CN109628558A - A kind of capture probe and its application for high-flux sequence detection gene mutation - Google Patents

Abstract

The present invention relates to technical field of gene detection, and in particular to a kind of capture probe and its application for high-flux sequence detection gene mutation.The present invention provides a kind of capture probe for high-flux sequence detection gene mutation, it is single-stranded probe mixture, including since one end of DNA to be measured, respectively for the nucleotide sequence of the positive-sense strand of DNA to be measured and antisense strand, the probe for positive-sense strand and the probe for antisense strand successively designed；In every two adjacent single-stranded probe, one is the single-stranded probe for the design of the nucleotide sequence of positive-sense strand, another is the single-stranded probe for the nucleotide sequence design of antisense strand；The targeted DNA to be measured of every two adjacent single-stranded probe is not overlapped；Single-stranded probe mixture can cover all DNA to be measured.Capture probe provided by the invention can significantly improve storage capacity, capture rate and the sequencing data effective depth in capture library, reduce the repetitive rate of sequencing data, have very high application value.

Description

A kind of capture probe and its application for high-flux sequence detection gene mutation

Technical field

The present invention relates to technical field of gene detection, and in particular to a kind of catching for high-flux sequence detection gene mutation Obtain probe and its application.

Background technique

Genome sequencing can obtain mutation, insertion, missing and the structure variation of full-length genome horizontal extent.So And since gene pool-size is larger, with 30 × carry out sequencing and will generate data volume close to 100G.And tumour etc. is relevant low Frequency of mutation sequencing then needs at least 1000 × coverage can generate up to 3000G if carrying out genome sequencing Data volume.The data volume of scale can also generate huge other than it can cause greatly difficulty to the analysis work of data in this way Sequencing cost.

Target area solution hybridization capture technique is to design the spy complementary with target sequence according to DNA sequence dna complementarity principle Specific probes, and synthesize in solid phase or liquid-phase chip, sample gene group DNA is interrupted, in addition hybridize after sequence measuring joints with probe, Finally target area is captured, it is subsequent by recycling, constructing library and be directly used in high-flux sequence.It mentions both at home and abroad at present Have much for the company of liquid phase hybrid capture technology, such as the external chemical reagent work of Agilent, Roche, IDT, TWIST bioscience The domestic manufacturers such as quotient and Ai Jitaikang, Mai Jinuo.

The probe of the most critical applied to hybrid capture of above-mentioned manufacturer is had nothing in common with each other, and is also had their own characteristics each.Such as: Agilent It is rna probe, Roche and IDT using DNA single-stranded probe with Ai Jitaikang use.Rna probe and the design of DNA single-stranded probe Principle be substantially based on the design method of stacked tile type, keep consistent with genomic sense strand is referred to or on the contrary, can only catch It receives and is then dropped with the positive-sense strand or the consistent library part of antisense strand, another chain for referring to genome, in such cases, If capture effect is bad, two chains that will lead to library are all lost, and reduce the richness in library.TWIST Bioscience, Mai Jinuo carry out library capture using double-chain probe, can guarantee that two chains in library are all captured in this way, still Since double-chain probe is reverse complemental structure, has and hybridized mutually between many probes, result in the need for the dosage of probe compared with Greatly or cause capture rate relatively low.Therefore, it develops capture rate height, can be avoided the interaction between probe, and can The capture probe for improving library richness is of great significance.

Summary of the invention

In order to solve the technical problems existing in the prior art, the object of the present invention is to provide one kind to be used for high-flux sequence Detect capture probe and its application of gene mutation.

To solve single-stranded capture probe and double-strand capture probe in the prior art, it may be assumed that (1) single-stranded Probe can only capture a chain of target fragment, cause the template of capture few, since probe is only directed to the probe of antisense strand, only Having two antisense strands of segment 1 and segment 2 can be captured, and two positive-sense strands can not be captured due to identical as probe sequence And lose (as shown in Figure 1).(2) although positive-sense strand and antisense strand that double-chain probe can capture target fragment 1 and segment 2 (such as Shown in Fig. 2), but since double-chain probe is reverse complemental structure, it has and is hybridized mutually between many probes, result in the need for visiting The dosage of needle is larger or causes capture rate relatively low.The present invention creatively proposes a kind of completely new capture probe design side Method, the present invention use the single-stranded probe for positive-sense strand and the single-stranded probe for antisense strand to be staggered, cover whole purposes The form in region had both avoided the mutual hybridization between probe, caused capture rate and probe utilization rate to reduce, and can be simultaneously Two chains of target fragment in library are captured, library total amount and richness, probe design diagram such as Fig. 3 institute of the invention are improved Show.

