agcagaaaga atcatgagca actggaaaaa ataataaaat gtaataggtc tacagaaata 120

<210> 168

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 168

atcttcagaa actgggagtg atttttccat gtttgaagct ttgcgagata ctatttattc 60

tgaagtagct acattaattt ctcaaaatga atctcgtcca cattttctta ttgaactctt 120

<210> 169

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 169

tccatgagct gcagctacta aacacagact acttgagaca gagggcttta tatgcattgc 60

aggacatagt atccagacat atttctgaga gccatgaaaa aggagaaaat gtaaagtcag 120

<210> 170

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 170

gtaaactctg gtacttggat agcatcaaac tcagaactta ctcctagtga gagccttgct 60

actactgatg atgaaacttt tgagaagaac tttgaaagag aaacccataa aataagtgag 120

<210> 171

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 171

gcaaaatgat gctgataatg ctagtgtcct gtctgtatca tcaaattttg agccttttgc 60

aacagatgat ctaggtaaca ccgtgattca cttagatcaa gcattagcca gaatgagaga 120

<210> 172

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 172

taaatagtgc tgcccataag gagtcacctc ctactgttga ttcaactcaa cagcctaacc 60

ctttgccgtt acgtttacct gaaatggaac ccttagtgcc tagagtcaaa gaagttaaat 120

<210> 173

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 173

tctgctcagg aaactcctga aagctctctg gctggaagtc ctgatactga atctccagtg 60

ttagtgaatg actatgaagc agaatctggt aatataagtc aaaagtctga tgaagaagat 120

<210> 174

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 174

ttttgtaaaa gttgaagatt taccactgaa actgacaata tattcagagg cagatctaag 60

aaagaaaatg gtagaagaag aacagaaaaa ccatttatct ggtgaaatat gtgaaatgca 120

<210> 175

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 175

atgcagctga tctggtggta caaggcagag ggaaatttga ggagctaatg gtgtgttctc 60

atgaaattgc tgctagcaca gcccagcttg tggctgcatc caaggtgaaa gctgataagg 120

<210> 176

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 176

ggacagcccc aacctagccc agctgcagca ggcctctcgg ggagtgaacc aggccactgc 60

cggcgttgtg gcctcaacca tttccggcaa atcacagatc gaagagacag acaacatgga 120

<210> 177

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 177

gacttctcaa gcatgacgct gacacagatc aaacgccaag agatggattc tcaggttagg 60

gtgctagagc tagaaaatga attgcagaag gagcgtcaaa aactgggaga gcttcggaaa 120

<210> 178

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 178

aagcgagtag aagccatgaa acagtatcag gaagaaattc aagaacttaa tgaagtcgcg 60

agacatcggc cacggtccac gttagttatg ggaatccagc aagaaaacag acaaatcaga 120

<210> 179

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 179

cagagagttg caacaagaaa acaaagaatt acgtacatct ctggaagaac atcagtcggc 60

cttggaactt ataatgagca agtaccgaga acaaatgttt agattgctaa tggctagcaa 120

<210> 180

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 180

gcaaaaaaga tgatccgggt ataataatga agttaaaaga gcagcactcc aagattgaca 60

tggtacatcg taacaagtcc gaaggattct tccttgatgc atctcgacac atccttgaag 120

<210> 181

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 181

gaaagtgcag ctgcagattt cagtgatgaa gatgaagatg atgatgttga tggaagagag 60

aaaagcgtca ttgatacttc aacaattgtt aggaaaaaag cattgcctga cagcggtgaa 120

<210> 182

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 182

gatcgagata caaaagaagc tctaaaggag tttgacttct tggttacatc agaggaagga 60

gacaatgaat ctagaagtgc aggcgatgga acagactggg aaaaggaaga ccagtgtctc 120

<210> 183

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 183

atgcctgaag cctggaatgt ggaccaggga gtaattacca aactcaagga acaatacaaa 60

aaggagagaa aggggaaaaa gggggtgaag aggcccaata ggtcaaaact acaagatatg 120

<210> 184

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 184

ggcgcgtcag gcaggagctg gaggataaga cagagcagct tgtggacacc agacatgagg 60

tggaccagct ggtgctggaa ctgcagaaag ttaagcagga gaacatccag ctagcggcag 120

<210> 185

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 185

agacgcccgg tctgctcgtg cctatcgaga cgagctggat tccctgcggg agaaggcgaa 60

ccgcgtggag aggctggagc tggagctgac ccgctgcaag gagaagctgc acgacgtgga 120

<210> 186

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 186

gacttctaca aggcccgcat ggaggagctg agagaagata atatcatttt aattgaaacc 60

aaggccatgc tggaggaaca gctgactgct gctcgggccc ggggcgataa agtccatgag 120

<210> 187

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 187

aggtgatgtg ctctatgaac tccttcagca tattctgaag cagaggaaac ctcggattct 60

tttttcacca ttcttccacc ctggaaactc tatacacaca cagccggagg tcatactgca 120

<210> 188

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 188

tcagaaccat gaagaagata actgtgtcca gaggaccccc aggccatccg tggataatgt 60

