CN114561467B - MET fusion gene detection method, kit and probe library - Google Patents

Abstract

The invention develops a method for capturing a specific MET fusion gene sequence based on hybridization selection, by adopting the method, thousands of times of enriched MET fusion gene DNA fragments can be obtained, the enriched MET fusion gene fragment sample can be selectively applied to various gene detection technologies, and particularly can be applied to detection of aspects of gene mutation, deletion, amplification, fusion and the like by using a next generation sequencing technology, so that high-efficiency and accurate results can be obtained.

Description

MET fusion gene detection method, kit and probe library

Technical Field

The invention relates to a detection method, a kit and a probe library of a MET fusion gene, belonging to the technical field of gene sequencing.

Background

The MET gene is located on the long arm of human chromosome 7, encodes hepatocyte growth factor receptor HGF, and has tyrosine kinase activity. After MET binds HGF, autophosphorylation occurs and activates multiple signaling pathways such as downstream PI3K/ALK, RAS-MAPK, β -catenin signaling pathways. The MET gene is associated with a variety of oncogene products and regulatory proteins, and is involved in the regulation of cell information transduction, cytoskeletal rearrangement, an important regulator of cell proliferation, differentiation, motility, angiogenesis and tissue regeneration. In healthy adult cells, HGF/MET expression levels are low, except at tissue injury, with little activity. In cancer cells, however, HGF/MET is often overexpressed, and this overexpression is associated with tumorigenesis, metastasis, and a poor overall prognosis.

Oncogenic activation of MET signaling is found in many malignancies, including digestive system tumors, hematopoietic system tumors, osteosarcomas, melanomas, gliomas, and the like. HGF/MET pathway abnormalities are associated with the development and metastasis of tumors and induce the development of drug resistance, including MET gene rearrangement, gene mutation, gene amplification, and protein overexpression. MET exists predominantly in a fused form in gliomas. Four MET inhibitors, respectively, teprotinib, carbamazepine, cabotinib, and crizotinib, have been approved by the FDA at present. It is reported in the literature that glioblastoma patients with PTPRZ1-MET fusion were detected with a2 month response to treatment with the MET inhibitor crizotinib and that tumors continued to shrink.

MET fusion occurs at about 2.8-5% in gliomas and about 10% in recurrent childhood gliomas. There are several MET fusion partner genes in brain gliomas, with PTPRZ1 accounting for about 36-60%; secondly, CAPZA2, accounting for about 18-33%; further, ST7, TPR, PTN are 27%, 9%, 5%, respectively; in addition, TRIM24, TFG, CLIP2, DNAH14, PHTF2, HLA-DRB1, CFTR, HLA-DRB5, RRBP1, etc. have been reported. The incidence of PTPRZ1-MET is about 10-16% from the high grade adult glioma overall.

Disclosure of Invention

The invention provides a detection method of MET fusion genes based on NGS sequencing, which can accurately detect the occurrence of MET fusion genes under the condition of low sample size, can detect and obtain reported fusion forms and can detect and obtain unreported new fusion.

The probe library for MET family fusion gene mutation includes one probe with nucleotide sequence as shown in SEQ ID No.1-197 or one probe with the same function as the probe.

Preferably: the probe library includes all of the probes described above.

Preferably: the probes with the same function refer to probes with the same hybridization capturing function by replacing and/or deleting and/or adding one or more nucleotides on any one of the probes with SEQ ID NO. 1-197.

Preferably: the probe having the same function has 80% or more of the same base as the original probe, more preferably 90% or more of the same base, still more preferably 95% or more of the same base.

A method for detecting a MET fusion gene, comprising:

step 1, obtaining DNA of a sample to be detected;

step 2, hybridizing the DNA of the sample to be detected with the probe, capturing, performing NGS sequencing, comparing the on-machine data with the reference genome data, and analyzing to obtain the information of the MET fusion gene;

wherein the probe refers to the probe library.

The sample to be tested is derived from a cell, tissue or body fluid sample.

The DNA of the sample to be detected is cDNA obtained by direct extraction or synthesis after mRNA extraction.

The DNA of the sample to be detected is fragmented and has a length of 150-600 bp.

The detection method is used for non-therapeutic and diagnostic applications.

A detection kit comprises the probe library.

The kit also comprises: and the DNA extraction reagent is used for extracting the DNA of the sample to be detected.

The kit also comprises: the RNA extraction reagent is used for extracting mRNA of a sample to be detected; a reverse transcription kit for reverse transcribing the extracted mRNA into cDNA.

Advantageous effects

Compared with traditional detection means such as FISH, the detection method based on the NGS for detecting the MET fusion has the advantages that the NGS technology can detect the MET fusion in a tissue sample, and can detect the MET fusion state through liquid samples such as blood plasma and hydrothorax;

in addition to detecting common MET fusion types, NGS technologies can also detect rare MET fusion types; meanwhile, compared with the traditional detection means, the NGS can also clearly fuse partner genes, and can detect a plurality of other MET gene variation types, thereby assisting in the formulation of clinical targeted treatment schemes.

Drawings

FIG. 1 is an exemplary process flow diagram of the present invention wherein target gene clusters are enriched and used for gene fusion detection based on next generation sequencing technology.

FIG. 2 is a schematic representation of the design of an exon-adjacent intron probe of the present invention.

FIG. 3 is a graph comparing sequencing results of intron region probe design with conventional probe design.

FIG. 4 is a graph comparing the sequencing results of exon adjacent intron design with conventional probe design.

Detailed Description

The invention discloses mutation information of MET fusion genes to be detected, wherein 'fusion mutation' refers to chimeric genes formed by connecting coding regions of two or more genes end to end under the control of the same set of regulatory sequences (including promoters, enhancers, ribosome binding sequences, terminators and the like).

Common MET fusion partner genes comprise PTPRZ1, CAPZA2, ST7, TPR, PTN, TRIM24, TFG, CLIP2 and the like, and if the rare fusion is likely to be missed by using a traditional detection means, the fusion can not be distinguished by the traditional FISH and IHC detection. IHC diagnostic kit of MET is still in clinical research stage, and the fusion advantage of NGS detection MET is prominent. At present, MET fusion studies are relatively few, fusion cleavage sites are not fixed, but a few introns are often generated in front of the gene kinase domain, and the traditional probe coverage pattern is incomplete for the introns and uneven for the introns, which may also cause missed detection of MET genes. The probe designed by the research optimizes on the basis of the traditional probe, and not only covers the whole area of the exon region of the gene, but also covers the whole area of the important intron region of the gene uniformly so as to avoid common and rare fusion omission.

The present invention provides a method for enriching MET fusion gene fragments. Specifically, the method of the present invention comprises: extracting genomic DNA or mRNA from a cell, body fluid or tissue sample of a mammal such as a human, and treating or synthesizing cDNA to obtain fragmented double-stranded DNA as a DNA sample library; in addition, designing DNA probes hybridized with the MET fusion gene aiming at the MET fusion gene fragments to be enriched, and screening a plurality of probes from the DNA probes as a DNA probe library; then, the DNA sample library is hybridized with a DNA probe library, so that MET fusion gene DNA fragments are enriched from the DNA sample library. According to an embodiment of the present invention, each probe in the DNA probe pool may be biotinylated first, then after hybridization, the hybridization product is adsorbed with streptavidin magnetic beads, and then the enriched MET fusion gene fragment is released from the magnetic beads. After the adaptation treatment, the next generation sequencing gene can be adopted to detect the gene structure mutation of the MET fusion gene segment so as to confirm various mutations of the MET fusion gene.