Firstly, the present invention provides a kind of capture probe for high-flux sequence detection gene mutation, the capture probe For single-stranded probe mixture, the single-stranded probe mixture includes since one end of DNA to be measured, respectively for DNA's to be measured The nucleotide sequence of positive-sense strand and antisense strand, the probe for positive-sense strand and the probe for antisense strand successively designed；Every two In a adjacent single-stranded probe, one is the single-stranded probe for the design of the nucleotide sequence of positive-sense strand, another is for anti- The single-stranded probe of the nucleotide sequence design of adopted chain；The targeted DNA to be measured of every two adjacent single-stranded probe is not overlapped；Institute All DNA to be measured can be covered by stating single-stranded probe mixture.

Suitable probe length and mixed proportion can preferably improve probe capture rate, described single-stranded in the present invention The length of probe is 90~120nt；Each single-stranded probe is mixed in the single-stranded probe mixture with identical molar ratio.

Preferably, in the present invention, the length of the single-stranded probe is 110~120nt.

On this basis, the present invention also provides the capture probes examines in gene order-checking library construction or high-flux sequence Application in cls gene mutation.

The present invention also provides a kind of construction methods of gene mutation sequencing library, to interrupt genomic DNA, using this The invention capture probe constructs sequencing library.

Specifically, the construction method of gene mutation sequencing library includes the following steps:

(1) genomic DNA is extracted, is interrupted as small fragment；

(2) library preparation is carried out to the small fragment DNA that step (1) obtains；

(3) the small fragment DNA library that step (2) obtains is hybridized with capture probe of the present invention, captures mesh Segment；

(4) segment of step (3) capture is expanded, machine on amplified production is sequenced.

Under the guidance of above-mentioned capture probe and preparation method thereof, the present invention is by taking EGFR genetic mutation detects as an example, exploitation For the capture probe of EGFR gene.

The present invention provides a kind of specific capture probe of EGFR gene comprising such as SEQ ID NO.172~SEQ ID Single-stranded probe shown in NO.228.

In the present invention, the capture probe can be marked using the biomarker that any this field allows, as One embodiment of the present invention, the present invention use the above-mentioned single-stranded probe of biotin labeling.

On the basis of above-mentioned capture probe, the present invention provides a kind of kit for detecting EGFR genetic mutation comprising The specific capture probe of the EGFR gene.

Preferably, the capture probe is rubbed as single-stranded probe as shown in SEQ ID NO.172~SEQ ID NO.228 etc. You form in ratio mixing.

It is highly preferred that the working concentration of the capture probe is 0.1pM~0.75pM.

For the high-flux sequence for being easy to implement EGFR genetic mutation, in the present invention, the kit further includes Hybridization Buffer Liquid, elution buffer, positive quality control product, negative quality-control product.

Further, the present invention also provides the specific capture probe of the EGFR gene or include the special of EGFR gene Property capture probe kit high-flux sequence detect EGFR genetic mutation or building detection EGFR genetic mutation sequencing text Application in library.

As one embodiment of the present invention, using capture probe of the present invention carry out sequencing library building and The method of high-flux sequence includes the following steps:

(1) probe design, synthesis and label；

(2) library construction before capturing: genomic DNA is interrupted as small fragment, progress sequencing library building；

(3) library of step (2) building hybridizes with capture probe, carries out the building in capture library；

(4) machine sequencing and data analysis on.

The beneficial effects of the present invention are: the present invention provides a kind of completely new capture probe preparation methods, will be for just It the probe of adopted chain and is interspersed for the probe of antisense strand, covers whole destination regions, both avoided due between probe Capture rate caused by hybridizing mutually and probe utilization rate reduce, and can capture two chains of target fragment in library simultaneously, mention High library total amount and richness can guarantee the richness and capture rate that capture library simultaneously.It is caught using provided by the invention The EGFR specificity capture probe for obtaining probe design process preparation can when carrying out sequencing library building and high-flux sequence The technical effect that library storage capacity is big, capture rate is high, Data duplication rate is low and data effective depth is high is realized simultaneously, is conducive to mention The working efficiency of high high-flux sequence has very high application value.