gcaccataac cctcccacca ttgaactgtt gcaccgctcc aggtcaccta tcacgacaaa 120

<210> 189

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 189

tcaccggcct tctcctgacc ccgagcagcg gcccctccgg tcccccctgg acaacatgat 60

ccgccgcctc tccccggctg agagagctca gggacccagg ccgcaccagg agaacaacca 120

<210> 190

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 190

ccaggagtcc taccctctgt cagtgtctcc catggagaat aatcactgcc cagcgtcctc 60

cgagtcccac ccgaagccat ccagcccccg gcaggagagc acacgcgtga tccagctgat 120

<210> 191

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 191

gcccagcccc atcatgcacc ctctgatcct gaacccccgg cactccgtgg atttcaaaca 60

gtccaggctc tccgaggacg ggctgcatag ggaagggaag cccatcaacc tctctcatcg 120

<210> 192

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 192

ggaagacctg gcttacatga accacatcat ggtctctgtc tccccgcctg aagagcacgc 60

catgcccatt gggagaatag cagactgtag actgctttgg gattacgtct atcagttgct 120

<210> 193

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 193

ttctgacagc cggtacgaaa acttcatccg atgggaggac aaagaatcca aaatattccg 60

gatagtggat cccaacggac tggctcgact gtggggaaac cataagaaca gaacaaacat 120

<210> 194

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 194

gacctatgag aaaatgtcca gagccctgcg ccactactac aaactaaaca ttatcaggaa 60

ggagccagga caaaggcttt tgttcaggtt tatgaaaacc ccagatgaaa tcatgagtgg 120

<210> 195

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 195

ccgaacagac cgtctggagc acctagagtc ccaggagctg gatgaacaaa tataccaaga 60

agatgaatgc tgaaggaacc aacagtccac ctcagcgggc cagcagccca gggaacccct 120

<210> 196

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 196

tgaagtctca tttgcagggt gagaagcaga aagccacaga ggccagtgct gtggagcaga 60

cggcagagag ctgcgaagtt caagaaatgt tgaaagtagc ccgagcagag aaagatctac 120

<210> 197

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 197

actggaactg tcttgcaatg agctcagaca agaattacta aaggcaaacg gtgaaattaa 60

acatgtttcc agtctgctgg ccaaggtgga aaaggattat tcatacctga aggagatatg 120

<210> 198

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 198

tgtgatcacc aagccgaaca gctgagcaga accagcctaa agctgcaaga aaaagcatca 60

gagagtgatg cagagatcaa agacatgaaa gaaaccatat ttgaattgga agatcaggtg 120

<210> 199

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 199

ggccaatcac tccctccagc tggcctcaca gatccagaag gcaccagacg tggccataga 60

ggtgctgacc cgctccagcc tagaagttga gttggccgcc aaggagcggg agatcgcaca 120

<210> 200

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 200

acagctggtg gaggacgtgc agagactcca ggccagcctc accaagctgc gggagaattc 60

ggccagccag atctcacagc ttgagcagca gctgagcgcc aaaaacagca cactcaaaca 120

<210> 201

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 201

acaactggaa gaaaaactca aaggccaggc tgactatgaa gaggtgaaga aagagctgaa 60

cattctgaag tccatggagt ttgcaccgtc cgagggcgct gggacacagg atgcggccaa 120

<210> 202

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 202

ggctgcgctg cagaccgggc agaggagcag cgccggcacc aagatgggct gccctacatt 60

gatgactcgc cctcctcatc gccccacctc agcagcaagg gcaggggcag ccgggatgcg 120

<210> 203

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 203

gctggtctcg ggagccctgg agtccactaa agcgagtgag ctggacttgg aaaagggctt 60

ggagatgaga aaatgggtcc tgtcgggaat cctggctagc gaggagactt acctgagcca 120

<210> 204

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 204

acctggaggc actgctgctg cccatgaagc ctttgaaagc cgctgccacc acctctcagc 60

cggtgctgac gagtcagcag atcgagacca tcttcttcaa agtgcctgag ctctacgaga 120

<210> 205

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 205

atccacaagg agttctatga tgggctcttc ccccgcgtgc agcagtggag ccaccagcag 60

cgggtgggcg acctcttcca gaagctggcc agccagctgg gtgtgtaccg ggccttcgtg 120

<210> 206

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 206

ctgaatgtca tcgtccactc agccactgga tttaagcaga gttcaaatct gtactgcacc 60

ctggaggtgg attcctttgg gtattttgtg aataaagcaa agacgcgcgt ctacagggac 120

<210> 207

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 207

cacagctgag ccaaactgga acgaggaatt tgagatagag ctggagggct cccagaccct 60

gaggatactg tgctatgaaa agtgttacaa caagacgaag atccccaagg aggacggcga 120

<210> 208

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 208

agagcacgga cagactcatg gggaagggcc aggtccagct ggacccgcag gccctgcagg 60

acagagactg gcagcgcacc gtcatcgcca tgaatgggat cgaagtaaag ctctcggtca 120

<210> 209

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 209

cagatctctg cactcgtcct aagagcccag gcctccgaga tcttacttga agagttacag 60