Regarding the design of the probe pool in this patent:

the capture specificity is affected by various factors, such as poor design of capture probes, poor capture conditions, insufficient blocking of repetitive sequences in genomic DNA, and improper ratio of genomic DNA to capture probes, which can affect the capture specificity, sensitivity, sequencing coverage, and other results. In order to achieve high enrichment and low off-target rate of the target gene, a person skilled in the art needs to make a lot of experiments about the type, length, sequence, hybridization conditions, etc. of the probe, and needs to obtain the optimal parameter combination through creative exploration work, and whether the same effect can be achieved is not proved by corresponding evidence, which is unexpected by the person skilled in the art. Meanwhile, when a sample with mutation is detected, the proportion of the mutation sample in the tissue sample can be different from one individual to another, so that if the abundance of the mutation sample is low, the problem that the probe cannot be hybridized with the mutated fragment accurately is easily caused, the sensitivity of detection is low, and the probe sequence needs to be tested and searched.

In addition to all gene probe designs involving commonality factors, the characteristics of the MET fusion gene itself have led to additional considerations in the process of probe design. According to the statistics of the commemorative Stonex-Katelin cancer center, the MET fusion incidence rate of the brain glioma is about 0.1%, wherein MET gene fusion breaking sites are mainly located in the exon1-exon2 and exon13-exon15 intervals, and on the basis of ensuring the specificity of probes, probes located near the breaking sites are required to be optimally designed so as to improve detection accuracy.

The steps of processing the sample used in the present invention are as follows:

extraction and fragmentation of DNA samples

The commercial DNA extraction kit is adopted to extract DNA from tissue samples, plasma samples, ascites samples or hydrothorax samples, quantitative quality control is carried out through Nanodrop and Qubit, and for the tissue and hydrothorax DNA samples, ultrasonic crushing is needed due to longer DNA fragments.

Purification and end repair of DNA samples

Adding enzyme-free water into the DNA sample obtained in the steps to fill up to 100ul, adding 150ul of Axygen beads, magnetically separating to remove supernatant after resuspension, adding 200ul of 80% ethanol, washing on a magnetic rack, incubating, removing ethanol, and drying until all ethanol volatilizes completely; adding 28ul of anhydrous enzyme water, mixing uniformly, eluting DNA from the magnetic beads, taking 1ug into terminal repair, and adding anhydrous enzyme to 25ul. Adding a terminal repair reagent to perform terminal repair under proper conditions.

Joint ligation and product purification

Universal adaptors at a concentration of 15uM were used and adaptor dilution work was performed based on the total amount of DNA entering the end repair. The following reagents were added to the linker ligation reaction:

after the reaction solution is briefly centrifuged, the reaction solution is incubated on a PCR instrument at 20 ℃ for 15 minutes, 9.5ul of Axygen beads are added to the reaction solution for resuspension, the supernatant is removed after magnetic separation, 200ul of 80% ethanol is added for cleaning, the reaction solution is incubated at room temperature for removing ethanol, and the reaction solution is dried and 20ul of enzyme-free water is added for eluting magnetic beads.

Library amplification

The purified product is subjected to PCR amplification by adopting a reaction system shown in the specification, so that the product meets the sequencing requirement.

The amplification conditions were: pre-denaturation at 98 ℃ for 45 seconds, denaturation at 98 ℃ for 15 seconds, annealing at 65 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and circulation for 12 times; extension was carried out at 72℃for 1 minute.

According to the above-described method and idea for designing a probe, a probe was designed and synthesized for testing, and Biotin (Biotin) was present at the 5' end.

The DNA sample pool was mixed with hybridization buffer (10 mM Tris-HCl,2% bovine serum albumin, pH 8.0) (after mixing, the DNA sample pool concentration was not more than 50ng/ul at most), the reaction conditions were 95℃for 5 minutes, and then kept at 65 ℃. The reaction was performed in a PCR amplification instrument. Then in DNA sample library: the probe pool was added to the above mixture at a molar ratio of 1:100, and the reaction conditions were 65℃for 5 minutes. The hybridization reaction was placed in a PCR amplification apparatus and incubated at 65℃for 24 hours.

50ul of Dynabeads magnetic beads were mixed with 200ul of binding buffer (10 mM Tris-HCl,2% BSA) and the beads were separated by a magnetic separator and repeated 3 times; mixing the hybridization reaction product with streptavidin magnetic beads, separating in a magnetic separator, adding 500ul of washing buffer (containing 0.1% Tween-20 and 0.1% SDS) into the magnetic beads, incubating at 60 ℃ for 15min, and repeating for 3 times; the magnetic beads were mixed with 50ul of elution buffer (containing 10mM NaOH), incubated at 20℃for 15min, and the magnetic beads were separated by a magnetic separator, and a detection library was obtained from the supernatant.

The library is amplified by PCR by adopting a reaction system shown in the specification, so that the product reaches the sequencing requirement.

The amplification conditions were: pre-denaturation at 98 ℃ for 45 seconds, denaturation at 98 ℃ for 20 seconds, annealing at 65 ℃ for 30 seconds, extension at 72 ℃ for 25 seconds, and circulation for 5 times; extension is carried out at 72℃for 3 minutes.

Sequencing and analysis

Sequencing by using an Illumina HiSeq2000 next generation sequencing system, and carrying out data analysis to obtain gene mutation information. According to the method, the test results using different probes are as follows:

1. comparison of detection data of different probe lengths

The length of the probe has larger influence on the specificity and the middle target rate of detection, so 4 probes with different lengths are designed, the lengths of the probes are 80bp, 100bp, 120bp and 140bp respectively, NGS capture sequencing is carried out by using the probes, quality control is carried out on sequencing data, and the influence of different probe lengths on sequencing results is determined by the middle target rate, the coverage rate of a target area and the economy. The results of physical control are shown in the following table:

from the sequencing results, the target rate and the coverage rate of the target area in the 120bp probe are significantly higher than those of the 80bp probe and the 100bp probe. When the probe length is continuously increased to 140bp, the middle target rate and the coverage rate of the target area are not obviously improved, however, the increase of the probe length tends to cause the improvement of the probe synthesis cost, and the probe with the length of 120bp is comprehensively considered.

2. Comparison of different probe overlap lengths

The connection mode between adjacent probes is possibly related to the uniformity of probe coverage, so three connection modes, namely 10bp gap, 5bp overlap and 20bp overlap, are designed, NGS sequencing is carried out after capturing, and quality control results are as follows:

the coverage rate of 5bp overlap between adjacent probes is significantly increased compared with that of 10bp gap, and there is no significant difference from 20bp overlap.