Detailed description of the invention

Fig. 1 is the design diagram of traditional single-stranded probe in summary of the invention of the present invention.

Fig. 2 is the design diagram of traditional double-chain probe in summary of the invention of the present invention.

Fig. 3 is to show in summary of the invention of the present invention for the probe design that the single-stranded probe of positive-sense strand and antisense strand is interspersed It is intended to.

Specific embodiment

The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Embodiment 1

The present embodiment verifies the design side of capture probe provided by the invention by taking EGF-R ELISA EGFR as an example Method.For the probe of EGFR full exon region distribution three kinds of forms of design: traditional single-stranded probe (control group 1), double-strand are visited Needle (control group 2) and positive-sense strand probe provided by the invention and the staggered single-stranded probe of antisense strand probe (experimental group), And the sequencing analysis of human genome library construction and EGFR hybrid capture library is carried out, library storage capacity, capture to building Efficiency, repetitive rate, data effective depth etc. are analyzed, the capture effect of more different capture probes.

One, probe designs

(1) using the design method of traditional single-stranded probe, single-stranded probe is designed, for the single-stranded probe such as SEQ ID of EGFR Shown in NO.1~SEQ ID NO.57.

(2) using the design method of traditional double-chain probe, double-chain probe is designed, double-chain probe increases than above-mentioned single-stranded probe Reverse complemental probe, for EGFR double-chain probe as shown in SEQ ID NO.58~SEQ ID NO.171.

(3) capture probe being interspersed using probe design process design positive-sense strand of the invention and antisense strand, probe As shown in SEQ ID NO.172~SEQ ID NO.228.

Above-mentioned probe carries out subregion by full exon region to EGFR gene, design a plurality of length be 120nt or The probe of 120bp guarantees that each region of EGFR gene exon is covered by probe.Use the online probe design software of IDT (https: //sg.idtdna.com/site/order/ngs) design of probe is carried out, preset parameter is as follows: probe length 120bp, multiplier 1 ×, behave with reference to genome Human (Feb.2009GRCh37/hg19) with reference to genome, complete outer (do not include UTR).The exon of EGFR gene is separated by multiple intrones generates the different exon subregion of 28 length, in above-mentioned (3) When the design for the capture probe that positive-sense strand and antisense strand are interspersed, when the length of exon subregion is just covered by a probe When, then there is the subregion and only has the case where positive-sense strand probe.5 ' ends of above-mentioned all probes are all made of biotin labeling.

Two, library construction

1, library construction before capturing: using the whole blood cells of people as sample, genomic DNA is extracted, is interrupted as 200bp's Segment.9 parts of above-mentioned genomic DNAs for interrupting segment as 200bp are taken, every part of 1 μ g carries out routine illumina library construction, tool Body method is as follows:

(1) end repairs and adds A: configure reaction solution by reaction system shown in table 1 in PCR pipe, with rifle gently on Lower pressure-vaccum mixes.Above-mentioned reaction system is put into PCR instrument, program is set by conditional parameter shown in table 2, is reacted.

Repair and add A reaction system in 1 end of table

Repair and add A response procedures in 2 end of table

(2) adaptor connection: configuring reaction solution by reaction system shown in table 3 in PCR pipe, gently upper and lower with rifle Pressure-vaccum mixes.Above-mentioned reaction system is divided into 2 pipes, is put in PCR instrument, 20 DEG C of warm bath 15min.With the AMPure of 1 times of volume XP magnetic beads for purifying DNA sample, with the water elution of 23 μ l nuclease frees.

Table 3adaptor coupled reaction system

(3) library of PCR amplification connection adaptor: reaction solution is configured by reaction system shown in table 4 in PCR pipe, is used Gently pressure-vaccum mixes rifle up and down.Above-mentioned reaction system is put into PCR instrument, is carried out by the setting program of response procedures shown in table 5 Reaction.With 1.8 times of AMPure XP magnetic beads for purifying amplified productions, the water elution of 30 μ l nuclease frees, by obtain 9 libraries point It Bian Hao not be Y1-Y9.