caggggcttt cccaggcaaa gagggatgtt caggaacaga tggcggtgct gatgcagtca 120

<210> 210

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 210

cacgggaaca ggtttcagaa gagctggtga ggttacagaa agataatgac agtctccagg 60

gaaagcacag cctgcatgtg tcattacagc aagcagaaga cttcatcctc ccagacacta 120

<210> 211

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 211

tacagaggca ctgcgggagt tggtattaaa ataccgtgag gacatcatta atgtgcggac 60

agcagcagac cacgtagaag aaaagctgaa ggctgagata cttttcctaa aagagcagat 120

<210> 212

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 212

gagcaagaaa aagtagagaa caaaactcct ttagttaatg agagcctgaa aaagttttta 60

aataccaaag acggtcgttt agtggctagt cttgttgcag aatttcttca gttttttaac 120

<210> 213

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 213

aaccttgact ttactttggc tgtttttcaa cctgaaacta gcacactgca aggtctcgaa 60

ggtcgagaga atttagcccg agatttaggt ataattgaag cagaaggtac tgtgggtgga 120

<210> 214

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 214

ggacccttat tattagaagt gatcaggcgc tgtcaacaga aagaaaaagg gccaaccact 60

ggggaaggtg cacttgatct atctgatgta cattctccac caaagtcacc agagggaaaa 120

<210> 215

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 215

aaaacaagtg cacagacaac accaagtaag ataccaaggt ataaaggaca aggtaagaag 60

aagacaagcg ggcagaaggc tggtgacaag aaggccaatg atgaggccaa tcagagtgat 120

<210> 216

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 216

gatacaagtg tctccttgtc agaacccaag agcaaaagca gccttcactt actgtcccat 60

gaaacaaaaa ttggatcttt tctaagcaac agaactttag atggcaaaga caaagctggc 120

<210> 217

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 217

acgacctgga aagagccaag cgcgccacga tcatgtctct cgaagacttt gagcagcgct 60

tgaatcaggc catcgaaaga aatgccttcc tggaaagtga acttgatgaa aaagagaatc 120

<210> 218

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 218

ctcctggaat ctgttcagag actgaaggat gaagccagag atttgcggca ggaactggcc 60

gtgcagcaga agcaggagaa acccaggacc cccatgccca gctcagtgga agctgagagg 120

<210> 219

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 219

gacagacaca gctgtgcagg ccacgggctc cgtgccgtcc acgcccattg ctcaccgagg 60

acccagctca agtttaaaca cacctgggag cttcagacgt ggcctggacg actccaccgg 120

<210> 220

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 220

ggatatgatg gaaaggcttt tggttcacct atgatcgatt tgagctcacc agtgggaggg 60

tcttataatc ttccctctct tccggatatt gactgttcaa gtactattat gctggacaat 120

<210> 221

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 221

atattgtgag gaaagatact aatatagatc atggccagcc aagaccaccc tcaaacagaa 60

cggtccagtc accaaattca tcagtgccat ctccaggcct tgcaggacct gttactatga 120

<210> 222

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 222

gactagtgta caccccccaa tacgttcacc tagtgcctcc agcgttggaa gccgaggaag 60

ctctggctct tccagcaaac cagcaggagc tgactctaca cacaaagtcc cagtggtcat 120

<210> 223

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 223

acaggacctc cgaaaccaac acaccacagg gaaaccagga acaactggta actgtcatgg 60

aggaacgaat gatagaagtt gaacagaaac tgaaactagt gaaaaggctt cttcaagaga 120

<210> 224

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 224

aagtgaatca gctcaaagaa caactctgca agaacactaa ggcagacgca atggtgaagg 60

acttgtatgt tgaaaatgcc cagttgttga aagctctgga agtgactgaa cagcgacaga 120

<210> 225

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 225

aaacagcaga gaagaaaaat tacctcctgg aggagaagat tgccagcctc agtaatatag 60

ttaggaatct gacaccagcg ccattgactt ctacacctcc tttgaggtca tagccaaacc 120

<210> 226

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 226

cccctactgc ctccagtagc agcagcacaa cccctacccg aaagatcaca gaaagtcctc 60

gtgcctccat gggagttctc tcaggcaaaa gaaaacttat cacttctgaa gaggaacggt 120

<210> 227

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 227

cccctgccaa gcgaggtcgc aagtctgcca cagtaaaacc tggtgcagta ggggcaggag 60

agtttgtgag cccctgtgag agtggagaca acaccggtga accctctgcc ctggaagagc 120

<210> 228

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 228

agagagggcc tttgcctctc aacaagacct tgtttctggg ctacgcattt ctccttacca 60

tggccacaac cagtgacaag ttggccagcc gctccaaact gccagatggt cctacaggaa 120

<210> 229

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 229

ctaagcataa ctaaaggtga aaagctccgg gtcttaggct ataatcacaa tggggaatgg 60

tgtgaagccc aaaccaaaaa tggccaaggc tgggtcccaa gcaactacat cacgccagtc 120

<210> 230

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 230

aacagtctgg agaaacactc ctggtaccat gggcctgtgt cccgcaatgc cgctgagtat 60