3. Comparison of conventional coverage and optimized coverage patterns for intron regions

After the optimization, experiments are carried out by adopting probes shown as SEQ ID NO.1-197, and as most of gene fracture sites are positioned in an intron region of the gene, the high-frequency fracture interval of the MET gene intron is fully covered. Compared with the traditional coverage form (probes are designed on the area only close to the exons), the probe coverage situation of the introns of the coverage hot spot is shown in fig. 3, the upper diagram is the probe coverage situation designed by adopting the optimization technology of the patent, the lower diagram is the probe coverage situation after capturing by adopting the traditional probes, and the coverage of the optimized probe design on the introns is obviously superior to that of the traditional probe design. The number of intron region probes marked in FIG. 3 was increased by 25 over control 1, SEQ ID NOS.62-86, and the remaining parameter experiment groups were identical to control 1. If the breaking site of MET gene fusion is located in the hot spot intron region, the optimized probe is adopted to cover the breaking site, the sequencing depth is 1000X, the mutation abundance is 10%, and if the conventional probe is not adopted to cover the breaking site, the fusion omission is directly caused. Fig. 3 shows the experimental group in the upper half and the control group 1 in the lower half.

4. Comparison of conventional coverage and optimized coverage patterns of exons adjacent to introns

The coverage of intronic regions is extremely important for fusion gene detection, so we also designed specific probes for intronic regions adjacent to exons, and compared to conventional probe designs (probes are not designed in regions adjacent to introns) the coverage of intronic regions is shown in FIG. 4, where the upper panel is the optimized probe covering intronic regions adjacent to introns, and the lower panel is the conventional probe intron coverage. The number of probes in the exon adjacent to the intron region marked in FIG. 4 was increased by 1 compared with that in control group 2, SEQ ID NO.59, and the rest of the parameters were the same as those in control group 2. The capturing effect of the optimized probe intron region is obviously better than that of the traditional probe. Fig. 4 shows the experimental group in the upper half and the control group 1 in the lower half.

5. Verification of detection sensitivity

In the prior art, the problem of low detection sensitivity exists in the fusion gene detection, and the sensitivity is generally about 10% or more, so how to improve the detection sensitivity is a problem to be solved.

For PTPRZ1: exon8-MET: exon2 fusion mutation, mutant plasmids and wild plasmids are constructed, samples with different abundances are mixed according to the copy number proportion of the mutant in the wild, the probe library is adopted for capturing and sequencing to examine sensitivity, the probe of SEQ ID NO.198-199 is used as a control of the probe of SEQ ID NO.22, the detection sensitivity is examined, and each sample is repeatedly tested for 3 times, and the result is as follows:

as can be seen from the table, the detection probe library and the detection method provided by the invention can have better detection sensitivity to low-abundance samples, and can reach the detection sensitivity level of about 0.5%.

6. Verification of detection accuracy

The optimized probe library is adopted to perform MET gene fusion detection on 10 patient tumor tissue samples which are subjected to FISH and IHC detection and are subjected to MET fusion positive and 5 patient tumor tissue samples which are subjected to detection and negative respectively, and sequencing is performed through the optimized probe and the method, and the results show that the MET gene fusion can be better detected through the detection probe library and the detection method provided by the invention, and the detection results are the same as the detection results of FISH and IHC, so that the reliability of the method is proved, and meanwhile, fusion partner genes and specific fusion forms can be definitely detected through NGS.

Sequence listing

<110> Jiangsu province middle hospital

Nanjing Shihe gene Biotechnology Co.,Ltd.

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tcgatcagga ccatcaaccc ctcatgtgaa ttttctcctg gactcccatc cagtgtctcc 120