4 PCR amplification of table connects the reaction system in the library of adaptor

5 PCR amplification of table connects the response procedures in the library of adaptor

2, Library hybridization capture and elution

It takes the library Y1-Y9 of the above-mentioned building of 500ng in a new centrifuge tube respectively, drains machine with vacuum and be concentrated Until library is evaporated.Using IDT hybrid capture kit, method to specifications carries out hybrid capture and elution.Hybridized It must guarantee that pipe lid covers tightly in journey, minimize the evaporation for reducing hybrid mixed liquid product, otherwise will will affect crossbreeding effect.Its In, single-stranded probe (SEQ ID NO.1~SEQ ID NO.57) the progress hybrid capture of the library Y1, Y2, Y3 use synthesis, Y4, The library Y5, Y6 carries out hybrid capture, Y7, Y8, Y9 using the double-chain probe (SEQ ID NO.58~SEQ ID NO.171) of synthesis Library uses the probe (SEQ ID NO.172~SEQ ID NO.228) synthesized using design method of the invention to be captured. The response procedures of hybrid capture are as shown in table 6.

6 hybrid capture response procedures of table

3, PCR amplification captures library

PCR reaction solution is prepared according to reaction system shown in table 7 on ice, confirmation mixes the reaction solution containing magnetic bead Afterwards, pipe is put into PCR instrument and is expanded, response procedures are as shown in table 8:

Table 7 captures Library PCR amplification reaction system

Table 8 captures Library PCR amplification response procedures

The library obtained using the above-mentioned PCR amplification of AMPure XP magnetic beads for purifying of 1.8 times of volumes, with 20 μ l nuclease frees Water elution, whole library is respectively designated as Z1-Z9 (corresponding respectively to the library Y1-Y9), the library Z1-Z9 outbound situation such as 9 institute of table Show, the results showed that, the storage capacity for carrying out the library of hybrid capture acquisition using single-stranded probe is minimum, is hybridized using double-chain probe Capture and using positive anti-chain of the invention interlock the library that single-stranded probe carries out hybrid capture acquisition storage capacity it is close.

Table 9Z1-Z9 library outbound situation

Bank number	Library amount
		Z1	15ng
Z2	17ng
		Z3	13ng
Z4	30ng
		Z5	32ng
Z6	29ng
		Z7	25ng
Z8	27ng
		Z9	30ng

4, the sequencing in the library Z1-Z9 is carried out using 500 sequenator of Illumina nextseq, sequencing data analyzes result As shown in table 10, final sequencing data analysis the result shows that, in terms of capture rate, (Z1- is captured using single-stranded probe Z3) suitable with the capture rate of (Z7-Z9) is captured using probe of the invention, and (Z4- is captured using double-chain probe Z6 efficiency) is minimum；In terms of repetitive rate, the weight for capturing with being captured using probe of the invention is carried out using double-chain probe Rate is suitable again, and the repetitive rate highest captured using single-stranded probe；In terms of data effective depth, using single-stranded probe into Row capture number suitable, and being captured using probe of the invention with the data effective depth captured using double-chain probe It is significantly improved according to effective depth.

10 library sequencing data of table analyzes result

In conclusion the EGFR specificity capture probe prepared using capture probe design method provided by the invention, It carries out sequencing library building and when high-flux sequence, can be achieved at the same time that library storage capacity is big, capture rate is high, Data duplication rate is low The high technical effect with data effective depth, solves existing for traditional single-stranded probe and double-chain probe that library storage capacity is low, catches Low efficiency and the low problem of data effective depth are obtained, there is very high application value.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications Also it should be regarded as protection scope of the present invention.