ctgctgagca gcgggatcaa tggcagcttc ttggtgcgtg agagtgagag cagtcctggc 120

<210> 231

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 231

cagaggtcca tctcgctgag atacgaaggg agggtgtacc attacaggat caacactgct 60

tctgatggca agctctacgt ctcctccgag agccgcttca acaccctggc cgagttggtt 120

<210> 232

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 232

catcatcatt caacggtggc cgacgggctc atcaccacgc tccattatcc agccccaaag 60

cgcaacaagc ccactgtcta tggtgtgtcc cccaactacg acaagtggga gatggaacgc 120

<210> 233

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 233

acggacatca ccatgaagca caagctgggc gggggccagt acggggaggt gtacgagggc 60

gtgtggaaga aatacagcct gacggtggcc gtgaagacct tgaaggagga caccatggag 120

<210> 234

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 234

gtggaagagt tcttgaaaga agctgcagtc atgaaagaga tcaaacaccc taacctggtg 60

cagctccttg gggtctgcac ccgggagccc ccgttctata tcatcactga gttcatgacc 120

<210> 235

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 235

tacgggaacc tcctggacta cctgagggag tgcaaccggc aggaggtgaa cgccgtggtg 60

ctgctgtaca tggccactca gatctcgtca gccatggagt acctggagaa gaaaaacttc 120

<210> 236

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 236

tgaatctagg cggcagtacg aagagaaaaa caaagaattt gaaagggaaa aacacgccca 60

cagtatactg caatttcagt ttgctgaagt caaggaggcc ctgaagcaaa gagaggaaat 120

<210> 237

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 237

gctcgagaaa catggaataa tcctaaattc agaaatagct accaatggag agacttccga 60

caccctcaat aatgttggat accaaggtcc taccaagatg acaaaagaag agttaaatgc 120

<210> 238

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 238

cctcaagtcg acaggggatg ggaccctagg aagagccagt gaagtggagg tgaaaaatga 60

aatcgtggcg aatgtgggga aaagagaaat cttgcacaat actgagaaag aacaacacac 120

<210> 239

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 239

ttgtacagac ctaagaaacc ctgacattcg ggtgctaatt tgtgaagaaa aagcaccagt 60

tctcaagatg gagcttgata gatcagctga tcctcctcct gcaatttggg ttgcaacaac 120

<210> 240

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 240

taagtctaca gtaaataaat ggactttgaa aggaattcat aattttagag cctctggaga 60

ttatgacaat gactgtacaa atcctataac acctctttgt acacaacctg accaggttat 120

<210> 241

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 241

taaagggggt gctagtatta ttcagtgcca cattcttaat gataagagac atatattaac 60

caaagatacc aataataatg tggcatattg ggatgtattg aaggcatgta aagttgaaga 120

<210> 242

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 242

acctttaaca atgacaaaag ctacttactg taaacctcac atgcagacca aatcttgtca 60

gacagatgat acttggagga cagaatatgt tccagtgcct atccctgtgc ctgtgtatat 120

<210> 243

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 243

tcccagttcc tatgcacatg tacagtcaga atattcctgt tcctactaca gttcctgttc 60

ctgtgccagt tcctgttttt ctgcctgctc cattggacag cagtgagaag attcctgcag 120

<210> 244

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 244

gcaattgagg agctaaaaag caaggtttct tcagatgctc ttgatacaga gttgcttaca 60

atgacggata tgatgagtga agacgagggg aaaacagaga caaccaacat caacagtgta 120

<210> 245

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 245

aggcgacggg caaatgagaa cagcaacata caggtccttt ctgaaagatc tgctactgaa 60

gtagacaaca attttagcaa accacctccg tttttccctc caggagctcc tcccactcac 120

<210> 246

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 246

caccttccac ctcctccatt tcttccacct cctccgactg tcagcactgc tccacctctg 60

attccaccac cgggttttcc tcctccacca ggcgctccac ctccatctct tataccaaca 120

<210> 247

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 247

acaatagaaa gtggacattc ctctggttat gatagtcgtt ctgcacgtgc atttccatat 60

ggcaatgttg cctttcccca tcttcctggt tctgctcctt cgtggcctag tcttgtggac 120

<210> 248

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 248

gacaccagca agcagtggga ctattatgcc agaagagaga aagaccgaga tagagagaga 60

gacagagaca gagagcgaga ccgtgatcgg gacagagaaa gagaacgcac cagagagaga 120

<210> 249

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 249

agagagaggg agcgtgatca cagtcctaca ccaagtgttt tcaacagcga tgaagaacga 60

tacagataca gggaatatgc agaaagaggt tatgagcgtc acagagcaag tcgagaaaaa 120

<210> 250

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 250

aaagaagaac gacatagaga aagacgacac agggagaaag aggaaaccag acataagtct 60

tctcgaagta atagtagacg tcgccatgaa agtgaagaag gagatagtca caggagacac 120

<210> 251

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 251

ccagtgctgg tgattatggg agtgctattg agacactggt aactgcaatt tctttaatta 60