<210> 36

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 36

gtctccagaa gtgattgtgg agcatacatt aaaccaaaat ggctacacac tggttatcac 60

tgggaagaag gtaagctgtt cccacaggga atttccatag acgtggtttt tcccaaatgc 120

<210> 37

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 37

acaaactaga tacccctctg gaagctcttt ccaccccttc tcttcacaga tcacgaagat 60

cccattgaat ggcttgggct gcagacattt ccagtcctgc agtcaatgcc tctctgcccc 120

<210> 38

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 38

accctttgtt cagtgtggct ggtgccacga caaatgtgtg cgatcggagg aatgcctgag 60

cgggacatgg actcaacaga tctgtctgcc tgcaatctac aaggtaggaa tctctaacag 120

<210> 39

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 39

atgtgtgcga tcggaggaat gcctgagcgg gacatggact caacagatct gtctgcctgc 60

aatctacaag gtaggaatct ctaacagctg gcatacatgt ttttgtttgg tgtttttttt 120

<210> 40

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 40

aaattataat atattgggtt tttttaaaag ttctatgttg tccttgtagg ttttcccaaa 60

tagtgcaccc cttgaaggag ggacaaggct gaccatatgt ggctgggact ttggatttcg 120

<210> 41

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 41

gaggaataat aaatttgatt taaagaaaac tagagttctc cttggaaatg agagctgcac 60

cttgacttta agtgagagca cgatgaatac gtaaggatct taaaatgctt tgctggggtg 120

<210> 42

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 42

taaagaaaac tagagttctc cttggaaatg agagctgcac cttgacttta agtgagagca 60

cgatgaatac gtaaggatct taaaatgctt tgctggggtg tgcttggaaa ataggttttg 120

<210> 43

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 43

gaaaattcct tggatttgtc atgtattaaa ctttgggttt tttttccaga ttgaaatgca 60

cagttggtcc tgccatgaat aagcatttca atatgtccat aattatttca aatggccacg 120

<210> 44

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 44

ggacaacaca atacagtaca ttctcctatg tggtaaggaa gattctatcc tatcatgttt 60

gatttttact taatctattt aaattataag atgaacaagt tactttgttt tgtttttatc 120

<210> 45

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 45

tcccctccag gatcctgtaa taacaagtat ttcgccgaaa tacggtccta tggctggtgg 60

cactttactt actttaactg gaaattacct aaacagtggg aattctagac acatttcaat 120

<210> 46

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 46

ctggaaatta cctaaacagt gggaattcta gacacatttc aattggtgga aaaacatgta 60

ctttaaaaag gtgttgtaaa tttatttttt gttgcatctg tcaatttgaa ttaatatctg 120

<210> 47

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 47

tgtgtatctc taatagctaa aattcacttc cttaattttt tttgttcagt gtgtcaaaca 60

gtattcttga atgttatacc ccagcccaaa ccatttcaac tgagtttgct gttaaattga 120

<210> 48

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 48

aaattgactt agccaaccga gagacaagca tcttcagtta ccgtgaagat cccattgtct 60

atgaaattca tccaaccaaa tcttttatta ggtaagtaga agcttctgat gggtataaga 120

<210> 49

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 49

agacaagcat cttcagttac cgtgaagatc ccattgtcta tgaaattcat ccaaccaaat 60

cttttattag gtaagtagaa gcttctgatg ggtataagaa aacaatgaat acaaggatga 120

<210> 50

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 50

ggtggaaaga acctctcaac attgtcagtt ttctattttg ctttgccagt ggtgggagca 60

caataacagg tgttgggaaa aacctgaatt cagttagtgt cccgagaatg gtcataaatg 120

<210> 51

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 51

aaacctgaat tcagttagtg tcccgagaat ggtcataaat gtgcatgaag caggaaggaa 60

ctttacagtg gtaagtcctt tgagcaatgg ttctactcag agctctgcat ctttgcctct 120

<210> 52

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 52

gttgccaagc tgtattctgt ttacagtgga taattgtgtc tttctctagg catgtcaaca 60

tcgctctaat tcagagataa tctgttgtac cactccttcc ctgcaacagc tgaatctgca 120

<210> 53

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 53

actccccctg aaaaccaaag cctttttcat gttagatggg atcctttcca aatactttga 60

tctcatttat gtacataatc ctgtgtttaa gccttttgaa aagccagtga tgatctcaat 120

<210> 54

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 54

tcctgtgttt aagccttttg aaaagccagt gatgatctca atgggcaatg aaaatgtact 60

ggaaattaag gtaagaaatg ctttaaacac tgtcttaaat catcagctca aacttaattg 120

<210> 55

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 55

tgcagaactg tgaagtgtta acaacctttt tttttttttt tcctttcagg gaaatgatat 60

tgaccctgaa gcagttaaag gtgaagtgtt aaaagttgga aataagagct gtgagaatat 120

<210> 56

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 56

acacttacat tctgaagccg ttttatgcac ggtccccaat gacctgctga aattgaacag 60

cgagctaaat atagaggtgg gattcctgca ttcctctcat gatgtaaata aggaagccag 120

<210> 57

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 57

acattctgaa gccgttttat gcacggtccc caatgacctg ctgaaattga acagcgagct 60

aaatatagag gtgggattcc tgcattcctc tcatgatgta aataaggaag ccagtgtaat 120

<210> 58

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 58

aatatctatc atggctaaat gctgactttt ctttatttgt catttttagt ggaagcaagc 60

aatttcttca accgtccttg gaaaagtaat agttcaacca gatcagaatt tcacaggatt 120

<210> 59

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 59

gattgctggt gttgtctcaa tatcaacagc actgttatta ctacttgggt ttttcctgtg 60

gctgaaaaag agaaagcaaa ttaaaggtgc atttttgtta ctgttcattt ttagaagtta 120

<210> 60

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 60

ccttaagaac acagtcatta cagtttaaga ttgtcgtcga ttcttgtgtg ctgtcttata 60

tgtagtccat aaaacccatg agttctgggc actgggtcaa agtctcctgg ggcccatgat 120

<210> 61

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 61

agccgtcttt aacaagctct ttctttctct ctgttttaag atctgggcag tgaattagtt 60

cgctacgatg caagagtaca cactcctcat ttggataggc ttgtaagtgc ccgaagtgta 120

<210> 62

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 62

agcccaacta cagaaatggt ttcaaatgaa tctgtagact accgagctac ttttccagaa 60

ggtatatttc agtttattgt tctgagaaat acctatacat atacctcagt gggttgtgac 120

<210> 63

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 63

attgttgttt atttttggtt ttgcatttat atttttataa aaacctaaag gaagtattta 60

cctctgccaa gtaagtattt gacacaaaat tacatggctc ttaattttaa aagaacccat 120

<210> 64

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 64

gtatatatta cattatgatt ttagagtcca taagctctca tttcacaaaa aggttaattt 60

gagcaaaagt aatttgttta tcatctaagt gcaatagtaa gaaattgcga agctctcttt 120

<210> 65

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 65

tacaatccag gaagagttaa gttacaaaat atacttattt aaatgtaagt tggaactgct 60

acatttttta cctgttgaag cccaaacatt gaaattatac tgttagtaat tcttcgaagt 120

<210> 66

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 66

gttttcaatg aactgttagt acacagcctt tttcccacca tattctagga cttgaatgta 60

ttttgagact tagccaagga aaaccttcaa ttatgccatg aaaaaaagga ggggtcaata 120

<210> 67

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 67

tcatcagctt tgtaaaacac tatgcctagt aatgttcagg ttaatcagag ttttcatgtt 60

gttttattta aatctcctgg taaaagcaaa aggtctgtat tgtatcagct ccattatctt 120

<210> 68

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 68

tagaagttac aggatgtgag tcaagtacaa gcatttcctt ggttgaatat ttaccattgg 60

acaaataaaa tgagtcacag atcattgagg atactggaaa agttagaagt tgctcatcca 120

<210> 69

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 69

aacaagttca agagcaatga agcacttaac attttaacat tttcaacact tactacctct 60