Sequence table

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<213>artificial sequence (Artificial Sequence)

<400> 36

ttagaagcta catagtgtct cactttccaa gatcattcta caagatgtca gtgcactgaa 60

acatgcaggg gcgtgttgag tgccaaggcc atggaatctg tcagcaacct cacccttcct 120

<210> 37

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 37

ggaatctgtc agcaacctca cccttccttg ttcctccacc tcattccagg cctaagatcc 60

cgtccatcgc cactgggatg gtgggggccc tcctcttgct gctggtggtg gccctgggga 120

<210> 38

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 38

tcggcctctt catgcgaagg cgccacatcg ttcggaagcg cacgctgcgg aggctgctgc 60

aggagaggga ggtgagtgcc agtcctgggt gggctcagga gccctcgcac cccgacagga 120

<210> 39

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 39

ctttccagca tggtgagggc tgaggtgacc cttgtctctg tgttcttgtc ccccccagct 60

tgtggagcct cttacaccca gtggagaagc tcccaaccaa gctctcttga ggatcttgaa 120

<210> 40

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 40

ggaaactgaa ttcaaaaaga tcaaagtgct gggctccggt gcgttcggca cggtgtataa 60

ggtaaggtcc ctggcacagg cctctgggct gggccgcagg gcctctcatg gtctggtggg 120

<210> 41

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 41

tctgtcatag ggactctgga tcccagaagg tgagaaagtt aaaattcccg tcgctatcaa 60

ggaattaaga gaagcaacat ctccgaaagc caacaaggaa atcctcgatg tgagtttctg 120

<210> 42

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 42

cactgacgtg cctctccctc cctccaggaa gcctacgtga tggccagcgt ggacaacccc 60

cacgtgtgcc gcctgctggg catctgcctc acctccaccg tgcagctcat cacgcagctc 120

<210> 43

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 43

atgcccttcg gctgcctcct ggactatgtc cgggaacaca aagacaatat tggctcccag 60

tacctgctca actggtgtgt gcagatcgca aaggtaatca gggaagggag atacggggag 120

<210> 44

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 44

catgatgatc tgtccctcac agcagggtct tctctgtttc agggcatgaa ctacttggag 60

gaccgtcgct tggtgcaccg cgacctggca gccaggaacg tactggtgaa aacaccgcag 120

<210> 45

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 45

catgtcaaga tcacagattt tgggctggcc aaactgctgg gtgcggaaga gaaagaatac 60

catgcagaag gaggcaaagt aaggaggtgg ctttaggtca gccagcattt tcctgacacc 120

<210> 46

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 46

cctcatctct caccatccca aggtgcctat caagtggatg gcattggaat caattttaca 60

cagaatctat acccaccaga gtgatgtctg gagctacggt gagtcataat cctgatgcta 120

<210> 47

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 47

ttcattcatg atcccactgc cttcttttct tgcttcatcc tctcaggggt gactgtttgg 60

gagttgatga cctttggatc caagccatat gacggaatcc ctgccagcga gatctcctcc 120

<210> 48

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 48

atcctggaga aaggagaacg cctccctcag ccacccatat gtaccatcga tgtctacatg 60

atcatggtca agtgtgagtg actggtgggt ctgtccacac tgcctagctg agccttggtg 120

<210> 49

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 49

tccttcccca ggctggatga tagacgcaga tagtcgccca aagttccgtg agttgatcat 60

cgaattctcc aaaatggccc gagaccccca gcgctacctt gtcattcagg tacaaattgc 120

<210> 50

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 50

cgggccattc taatagcctc aaaatctctg caccaggggg atgaaagaat gcatttgcca 60

agtcctacag actccaactt ctaccgtgcc ctgatggatg aagaagacat ggacgacgtg 120

<210> 51

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 51

gtggatgccg acgagtacct catcccacag cagggcttct tcagcagccc ctccacgtca 60

cggactcccc tcctgagctc tctggtatga aatctctgtc tctctctctc tctcaagctg 120

<210> 52

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 52

agcattccat gggcaacttc tctgtttctt tttcagagtg caaccagcaa caattccacc 60

gtggcttgca ttgatagaaa tggggtatgt atgaacacct tataagccag aatttacagc 120

<210> 53

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 53

ctgcagctgc aaagctgtcc catcaaggaa gacagcttct tgcagcgata cagctcagac 60