aacaatccaa agtatctgct gatgatcgtt gcaaagttct tattagttct ttgcaagatt 120

<210> 252

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 252

ttgccttcat ggaattgagt ccaagtctta tggttctgga tcaagacgtg aacgatcaag 60

agagagggac catagtagat cacgagaaaa gagtcgacgt cataaatccc gtagtagaga 120

<210> 253

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 253

gaccgtcatg acgattatta cagagagaga agcagagaac gagagaggca ccgggatcgt 60

gaccgagacc gtgaccgaga gcgtgaccga gagcgcgaat atcgtcatcg ttagaagctg 120

<210> 254

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 254

aagctctgca agaacaactg gatgaaagac ttcaagaact agaaaagata aaggaccttc 60

atatggccga gaagactaaa cttatcactc agttgcgtga tgcaaagaac ttaattgaac 120

<210> 255

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 255

cagcttgaac aagataaggg aatggtaatc gcagagacaa aacgtcagat gcatgaaacc 60

ctggaaatga aagaagaaga aattgctcaa ctccgtagtc gcatcaaaca gatgactacc 120

<210> 256

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 256

ccagggagag gaattacggg aacagaaaga aaagtccgaa agagctgctt ttgaggaact 60

tgaaaaagct ttgagtacag cccaaaaaac agaggaagca cggagaaaac tgaaggcaga 120

<210> 257

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 257

cccatgagtt tgccacgctg gtcattgaca ttctcagtga cgccaagagg agacagcagg 60

gcagttctct ctcgggttca aaagacaatg tggagctcat actgaaaacc atcaataacc 120

<210> 258

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 258

ccagcacagc gttgagagtc aagacaacga tcagcccgac tatgacagcg tggcatcaga 60

cgaagacaca gatttggaaa ccactgcaag caaaacaaac cggcagaaga gcctagattc 120

<210> 259

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 259

tcagatttat cagatggacc agtcactgta caggaattta tggaggtcaa aaacgctcta 60

gtggcttctg aggccaagat acagcagcta atgaaggtga ataacaactt gagtgacgag 120

<210> 260

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 260

cttcattgtc gttttaaccc tgctaatttg tcacaagtac aaaaagcaat ttaggtatga 60

aagccagcta cagatggtac aggtgaccgg ctcctcagat aatgagtact tctacgttga 120

<210> 261

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 261

ttgatttcag agaatatgaa tatgatctca aatgggagtt tccaagagaa aatttagagt 60

ttgggaaggt actaggatca ggtgcttttg gaaaagtgat gaacgcaaca gcttatggaa 120

<210> 262

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 262

ggaattagca aaacaggagt ctcaatccag gttgccgtca aaatgctgaa agaaaaagca 60

gacagctctg aaagagaggc actcatgtca gaactcaaga tgatgaccca gctgggaagc 120

<210> 263

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 263

ggcagatagg aagtatgaag aggtggctcg taagttggtg atcattgaag gagacttgga 60

acgcacagag gaacgagctg agctggcaga gtctaagtgt tctgagctgg aggaggagct 120

<210> 264

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 264

ctgaagaatg tcaccaacaa cctcaagtct cttgaggctc aggcggagaa gtactctcaa 60

aaagaagata aatatgagga agaaatcaag attcttactg ataaactcaa ggaggcagag 120

<210> 265

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 265

agacccgtgc tgagtttgct gagagatcgg tagccaagct ggaaaagaca attgatgacc 60

tggaagatga gctctatgcc cagaaactga agtacaaggc cattagcgag gagctggacc 120

<210> 266

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 266

cgccaatatt taggagggag agactccctg atgtcccaag cacccatctc taaccaacaa 60

gctgaagtta cccccactgg ccgtcttgga aaacagactg atcaaggttc aatgcagata 120

<210> 267

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 267

taccttccag agatgatagc acttcattga ctgccaaaga ggatgtcagc atacccagat 60

ccacattagg agacttggac acagttgcag ggctggaaaa agaactgagt aatgccaaag 120

<210> 268

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 268

agaggaactt gaactcatgg ctaaaaaaga aagagaaagt cagatggaac tttctgctct 60

acagtccatg atggctgtgc aggaagaaga gctgcaggtg caggctgctg atatggagtc 120

<210> 269

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 269

ggccaatcac tccctccagc tggcctcaca gatccagaag gcaccagacg tggccataga 60

ggtgctgacc cgctccagcc tagaagttga gttggccgcc aaggagcggg agatcgcaca 120

<210> 270

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 270

acagctggtg gaggacgtgc agagactcca ggccagcctc accaagctgc gggagaattc 60

ggccagccag atctcacagc ttgagcagca gctgagcgcc aaaaacagca cactcaaaca 120

<210> 271

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 271

acaactggaa gaaaaactca aaggccaggc tgactatgaa gaggtgaaga aagagctgaa 60

cattctgaag tccatggagt ttgcaccgtc cgagggcgct gggacacagg atgcggccaa 120

<210> 272

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 272

aagaaagtga gacactgaca gaacaggatt tacagttata ttgcgatttt cctaatatta 60