tatgttttga agtttatgtt atttctatgg agatacacat agtaaacatt gtctttgccc 120

<210> 70

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 70

tgattccatt cacctttaaa aatccattcg tttaaccgtg tggaaaaatc aaacctagtt 60

tattgttttg aaatttagat ctatttagta ttttatgtgc acatttagtg catctattta 120

<210> 71

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 71

gtattttata tgcacatttc atagttctaa tctgagatca ttaaaattta caaattttct 60

ttgaaaaaaa aacttaccta atcttctttg aacctcctta ctcaccaaag ctctgtcatc 120

<210> 72

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 72

attgctaaga aggttgagtt tcacactctt ttctccattg agcctgctcc ttggagacat 60

gaaaagaaaa caggtaaaag agggtcattt agagagaatg agaaaatagg tgcacagcca 120

<210> 73

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 73

aaacctaatg aagaggcaac tgcagagctt tcctctctac atctggtggg gacagcattc 60

tcatcagact ttttcacgga gacctagagt gctatgtggt gtgacatcag ggtggcacac 120

<210> 74

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 74

tgatggtttc aattggtttc tgcacatgtt ggaatttagc tgaagagtca cgttttcatg 60

ccaaagggct tttatccatg tctcaccaag gatttccctc aatctgtgca cccttaagca 120

<210> 75

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 75

tttagagccc tgatctccag atgcaaaggc tttaggaagt gagaatgaaa gacctgagtt 60

tagagaggct gattggcatt cccaatcccc tggggaaggt ttagagaccc tgactccttg 120

<210> 76

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 76

gaattaaggg agcaagtacc cagctaggct ccttccttcc tcactcaccc aacatttcag 60

gtacttcact gatgttccac atccttcttt aaaggttgct cttgtctttt ttctggctag 120

<210> 77

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 77

tgtctactat aactgtaatt gatgcccaac gcttttctgg aaccactttt ggccaagttc 60

atttattatt aatcaaactg tccactgtag aaaatactaa aaatgctcaa gtgggattag 120

<210> 78

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 78

gaatagtcaa ggtactaaca gcattctttt tatgcccttc tctcagattc tgattctcct 60

gcttatttgc aaacaaatga tacattttgg tgctaatgag gaacccccac ataaccttct 120

<210> 79

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 79

ccctgtgtta catactaata catttcaata ctatgcctag tttatcttca tgtcagttgc 60

tgtggctatg atgccccctc cttgatatgt gtgaattccc agtggaaaga gaaagggaaa 120

<210> 80

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 80

gtggaaatgc cctatttggc attaagaaat tgactatcag caccatttct tcccctgaaa 60

taaaaaaaaa aattctcctt gcaaaaggga actttgcctg aggttcttac agagctttgg 120

<210> 81

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 81

ttataaagat caacttataa agaatgctta cccctttcat agtgtcctta actaaacaac 60

aaggatggtc cactaaccga gatctaacct gccttctcta aacaacagta acactaaatc 120

<210> 82

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 82

cagtgccatc actgcacagt ggagaattta ccactaatgt gaaaagcttt cagttttggg 60

aatatagcca ttatttattt ctaatcatat gtgtattttt cccttggcca ggaatccata 120

<210> 83

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 83

ggttttgcac aatagtaatt aattccatta acaaatagta gtgtctcaaa aggcatcttt 60

ttcattttct tatatttgag ctggattttt gtgagacgag gcaattgctc aactaccttt 120

<210> 84

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 84

gctgctacca ctgcttccat tcttaaggac atagtatatt caaaaataaa ccataagcat 60

ggctttttgc tattgataaa gagagaaatg tctaaggaaa tgagggtaaa aagctttcaa 120

<210> 85

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 85

aattaatact tagtctactt aaatgaaaat ctgtaaacat ctaatgaaat gcttgtatat 60

ataacttagt atcttttccc aatttattat catttttatc aaactaattc cattataaaa 120

<210> 86

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 86

gctcttcctg tttcagtccc cattaaatga ggttttactg ttgttcttta ataattttcc 60

ttcatcttac agatcagttt cctaattcat ctcagaacgg ttcatgccga caagtgcagt 120

<210> 87

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 87

atcctctgac agacatgtcc cccatcctaa ctagtgggga ctctgatata tccagtccat 60

tactgcaaaa tactgtccac attgacctca gtgctctaaa tccagagctg gtccaggcag 120

<210> 88

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 88

tgcagcatgt agtgattggg cccagtagcc tgattgtgca tttcaatgaa gtcataggaa 60

gaggtaagta tttccactca gctttttgtt aaatacgatt ttccagtaag cattttatct 120

<210> 89

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 89

ttggcctttg cagattagga acttagacaa tggtgaaagc aactgacaga gcagtgataa 60

caagtgtact tgatttctgt tctatagaaa tgtagccctg taaatcatat ccgtggggat 120

<210> 90

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 90

ttgcccttgt gcatggaagc aattggataa tcccccaaat atattagaac taaatcacaa 60

ttcgtcctcg tcctgtgtgt actagcaatt atagtttctt caaaggtgcc atttactttc 120

<210> 91

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 91

ttctaaaact cagggccagg cgcagtggct cacacgtgta atcccagcac tttgggaggc 60

cgaggcaggt ggatcacctg aggtcaggag ttcaaaacca tcctggccaa catggcaaaa 120

<210> 92

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 92

ccccgtctct actaaaaaaa tacaaaaatt agccaggcgt ggtggcgggt gcctttaatc 60

ccaactactg gggaggctga agcaaggaga attgcttgaa cccaggaggt ggaagttgca 120

<210> 93

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 93

gtgagccaag gttgtgccac agcactccag cctgggcagc agagcgagac tccatgtcaa 60

aaaaataaat acttataaat aaataaatat cactccttta attttgagta tttttattca 120

<210> 94

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 94

atctctctcc agtctttctt taccctgaga acagtgacaa actctatgaa gcctggttta 60

tagcagtttg tacactgctg gacagcatca gaagacagag aatttatagc attctcctga 120

<210> 95

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 95

tctaaagcaa catatatcat ctctacaatg cactccaatt tctttataca aagataaatg 60

aatatttgta ataagctagc caaagcagca ataacagcta gcataaacag tatttgtgga 120

<210> 96

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 96

aatatttagc aggaaaagaa accaatcaaa aaccgggaaa tgaacttcat tcttcttgtt 60

gttttttttt ttctaaaaag cttttaccct taatactaaa tggcctctga tcccttttat 120

<210> 97

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 97

tctatatgtg ctgcaagagg ttgtacaggc atctgccagt gtgatacaag agaagctgat 60

ggcgtgattt cccttatatg aacacaaggc cttctcactt tcctgtggta gcatccacac 120

<210> 98

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 98

attcttctgt caaacacccc agagcgtaat cctttgtgtg ctccttcttc cccaagagtg 60

gcttcactgt taacaaacat ctttccattt ctcttcagag taatatatag ttctgtcata 120

<210> 99

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 99

ggcctataaa tcccagtaat tgcatcaatt ttagagtgtg tggactcact aattgtgttc 60

taattctaca catttggaaa actagaattt tttatcatga aaggtactaa aatggcaatt 120

<210> 100

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 100

atttctcttt tgaagttata atcattaaca gctctataaa tattagctac tatgtattta 60

tatgtttttc tgcaggagaa taggagcact caaaagcata agttttattt tattcttctt 120

<210> 101

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 101

tgcattagta gagggcagcc taatgcctga cactcacata ctaggcactc atggtgtctt 60

ggccagctgg atgaattaat tagtgaatga cttacacagc tatcagacat ttggcacctc 120

<210> 102

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 102

tgggggaaat tactgccact ggataaaagg ctaccattgg gaaaatgatg tggttaaagc 60

cagagagaac tggatgaagt gagtcagggt gaatttgctt catctgggca actgcctttc 120

<210> 103

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 103