cccacaggcg ccttgactga ggacagcata gacgacacct tcctcccagt gcctggtgag 120

<210> 54

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 54

atcctgcatg ggatggtgct ttgctgatta cttcacctct gatttctttc cactttcaga 60

atacataaac cagtccgttc ccaaaaggcc cgctggctct gtgcagaatc ctgtctatca 120

<210> 55

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 55

caatcagcct ctgaaccccg cgcccagcag agacccacac taccaggacc cccacagcac 60

tgcagtgggc aaccccgagt atctcaacac tgtccagccc acctgtgtca acagcacatt 120

<210> 56

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 56

cgacagccct gcccactggg cccagaaagg cagccaccaa attagcctgg acaaccctga 60

ctaccagcag gacttctttc ccaaggaagc caagccaaat ggcatcttta agggctccac 120

<210> 57

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 57

agctgaaaat gcagaatacc taagggtcgc gccacaaagc agtgaattta ttggagcatg 60

accacggagg atagtatgag ccctaaaaat ccagactctt tcgataccca ggaccaagcc 120

<210> 58

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 58

cgagacacgc ccttaccttt cttttcctcc agagcccgac tcgccgggca gagcgcagcc 60

agcagcgcca ggagcgctgc cccggccgtc ccggagggtc gcatcgctgc tccccgaaga 120

<210> 59

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 59

ctgttttaga gctcattcaa aaggagatgg gctttatatt tttgttttca tttgctgaaa 60

caccagacag gcccttttgt catggaaata aatgcaatcc taggcccaaa ggaatgatag 120

<210> 60

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 60

acttggttat ccggataacc ttctcagcgc ggagaggtcg cctgccggag ggaacccaca 60

gcaaatgcac aaccagccag cagcccagag ctccctcttg ttctgaagtc cagtggagaa 120

<210> 61

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 61

aacatcctct ggaggctgag aaaatgatct tcaaaagtgc ccaactgcgt gagcttgtta 60

ctcgtgcctt ggcaaactgg aagaaaaaga cagatatcac atgaaatact gagaaatgca 120

<210> 62

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 62

attttctcca attttatttg tgtaggaaaa tcaaagtcac caacctttaa gaaggaaaga 60

tcataattcc tctgcacata ggtaatttcc aaattcccaa ggaccacctc acagttattg 120

<210> 63

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 63

atatttcctc tgatgatctg caggttttcc aaaggaattc gctccactgt gttgagggca 60

atgaggacat aaccagccac ctcctggatg gtctaagaag agaaaacata aatgcgtgat 120

<210> 64

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 64

ccccagcctt ggcatcccag cctctcaccc tgtaaatttc tcatgggcag ctccttcagt 60

ccggttttat ttgcatcata gttagataag actgctaagg cataggaatt ttcgtagtac 120

<210> 65

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 65

ccactggatg ctctccacgt tgcacagggc agggttgttg ctgaaccgca cggcgccatg 60

caggatttct gcgggagaat ggaactgcgg tgagcactct ttccagctcc tttaaattcc 120

<210> 66

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 66

gagctgtccc ccataggagc tggaggcaga gatagtgtgt atgcgacact tacagctgcc 60

caggtggttc tggaagtcca tcgacatgtt gctgagaaag tcactgctga ctatgtcccg 120

<210> 67

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 67

ggtctgaggc tgttcactga cttacgtttc tggcagttct cctctcctgc accccagcag 60

ctcccattgg gacagcttgg atcacacttt tggcctgaaa tgtaaaagta atagatgcat 120

<210> 68

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 68

ccaggcagtc gctctcccgg gggcctgtgc agcctgcagc acactggttg tggcagcagt 60

cactggggga cttgccacgg cagcgcccgg agcactgctg ggcacagatg attttggtca 120

<210> 69

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 69

gtggggttgt agagcatgag tggggggcag gtgtccttgc acgtggcttc gtctcggaat 60

ttgcggcaga cctatggagg agagaggaca cgctgggcaa gtgaggtaag tacactcagc 120

<210> 70

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 70

cgggacaagg atgcctgacc agttagaggg cccacagagg aggactcacg gggacacttc 60

ttcacgcagg tggcaccaaa gctgtatttg ccctcggggt tcacatccat ctggtacgtg 120

<210> 71

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 71