ttgatgtcag tattaagcaa gcaaaccaag agggttcaaa tgaaagccga gttgtaacta 120

<210> 273

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 273

ctatccataa gcaagatggt aaaaatctgg aaattgaact tagctcatta agggaagctt 60

tgtctttcgt gtcattaatt gatggatatt atagattaac tgcagatgca catcattacc 120

<210> 274

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 274

acctctgtaa agaagtagca cctccagccg tgcttgaaaa tatacaaagc aactgtcatg 60

gcccaatttc gatggatttt gccattagta aactgaagaa agcaggtaat cagactggac 120

<210> 275

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 275

gactgtatgt acttcgatgc agtcctaagg actttaataa atattttttg acttttgctg 60

tcgagcgaga aaatgtcatt gaatataaac actgtttgat tacaaaaaat gagaatgaag 120

<210> 276

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 276

aagagtacaa cctcagtggg acaaagaaga acttcagcag tcttaaagat cttttgaatt 60

gttaccagat ggaaactgtt cgctcagaca atataatttt ccagtttact aaatgctgtc 120

<210> 277

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 277

gtcccccaaa gccaaaagat aaatcaaacc ttctagtctt cagaacgaat ggtgtttctg 60

atgtaccaac ctcaccaaca ttacagaggc ctactcatat gaaccaaatg gtgtttcaca 120

<210> 278

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 278

acaaaatcag aaatgaagat ttgatattta atgaaagcct tggccaaggc acttttacaa 60

agatttttaa aggcgtacga agagaagtag gagactacgg tcaactgcat gaaacagaag 120

<210> 279

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 279

aagttctttt aaaagttctg gataaagcac acagaaacta ttcagagtct ttctttgaag 60

cagcaagtat gatgagcaag ctttctcaca agcatttggt tttaaattat ggagtatgtg 120

<210> 280

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 280

gtgtctgtgg agacgagaat attctggttc aggagtttgt aaaatttgga tcactagata 60

catatctgaa aaagaataaa aattgtataa atatattatg gaaacttgaa gttgctaaac 120

<210> 281

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 281

aacagttggc atgggccatg cattttctag aagaaaacac ccttattcat gggaatgtat 60

gtgccaaaaa tattctgctt atcagagaag aagacaggaa gacaggaaat cctcctttca 120

<210> 282

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 282

tcatcaaact tagtgatcct ggcattagta ttacagtttt gccaaaggac attcttcagg 60

agagaatacc atgggtacca cctgaatgca ttgaaaatcc taaaaattta aatttggcaa 120

<210> 283

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 283

caacagacaa atggagtttt ggtaccactt tgtgggaaat ctgcagtgga ggagataaac 60

ctctaagtgc tctggattct caaagaaagc tacaatttta tgaagatagg catcagcttc 120

<210> 284

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 284

ttcctgcacc aaagtgggca gaattagcaa accttataaa taattgtatg gattatgaac 60

cagatttcag gccttctttc agagccatca tacgagatct taacagtttg tttactccag 120

<210> 285

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 285

cagattatga actattaaca gaaaatgaca tgttaccaaa tatgaggata ggtgccctgg 60

ggttttctgg tgcctttgaa gaccgggatc ctacacagtt tgaagagaga catttgaaat 120

<210> 286

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 286

aatttctaca gcaacttggc aagggtaatt ttgggagtgt ggagatgtgc cggtatgacc 60

ctctacagga caacactggg gaggtggtcg ctgtaaaaaa gcttcagcat agtactgaag 120

<210> 287

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 287

gcccaccagg attgctggaa gtgtgacgga gcaggcgggc tgaggagagt ggaaaaggaa 60

gcgacccaga aatggcaggg acacttctct tgcagaccaa gagggaccct ggagcacctt 120

<210> 288

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 288

attaaaaaat gctcaaaaag aaattgccac attgaaacag cacctcagta atatggaagt 60

ccaagttgct tctcagtctt cacagagaac tggtaaaggt cagcctagca acaaagaaga 120

<210> 289

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 289

atgtggatga tcttgtgagt cagctaagac agacagaaga gcaggtgaat gacttaaagg 60

agagactcaa aacaagtacg agcaatgtgg aacaatatca agcaatggtt actagtttag 120

<210> 290

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 290

gaagaatccc tgaacaagga aaaacaggtg acagaagaag tgcgtaagaa tattgaagtt 60

cgtttaaaag agtcagctga atttcagaca cagttggaaa agaagttgat ggaagtagag 120

<210> 291

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 291

ggaagcactg gtcgaagagt gtgatggaca aacagtacga gattctgtgt gtcagccagt 60

ttaccctcca gtgtgtcctg aagggaaaca agcctgattt ccacctagca atgcccacgg 120

<210> 292

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 292

cccacggagc aggcagaggg cttctacaac agcttcctgg agcagctgcg taaaacatac 60

aggccggagc ttatcaaaga tggcaagttt ggggcctaca tgcaggtgca cattcagaat 120

<210> 293

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 293

tcagaatgat gggcctgtga ccatagagct ggaatcgcca gctcccggca ctgctacctc 60