agtttctgcc aacctggatt acgtattaac cagtgactaa tggggaaatc cttattctat 60

aatactaatc ctatttttaa tggtaacatt tttatttcat ttcagcttat tagaatatag 120

<210> 104

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 104

agaacttatt ttattatagt tcttcatgtg ggttaacttt attttcatat tttaaaatac 60

tatgtcatcc tttaaaaaaa tttatttgat gaggccgatg ttactcacct tttcagtggt 120

<210> 105

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 105

aaaaaaattt atttgatgag gccgatgtta ctcacctttt cagtggttcc actgtggttg 60

aatattttat taggttaggc tttaatatga gtcatatcat ctacaactta ttcatgaatt 120

<210> 106

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 106

tcataattaa atgttacgca gtgctaacca agttctttct tttgcacagg gcattttggt 60

tgtgtatatc atgggacttt gttggacaat gatggcaaga aaattcactg tgctgtgaaa 120

<210> 107

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 107

tgtgtatatc atgggacttt gttggacaat gatggcaaga aaattcactg tgctgtgaaa 60

tccttgaaca gtaagtggca ttttatttaa ccatggagta tacttttgtg gtttgcaacc 120

<210> 108

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 108

attcactgtt ccataatgaa gttaatgtct ccaccactgg atttctcagg aatcactgac 60

ataggagaag tttcccaatt tctgaccgag ggaatcatca tgaaagattt tagtcatccc 120

<210> 109

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 109

aatgtcctct cgctcctggg aatctgcctg cgaagtgaag ggtctccgct ggtggtccta 60

ccatacatga aacatggaga tcttcgaaat ttcattcgaa atgagactca tgtaagttga 120

<210> 110

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 110

gtctccgctg gtggtcctac catacatgaa acatggagat cttcgaaatt tcattcgaaa 60

tgagactcat gtaagttgac tgccaagctt actaactggc aaactagctg taagccagcc 120

<210> 111

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 111

agaacagtag atgcttagtt tatgcttttc taactctctt tgactgcaga atccaactgt 60

aaaagatctt attggctttg gtcttcaagt agccaaaggc atgaaatatc ttgcaagcaa 120

<210> 112

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 112

tagccaaagg catgaaatat cttgcaagca aaaagtttgt ccacagagac ttggctgcaa 60

gaaactgtat gtaagtatca gaatctctgt gccacaatcc aaattaagtg acaaggagga 120

<210> 113

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 113

taaattattc tatttcagcc acgggtaata atttttgtcc tttctgtagg ctggatgaaa 60

aattcacagt caaggttgct gattttggtc ttgccagaga catgtatgat aaagaatact 120

<210> 114

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 114

atagtgtaca caacaaaaca ggtgcaaagc tgccagtgaa gtggatggct ttggaaagtc 60

tgcaaactca aaagtttacc accaagtcag atgtggtaat gtattggtta tctctgagtt 120

<210> 115

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 115

aaagctgcca gtgaagtgga tggctttgga aagtctgcaa actcaaaagt ttaccaccaa 60

gtcagatgtg gtaatgtatt ggttatctct gagtttctcc tcttttactt tcatatccaa 120

<210> 116

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 116

tgtatggtca catctctcac ctcatctgtc ctgtttcttg ttttactagt ggtcctttgg 60

cgtgctcctc tgggagctga tgacaagagg agccccacct tatcctgacg taaacacctt 120

<210> 117

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 117

tgatataact gtttacttgt tgcaagggag aagactccta caacccgaat actgcccaga 60

ccccttgtaa gtagtctttc tgtacctctt acgttcttta cttttacaga aatgcctgcc 120

<210> 118

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 118

ttcaaagggt ctcttacagc atgtctttct ttttggaaca gatatgaagt aatgctaaaa 60

tgctggcacc ctaaagccga aatgcgccca tccttttctg aactggtgtc ccggatatca 120

<210> 119

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 119

gcgatcttct ctactttcat tggggagcac tatgtccatg tgaacgctac ttatgtgaac 60

gtaaaatgtg tcgctccgta tccttctctg ttgtcatcag aagataacgc tgatgatgag 120

<210> 120

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 120

gtggacacac gaccagcctc cttctgggag acatcatagt gctagtacta tgtcaaagca 60

acagtccaca ctttgtccaa tggttttttc actgcctgac ctttaaaagg ccatcgatat 120

<210> 121

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 121

tctttgctct tgccaaaatt gcactattat aggacttgta ttgttattta aattactgga 60

ttctaaggaa tttcttatct gacagagcat cagaaccaga ggcttggtcc cacaggccac 120

<210> 122

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 122

ggaccaatgg cctgcagccg tgacaacact cctgtcatat tggagtccaa aacttgaatt 60

ctgggttgaa ttttttaaaa atcaggtacc acttgatttc atatgggaaa ttgaagcagg 120

<210> 123

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 123

aaatattgag ggcttcttga tcacagaaaa ctcagaagag atagtaatgc tcaggacagg 60

agcggcagcc ccagaacagg ccactcattt agaattctag tgtttcaaaa cacttttgtg 120

<210> 124

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 124

tgttgtatgg tcaataacat ttttcattac tgatggtgtc attcacccat taggtaaaca 60

ttccctttta aatgtttgtt tgttttttga gacaggatct cactctgttg ccagggctgt 120

<210> 125

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 125

agtgcagtgg tgtgatcata gctcactgca acctccacct cccaggctca agcctcccga 60

atagctggga ctacaggcgc acaccaccat ccccggctaa tttttgtatt ttttgtagag 120

<210> 126

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 126

acggggtttt gccatgttgc caaggctggt ttcaaactcc tggactcaag aaatccaccc 60

acctcagcct cccaaagtgc taggattaca ggcatgagcc actgcgccca gcccttataa 120

<210> 127

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 127

atttttgtat agacattcct ttggttggaa gaatatttat aggcaataca gtcaaagttt 60

caaaatagca tcacacaaaa catgtttata aatgaacagg atgtaatgta catagatgac 120

<210> 128

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 128

attaagaaaa tttgtatgaa ataatttagt catcatgaaa tatttagttg tcatataaaa 60

acccactgtt tgagaatgat gctactctga tctaatgaat gtgaacatgt agatgttttg 120

<210> 129

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 129

tgtgtatttt tttaaatgaa aactcaaaat aagacaagta atttgttgat aaatattttt 60

aaagataact cagcatgttt gtaaagcagg atacatttta ctaaaaggtt cattggttcc 120

<210> 130

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 130

aatcacagct cataggtaga gcaaagaaag ggtggatgga ttgaaaagat tagcctctgt 60

ctcggtggca ggttcccacc tcgcaagcaa ttggaaacaa aacttttggg gagttttatt 120

<210> 131

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 131

ttgcattagg gtgtgtttta tgttaagcaa aacatacttt agaaacaaat gaaaaaggca 60

attgaaaatc ccagctattt cacctagatg gaatagccac cctgagcaga actttgtgat 120

<210> 132

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 132

gcttcattct gtggaatttt gtgcttgcta ctgtatagtg catgtggtgt aggttactct 60

aactggtttt gtcgacgtaa acatttaaag tgttatattt tttataaaaa tgtttatttt 120

<210> 133

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 133

taatgatatg agaaaaattt tgttaggcca caaaaacact gcactgtgaa cattttagaa 60

aaggtatgtc agactgggat taatgacagc atgattttca atgactgtaa attgcgataa 120

<210> 134

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 134

ggaaatgtac tgattgccaa tacaccccac cctcattaca tcatcaggac ttgaagccaa 60

gggttaaccc agcaagctac aaagagggtg tgtcacactg aaactcaata gttgagtttg 120

<210> 135

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 135

gctgttgttg caggaaaatg attataacta aaagctctct gatagtgcag agacttacca 60

gaagacacaa ggaattgtac tgaagagcta ttacaatcca aatattgccg tttcataaat 120

<210> 136

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 136

gtaataagta atactaattc acagagtatt gtaaatggtg gatgacaaaa gaaaatctgc 60