cctttgcggc aaggcccttc gcacttctta cacttgcgga cgccgtcttc ctccatctca 60

tagctgtcgg ccccacaggc tcggacgcac gagccgtgat ctgtcaccac ataattacct 120

<210> 72

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 72

ttaatattcg tagcatttat ggagagtgag tctttaaatt caccaatacc tattccgtta 60

cacactgcag agataagaga gacgtgtatt ccgttcacat ttgttggatt aagaatagct 120

<210> 73

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 73

cagttaaaaa ggcagatttt gtttttcttt atacaagcaa ctgaacctgt gactcacccc 60

ctaaatgcca ccggcaggat gtggagatcg ccactgatgg aggtgcagtt tttgaagtgt 120

<210> 74

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 74

catgtgataa ttcagctcaa acctgtgatt tcctttacgg ttttcagaat atccagttcc 60

tgtggatcca gaggaggagt atgtgtgaag gagtcactga aacaaacaac agggtgatta 120

<210> 75

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 75

gaaaaatatc aaaacatcag ccattaacgg taaaatttca gaagaaatgt ttttattcca 60

agggaacagg aaatatgtcg aaaagttctc tctctaaaac actgatttcc catttatatt 120

<210> 76

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 76

ctgtggtcaa cttactgttg cttggtcctg ccgcgtatga tttctaggtt ctcaaaggca 60

tggaggtccg tcctgttttc aggccaagcc tgaatcagca aaaaccctgt aaggcagatg 120

<210> 77

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 77

gtttcctgaa attatcacat ctccatcact tatctccttg agggagcgta atcccaagga 60

tgttatgttc aggctgacga ctgcaagaga aaactgacca ctacattgga gagaagaaaa 120

<210> 78

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 78

aacagaaagc ggtgacttac tgcagctgtt ttcacctctg ttgcttataa ttttggtttt 60

ctgaccggag gtcccaaaca gttttttcca gtttattgta tttgcatagc acaaattttt 120

<210> 79

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 79

ccctgggctc cgggccccag cagccctcgg gggagcacaa ggcatggcag acctggcctg 60

tggcctctgc ggagacagag tgggtcacag cagtcagtga cagagcaggg acccgagggg 120

<210> 80

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 80

cctggagcct tatttttgat caacgcaagg ggattaaaga aataacctcc tacccctcca 60

gaaggttgca cttgtccacg cattccctgc ctcggctgac attccggcaa gagacgcagt 120

<210> 81

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 81

gcaaggggct cttacccgtc ctgtgcaggt gatgttcatg gcctgaggca ggcactctgg 60

gtggcactgt atgcactcag agttctccac aaactccctt ggctcactga gaggaggaag 120

<210> 82

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 82

ccatgactcc tgccgggcag gtcttgacgc agtgggggcc gtcaatgtag tgggcacact 60

ggatacagtt gtctggtccc tgcaccaagg tggagaaaaa tgttcatgca tgtctctttc 120

<210> 83

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 83

caagagagga aatgttctgt tctccttcac tttccactca cccgtaggtg cagtttggat 60

ggcacaggtg gcacacatgg ccggcgtctg cgtacttcca gaccagggtg ttgttttctc 120

<210> 84

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 84

atgtcaaatg aatagtgtca ggactttatt tgaagcaaag ggagaaattg aaattgtaga 60

aaggaaatgc agttttagtg ggagactaaa gtcagacagt gaagatttat taggacctgg 120

<210> 85

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 85

aaaggcagaa taacatggct ttgagactct catttcctgg cctggagaaa gatggacaag 60

tacatttcaa tatgtgagag gcctggggag ccctggtgta acactataat ctgatcctaa 120

<210> 86

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 86

gctgaaactc tgacggcaga agagaaggga tccatcctaa gcatgactcc tgagcagctg 60

ggctcccgct ctggtgggac acgctgccat cattactttg attacaggat gatagtttaa 120

<210> 87

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 87

agaaaggaaa agcgggggga acagacacgt gaaggcatga ggctcagtgg caggaggagg 60

ccgtgatgat cacagatgca gaaggccagg catggaggtg gctgcatcca ccccaaccca 120

<210> 88

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 88

gtcagtcctg ggggtgatgg ttagctacta gttggggtga gaggcactgg caaagggagt 60

ggaaggaagg aaatgggtag gatactcttg ggaacctggc ggcccacgtg caggggtggc 120

<210> 89

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 89