tgacccaaag cagctgtcaa agctcgaaaa acagcagcag aggaaagaaa agaccagagc 120

<210> 294

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 294

atgaagtgaa tgaaagagag aaagcaatgg aaaatcgtta caagagtctt ctgagtgaaa 60

gcaataaaaa attgcacaat caagagcaag tgatcaaaca tctaacagaa agtaccaatc 120

<210> 295

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 295

tcagaaggac gtgttgcttc agaaattcaa tgaaaaagat ttggaagtaa tacagcagaa 60

ctgctattta atggctgcag aggatcttga gctcaggagt gaaggcttaa taacagaaaa 120

<210> 296

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 296

aagtgctctt ctcaacagcc accaggcagc aaaaccatct tctctaagga aaagaaacaa 60

tcatcagact atgaagagct gattcaggtc ttaaagaaag agcaggacat ctatacccat 120

<210> 297

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 297

cctccgtgga aacaaacagt gtaggcatct cctgccagcc tgaatgtaag aataaagtcg 60

tagggcctga gctgcctatg aattggtgga ttgttaagga gagggtggaa atgcatgacc 120

<210> 298

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 298

cgatgtgctg ggaggtctgt ggaaatgtgt gacaagagtg tgagtgtgga agtcagcgtc 60

tgcgaaacag gcagcaacac agaggagtct gtgaacgacc tcacactcct caagacaaac 120

<210> 299

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 299

cttgaatctc aaagaagtgc ggtctatcgg ttgtggagat tgttctgttg acgtgaccgt 60

ctgctctcca aaggagtgcg cctcccgggg cgtgaacact gaggctgtta gccaggtgga 120

<210> 300

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 300

cagtggcaaa agaacttcag actttacaca acctgcgcaa actctttgtt caggacctgg 60

ctacaagagt taaaaagagt gctgagattg attctgatga caccggaggc agcgctgctc 120

<210> 301

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 301

tcagaagcaa aaaatctcct ttcttgaaaa taatcttgaa cagctcacta aagtgcacaa 60

acagttggta cgtgataatg cagatctccg ctgtgaactt cctaagttgg aaaagcgact 120

<210> 302

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 302

cttcgagcta cagctgagag agtgaaagct ttggaatcag cactgaaaga agctaaagaa 60

aatgcatctc gtgatcgcaa acgctatcag caagaagtag atcgcataaa ggaagcagtc 120

<210> 303

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 303

ataatctcag acaacaaaat gagactgcta gtaagataat agacagccaa caagatgaga 60

ttgacagaat gattttagaa attcagtcta tgcaaggaaa gctttctaaa gagaaactga 120

<210> 304

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 304

accactcaaa agatgatgga agagctggaa aagaaagaaa gaaatgtaca gagattaaca 60

aaagcattgc ttgaaaacca aagacaaaca gatgagacat gctctttatt ggatcagggc 120

<210> 305

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 305

ccaggaacct gaccaatcta ggcagcagac agttctttcc aaacggccac tatttgattt 60

gactgtgatt gatcagctgt tcaaggaaat gtcctgttgt ttgtttgact tgaaagcatt 120

<210> 306

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 306

ggtagcccag gacaaccatg agcgggccag gcgcctgatg aaggagctca accagatgca 60

gtatgagtac acggagctca agaaacagat ggcaaaccaa aaagatttgg agagaagaca 120

<210> 307

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 307

aatggaaatc agtgatgcaa tgaggacact taaatctgag gtgaaggatg aaatcagaac 60

cagcttgaag aatcttaatc agtttcttcc agaactacca gcagatctag aagctatttt 120

<210> 308

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 308

ggaaagaaac gaaaacctag aaggagaatt ggaaagcttg aaagagaacc ttccatttac 60

catgaatgag ggaccttttg aagaaaaact gaacttttcc caagttcaca taatggatga 120

<210> 309

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 309

acactggcgt ggagaagcac tccgggagaa actgcgtcac cgggaagacc gactcaaggc 60

ccaactccga cactgtatgt ccaagcaagc agaagtatta attaaaggaa agcggcagac 120

<210> 310

<211> 120

<212> DNA

<213> Artificial Synthesis

<400> 310

agagggcact ttacacagtt tgaggagaca agtagatgct ttaggggaat tggtcaccag 60

cacctctgca gattcagcgt catcacccag tctgtctcag ctggagtctt ccctcacaga 120

Claims (8)

1. A detection probe composition of a clonal eosinophilia-mediated disease fusion gene, wherein a target gene of the detection probe composition comprises a class I gene and a class II gene;

the target region of the detection probe composition is the full length of the transcript of the class I gene and the region within 100-300 bp near the fusion breakpoint of the class II gene;

the class I genes include FGFR1, PDGFRA and PDGFRB;

the class II genes include ABL1, BCR, CCDC88C, CDK5RAP2, CEP110, CEP85L, CPSF6, CUX1, DTD1, ETV6, FGFR1OP1, FGFR1OP2, FIP1L1, FLT3, GIT2, GOLGA4, HIP1, JAK2, KANK1, KIF5B, LRRFIP1, MYO18A, NDE1, NIN, PCM1, PDE4DIP, PRKG2, RABEP1, cc1, STRN, TPM3, TPR, TP53BP1, TRIM24, WDR48, and ZNF 198.