tctgtggaaa gaaagaactg tctctaccag ggtcaagagc atgaacgcat caatagaaag 120

<210> 137

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 137

aactcgggga aacatcccat caacaggact acacacttgt atatacattc ttgagaacac 60

tgcaatgtga aaatcacgtt tgctatttat aaacttgtcc ttagattaat gtgtctggac 120

<210> 138

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 138

agattgtggg agtaagtgat tcttctaaga attagatact tgtcactgcc tatacctgca 60

gctgaactga atggtacttc gtatgttaat agttgttctg ataaatcatg caattaaagt 120

<210> 139

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 139

tggtacttcg tatgttaata gttgttctga taaatcatgc aattaaagta aagtgatgca 60

acatcttgta tactgatagt ggttattgcc agtcatgctt gattacctgc atttgcataa 120

<210> 140

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 140

ccagcggagc ggtcctgcgc ccccgccccc tccaggagcc gcggcgcgca cagccgggct 60

ggagcgcgca cccggcactg aggcttcgct gctctcggag cgctcagacc gcggccgccg 120

<210> 141

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 141

cagccggcga aagaggcaaa gtcccgcacg ccggaggaca tgcgcctcgg ctagcggccc 60

cgggccccac caccgtgcgg ctttctccag attattcctc tctcgctgtc tctgactgtc 120

<210> 142

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 142

tctctctgtc tctgtctctg tctctctctc tctcacacac acacacacac acacaaacac 60

acatacgcac gcacgatctc acttcgatct atacactgga ggattaaaac aaacaaacaa 120

<210> 143

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 143

aaaaaacatt tccttcgctc cccctccctc tccactctga gaagcagagg agccgcacgg 60

cgaggggccg cagaccgtct ggaaatgcga atcctaaagc gtttcctcgc ttgcattcag 120

<210> 144

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 144

ctcctctgtg tttgccgcct gggtgagtga gaagagctcg gtggggtttc agctccggga 60

tcggtgggat gagggtggga gcatttcagc gtgtccgcga cttgccgccg ccgccgccat 120

<210> 145

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 145

ctagccagga ggaaaaaggg acagccaact gggctcccac ggagcgggcg gccgcgcggt 60

gcaggcagct gcgtgggatt tgcgcgcccc ctccccgccc cgcgccctcg ccccttccat 120

<210> 146

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 146

cggcccgcgg gcattcgtgc cccgggcccc aggagacgga gctactggag acggagggct 60

actcggatcc ctgccgccac cgttcttctc tcctcttttg caaagtgtag aaatcgcctt 120

<210> 147

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 147

ttagatatac ataggaagat aaatgaccag aaatacagct gtggtgcggg gctgtgtact 60

gtactgtctt ttttgctgtt catttttatg ctataggtat gatgggctta aatggaaagt 120

<210> 148

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 148

acacgtgtct ggtgtgaatt gcagacacac acacacacac acacacacac ctgaaaggta 60

gtaacaggca caagcaccaa cctgactttt cagggttttt tttattactt tcaatttagt 120

<210> 149

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 149

catattcaag tctgcgcaat tacttacaaa aaaaaaagtt ccatctaaat ctctaggttt 60

ggtcagtgct gccaaagtca gaggagagct gcaggttgat atacttattc acagatttca 120

<210> 150

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 150

tttttcatct gcaccctcca ggttacattt aaaactgaaa ttgcattcag caatagccag 60

tttaagcatt ccctttacct gcgatgcaga ctcaagtttt ccagtcattt tcgagaagtg 120

<210> 151

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 151

caagattgga cttttgcgtt ttattaaaga aatgcttttt ttccttcatg attttccatc 60

gcgatagaat ataatgggtt tacgcattaa cctgaaagtt acaaagtgtt gtacttcgct 120

<210> 152

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 152

cttcgctttg tgagtacaca tatttaattc ttttcagagg ctcccaattt ttcaatttta 60

gatggttcat agtagagtag atattaagcg tacattttca tccaatagat ggtgctgctc 120

<210> 153

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 153

ggacatatat ttttctacat actgattact tggtttttct tttttataga ttgggctaat 60

ggatactaca gacaacagag aaaacttgtt gaagagattg gctggtccta tacaggtaaa 120

<210> 154

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 154

catattactt atataatgag atttagcaga aacttttatt gttttgtatc tatttttata 60

tataaaagga agctttgtag cacaacacat catgtcaact tagctaaatg gatagttaga 120

<210> 155

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 155

ttaattaata gatatataaa atataaaacg atatcttatg ggctttccat aaacaaattt 60

ggtaatgtag ggcttattga catccaccca aactgtattt gaagctcagg cccaagctgt 120

<210> 156

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 156

ctatatacct tcaagaaata acataacctg gggctctgtt tttttcaacg gtgaaatggg 60

gggctgttct gagcattaaa tgagataaat aaagtgtgaa gcacttgtca cagagcctgc 120

<210> 157

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 157

ttctaaacag aaagctctca acaaatgtta gctgctggag ttgttatgat catgctaatg 60

attatttttg ttcatttaac aaatattgat gggattcttg ctctcagaag gggcattctt 120

<210> 158

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 158

ctgaattact gttgttaatt tattggtgtt agagtaaaaa atgattttgt cacattacta 60

gactttgcct agaacattat gaacatctga agttggcttg aaatagttac aaccaaaaat 120

<210> 159

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 159

catcagcaga aactctggga gagaaagttc tagccctgag tggaagaata tgagtggaat 60

ttgggtgaac catgacatgc ctagtaacca tgtgggatgg agttgagcat tagagttggg 120

<210> 160

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 160

tactcattag cagtaaactt gggttgataa gataaagtca gattacaagg gctttaccat 60

gagcctggcc aattttactt aatagatgaa gtgcagaagc atgctgaatt ttgtagataa 120

<210> 161

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 161

ggatgtattt tctttcttct ttttaattgc tattttaaaa gtagtaaata ttcattgtag 60

aaaatttaga aataggttca agtacaaaga aaaacataaa atcattattt tatttccctc 120

<210> 162

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 162

aagaaaaaca taaaatcatt attttatttc cctcaggaat agccacttaa aaaactgcta 60

tagtcctttt agacagtttt tattatccaa aatttatata ttttatttat agagttatgc 120

<210> 163

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 163

ttgcatggta atttaagaaa ctaaatttct tactccttct tttccctagg agcactgaat 60

caaaaaaatt ggggaaagaa atatccaaca tgtaatagcc caaaacaatc tcctatcaat 120

<210> 164

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 164

attgatgaag atcttacaca agtaaatgtg aatcttaaga aacttaaatt tcagggttgg 60

gataaaacat cattggaaaa cacattcatt cataacactg ggaaaacagg taaaatattt 120

<210> 165

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 165

gcattctgtt tgcctttaat atatttttca gacctggagt atgtttagac tagtttcatc 60

tcattttcct ttgaatagtg tactatgaac tagatagaag ttacatgtag aagaaaccaa 120

<210> 166

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 166

atggagcaga gaaaatggga aatttagaca atattttgac aatttgtaca aaaaaaaaat 60

gaccaatgtc atcatgtcat gttttcctac aaggtaacaa taatgatgct gcctctagaa 120

<210> 167

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 167

gatgtgataa tgtgatttac agctaatata atttagtcaa ttacttagtc aatcaggatt 60

tctttttctt gagcattata gtttgatctt ttctgcctag cctcatgtta tagtcagtat 120

<210> 168

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 168

ttggatatga aaaaagcaat tggctataat tgaaatttaa gaaaataaat tcctagctct 60

tctatataga aggtttaaag ttgtatccca tccttaggaa accctctcta agtgtctttt 120

<210> 169

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 169

tattgagcct atcttttttt ttttttaaaa aaaagcaaaa tataatacat taaaatttaa 60

gtacaaattc taataagcta tacatagata aataatttcc aaaaagtgac accttttctt 120

<210> 170