2. The detection probe composition of claim 1, wherein the fusion gene detected by the detection probe composition comprises: PCM1-JAK2, ETV6-JAK2, BCR-JAK2, ETV6-ABL 6, ETV6-FLT 6, ETV6-PDGFRA, FIP1L 6-PDGFRA, KIF5 6-PDGFRA, CDK5RAP 6-PDGFRA, STRN-PDGFRA, BCR-PDGFRA, ETV6-PDGFRB, WDR 6-PDGFRB, TPM 6-PDGFRB, MYO18 6-PDGFRB, PDE4DIP-PDGFRB, PRKG 6-PDGFRB, GOLGA 6-PDGFRB, HIP 6-PDGFRB, GIT 6-PDGFRB, NIN-PDGFRB, HIP 3653 BP 6-FRB, NDE 6-PDGB, RABEBEP 6-PDGFRB, RABEP 6-PDGFRB, PDGFR72-PDGFRB, PDGFRC 6-PDGFRB, PDGFR72-6-PDGFRB, PDGFRF 6-PDGFRB, PDGFRF 6-FGFR-6, PDGFR72-6, PDGFRB, PDGFRF 6-FGFR-6, PDGFRB, PDGFR72-6-FGFR-6, PDGFRB, PDGFR72-6-FGFR-6, PDGFRFGFR-6, PDGF-6, PDGFRFGFR-6, PDGFRFGFR-6, PDGF-6, PDGFRFGFR-6, PDGFRFGFR-6, PDGF-6, PDGFRB, PDGF-6, PDGFRFGFR-6, PDGF-FGFR-6, PDGFRFGFR-6, PDGF-6, PDGFRFGFR-6, PDGFRB, PDGFRFGFR-6, PDGFRFGFR-6, PDGF-6, FGFR-6, PDGF-6, PDGFRFGFR-6, PDGFR72-6, PDGFRFGFR-6, PDGFRFGFR-6, PDGF-6, PDGFRFGFR-6, FGFR-6, PDGFRFGFR-6, PDGF-6, PDGFR72, PDGF-6, FGFR-6, PDGFRB, FGFR-6.

3. The detection probe composition according to claim 1, wherein the detection probe composition comprises a nucleic acid sequence as set forth in SEQ ID No. 1-310;

and the probe in the detection probe composition modifies a marker molecule I, and the marker molecule I comprises biotin.

4. A kit for detecting a clonal eosinophilia fusion gene, comprising the detection probe composition of any of claims 1 to 3;

the detection kit also comprises any one or the combination of at least two of a reverse transcription reagent, a sequencing library construction reagent, a PCR reagent or a probe hybridization reagent.

5. The test kit according to claim 4, wherein the reverse transcription reagent comprises a single-stranded cDNA synthetase and/or a double-stranded cDNA synthetase;

the sequencing library construction reagent comprises any one or combination of at least two of RNA fragmentation enzyme, tail-end-added A-tailing enzyme, ligase or linker;

the PCR reagent comprises DNA polymerase and/or PCR primer;

the probe hybridization reagent comprises a hybridization buffer and/or an RNase inhibitor.

6. The test kit according to claim 4 or 5, wherein the test kit further comprises nucleic acid purification reagents;

the nucleic acid purification reagent comprises magnetic beads and/or a cleaning solution;

the magnetic beads modify marker molecules II, and the marker molecules II comprise streptavidin;

the label molecule II is combined with the label molecule I carried by the probe in the detection probe composition.

7. A device for detecting a clonal eosinophilia fusion gene, said device comprising:

a nucleic acid extraction module: for extracting RNA from a clonal eosinophilia sample;

a reverse transcription module: for reverse transcription of the extracted RNA into cDNA;

pre-library construction module: the method is used for carrying out terminal repair, A tail addition, linker connection and PCR amplification on the reverse transcription cDNA to construct a pre-library;

a hybrid capture module: a method for capturing a target fragment from a pre-library by using the detection probe composition of any one of claims 1 to 3, and performing PCR amplification to construct a probe capture library;

a sequencing module: for sequencing and data processing of the probe capture library.

8. Use of the detection probe composition of any one of claims 1 to 3, the detection kit of any one of claims 4 to 6 or the detection device of claim 7 for the preparation of a diagnostic product for clonal hypereosinophilia.

Images