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 170

ttttgatatt ctagtttttt taaaaattgt attttgggaa attatgtaaa ggcaaatata 60

cacgtaagac aaagattaga aaaatattca aaattataat agtagctcta tggggataag 120

<210> 171

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 171

ctttatcatt ggtgaatttt ttttatattt tctatattta ggtaatgtaa ttatttgaac 60

ataataattt aatttcaaaa ctcaagagag tagtagtcag gcactgtggc tcacgcctgt 120

<210> 172

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 172

gatcccagca cttcgggagg ccgaagtggc tggatcactt gaggtcaaga gtaccagact 60

agcctggcca atatggcaaa aacccatctc tactaaaaat acaaaaatta gctgggtgtg 120

<210> 173

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 173

aacccatctc tactaaaaat acaaaaatta gctgggtgtg gtggcacact cctgtaattc 60

cagatactca ggaggctgag acacaagaat cacttgaact caagagttgg aggctgcagt 120

<210> 174

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 174

aaaattttga ttttcagaag cttaggtgca attgtatttc tttttttagt ggaaattaat 60

ctcactaatg actaccgtgt cagcggagga gtttcagaaa tggtgtttaa agcaagcaag 120

<210> 175

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 175

ataacttttc actggggaaa atgcaatatg tcatctgatg gatcagagca tagtttagaa 60

ggacaaaaat ttccacttga ggtaagtcag gagatctgct gtgtactatt ttattttcta 120

<210> 176

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 176

ctggggaaaa tgcaatatgt catctgatgg atcagagcat agtttagaag gacaaaaatt 60

tccacttgag gtaagtcagg agatctgctg tgtactattt tattttctaa ataattgatg 120

<210> 177

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 177

ttttatgaag tctttgtgta tcataaaact atcattgtta cttttataga tgcaaatcta 60

ctgctttgat gcggaccgat tttcaagttt tgaggaagca gtcaaaggaa aagggaagtt 120

<210> 178

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 178

ccgattttca agttttgagg aagcagtcaa aggaaaaggg aagttaagag ctttatccat 60

tttgtttgag gtaatatata tacactttac actaatgtaa ttccttttta agtcacatta 120

<210> 179

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 179

ttatccaaat gttttattct ttttttagaa tgtgattctt ttttaacagg ttgggacaga 60

agaaaatttg gatttcaaag cgattattga tggagtcgaa agtgttagtc gttttggtaa 120

<210> 180

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 180

caggttggga cagaagaaaa tttggatttc aaagcgatta ttgatggagt cgaaagtgtt 60

agtcgttttg gtaagctact tggggaacta tctttcttca ggattctgct ttggatggat 120

<210> 181

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 181

aagttccagt gttgagattc cagctgagat gtatattatt cctttttagg gaagcaggct 60

gctttagatc cattcatact gttgaacctt ctgccaaact caactgacaa gtattacatt 120

<210> 182

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 182

tacaatggct cattgacatc tcctccctgc acagacacag ttgactggat tgtttttaaa 60

gatacagtta gcatctctga aagccaggta atcttagaaa ttcaaacaaa tcaagtaatt 120

<210> 183

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 183

atctcctccc tgcacagaca cagttgactg gattgttttt aaagatacag ttagcatctc 60

tgaaagccag gtaatcttag aaattcaaac aaatcaagta attcaaagcc atttaaaaaa 120

<210> 184

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 184

accaatgcct ttgaagcaga tatgttaaat tatttgatct ttttttcagt tggctgtttt 60

ttgtgaagtt cttacaatgc aacaatctgg ttatgtcatg ctgatggact acttacaaaa 120

<210> 185

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 185

caattttcga gagcaacagt acaagttctc tagacaggtg ttttcctcat acactggaaa 60

ggaagagatt catgaagcag gtatgtattt aaatataatc ttctacaact ctcatagatg 120

<210> 186

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 186

cagtgttata ggaggtaatt ttggtaaact gacaaatata gaggacttag agctttagaa 60

atgttttaat tattaatttt gtagctttag ataagctgtc taattttctg aatatcagat 120

<210> 187

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 187

accacatcta taaaatggca actaaaaaaa tctattcttc ccataagaat aaccccattt 60

tacagatgag aaaaactgag atttaataag gaaaatcatt tttccagaat acacagtaat 120

<210> 188

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 188

aagtagcaaa gcaaagtaca cacctccaag ttcactttct ggtatttgcc tgggctgaga 60

ggtatgagaa aggaatgaat aagaggctga ggtgttcaga gcttgggaga tcatttctgg 120

<210> 189

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 189

ttgggataat aaggaaaact ttatgggaga accgcctttg aactgagtct tgacacagta 60

tttgggtttt aaagtgctca tctttgtgtt ggcagccccc aagaaggagc ctagcacctt 120

<210> 190

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 190

atcagtactg aaaataaatg cactgagtta ttaatgaata agaagtgaat cttcttccca 60

gtttccttct ttggaggaaa agaaatttgc tctttttagg tacagccaag ggaaatgaca 120

<210> 191

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 191

ccagaggggt gtctctagaa tgccgtctgt tttttgaagc tgacacagta gttaaaagca 60

tttttaaatg tccagagaac acaatcagat acatctgctg acttgtcctt ttcagttttt 120

<210> 192

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 192

actgaggttg tcacttgttc attaaagaag tcgttgaagt gacctttgca ctggaaagga 60

cttctaacct ttccaagtag atggacctct ttgatatcag tcctgcaatg aattcaatga 120

<210> 193

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 193

tttttttttg ccatgtataa tattaattta ataatcatct gcttttgaag attaggcatt 60

aaaacaacaa gcttttattg ttttaatgtg atatttccaa gaatcgttta tcctttaaca 120

<210> 194

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 194

ttaggcatta aaacaacaag cttttattgt tttaatgtga tatttccaag aatcgtttat 60

cctttaacaa aaaaatagaa ttcaagacaa cagtgatgaa tagctatatt tactatgaca 120

<210> 195

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 195

gtcctcaagg ctaagttcta tcaacaactg tacttttttc tctctgaagt ttgtagttca 60

gaaccagaaa atgttcaggc tgacccagag aattatacca gccttcttgt tacatgggaa 120

<210> 196

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 196

agacctcgag tcgtttatga taccatgatt gagaagtttg cagttttgta ccagcagttg 60

gatggagagg accaaaccaa gcatgaattt ttgacagatg gctatcaaga cttggtaact 120

<210> 197

<211> 120

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 197

tttgtaccag cagttggatg gagaggacca aaccaagcat gaatttttga cagatggcta 60

tcaagacttg gtaactatat gatcagttgt tttacatagg gtaacattat aatttaattt 120

<210> 198

<211> 140

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 198

ccccgctgtg cttgcacctg gcatcctcgt gctcctgttt accttggtgc agaggagcaa 60

tggggagtgt aaagaggcac tagcaaagtc cgagatgaat gtgaatatga agtatcagct 120

tcccaacttc accgcggaaa 140

<210> 199

<211> 100

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 199

gtgaatatga agtatcagct tcccaacttc accgcggaaa cacccatcca gaatgtcatt 60

ctacatgagc atcacatttt ccttggtgcc actaactaca 100

Claims (3)

1. The application of the probe library in preparing a kit for detecting the fusion gene mutation of the MET family is characterized in that the probe library comprises all probes with nucleotide sequences shown in SEQ ID NO. 1-197.

2. The kit is characterized by comprising all probes with nucleotide sequences shown in SEQ ID NO. 1-197.

3. The kit of claim 2, further comprising: a DNA extraction reagent for extracting DNA of a sample to be detected; further comprises: the RNA extraction reagent is used for extracting mRNA of a sample to be detected; a reverse transcription kit for reverse transcribing the extracted mRNA into cDNA.

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