aaggctagaa gcatggactg gggccatgct aattacggag aaggctggca gcatggagtc 60

cctgcaggtt tctgacaaat tctgtgcttt gaacaatcat ctggcagcga ggaggaagag 120

<210> 90

<211> 119

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 90

gttctagcat tgaacaaagc agccagtttg ttattgagat gttttcacat ctggcttttg 60

acacagataa ttgtcccaca gctggctcta gaatttcaaa cagaggggca gaaggaacc 119

<210> 91

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 91

gcatatattt tggagatttt taattaaatc ataaagcaat atatgctaaa ctactcttaa 60

ataattaaaa tcactatgtg tctggatgtt ggattggtgg ttaattattt attatgcatt 120

<210> 92

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 92

tcttgacgag gtccatgtga cacagagctg tgaacactta cccattcgtt ggacagcctt 60

caagacctgg cccagtgcat ctgtaggaag tgaaagagaa atatacattt ttctcattct 120

<210> 93

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 93

aggaagggtg aggttgctga cagattccat ggccttggca ctcaacacgc ccctgcatgt 60

ttcagtgcac tgacatcttg tagaatgatc ttggaaagtg agacactatg tagcttctaa 120

<210> 94

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 94

tccccagggc caccaccagc agcaagagga gggcccccac catcccagtg gcgatggacg 60

ggatcttagg cctggaatga ggtggaggaa caaggaaggg tgaggttgct gacagattcc 120

<210> 95

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 95

tcctgtcggg gtgcgagggc tcctgagccc acccaggact ggcactcacc tccctctcct 60

gcagcagcct ccgcagcgtg cgcttccgaa cgatgtggcg ccttcgcatg aagaggccga 120

<210> 96

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 96

ttcaagatcc tcaagagagc ttggttggga gcttctccac tgggtgtaag aggctccaca 60

agctgggggg gacaagaaca cagagacaag ggtcacctca gccctcacca tgctggaaag 120

<210> 97

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 97

cccaccagac catgagaggc cctgcggccc agcccagagg cctgtgccag ggaccttacc 60

ttatacaccg tgccgaacgc accggagccc agcactttga tctttttgaa ttcagtttcc 120

<210> 98

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 98

cagaaactca catcgaggat ttccttgttg gctttcggag atgttgcttc tcttaattcc 60

ttgatagcga cgggaatttt aactttctca ccttctggga tccagagtcc ctatgacaga 120

<210> 99

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 99

gagctgcgtg atgagctgca cggtggaggt gaggcagatg cccagcaggc ggcacacgtg 60

ggggttgtcc acgctggcca tcacgtaggc ttcctggagg gagggagagg cacgtcagtg 120

<210> 100

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 100

ctccccgtat ctcccttccc tgattacctt tgcgatctgc acacaccagt tgagcaggta 60

ctgggagcca atattgtctt tgtgttcccg gacatagtcc aggaggcagc cgaagggcat 120

<210> 101

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 101

ctgcggtgtt ttcaccagta cgttcctggc tgccaggtcg cggtgcacca agcgacggtc 60

ctccaagtag ttcatgccct gaaacagaga agaccctgct gtgagggaca gatcatcatg 120

<210> 102

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 102

ggtgtcagga aaatgctggc tgacctaaag ccacctcctt actttgcctc cttctgcatg 60

gtattctttc tcttccgcac ccagcagttt ggccagccca aaatctgtga tcttgacatg 120

<210> 103

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 103

tagcatcagg attatgactc accgtagctc cagacatcac tctggtgggt atagattctg 60

tgtaaaattg attccaatgc catccacttg ataggcacct tgggatggtg agagatgagg 120

<210> 104

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 104

ggaggagatc tcgctggcag ggattccgtc atatggcttg gatccaaagg tcatcaactc 60

ccaaacagtc acccctgaga ggatgaagca agaaaagaag gcagtgggat catgaatgaa 120

<210> 105

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 105

caccaaggct cagctaggca gtgtggacag acccaccagt cactcacact tgaccatgat 60

catgtagaca tcgatggtac atatgggtgg ctgagggagg cgttctcctt tctccaggat 120

<210> 106

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 106

gcaatttgta cctgaatgac aaggtagcgc tgggggtctc gggccatttt ggagaattcg 60

atgatcaact cacggaactt tgggcgacta tctgcgtcta tcatccagcc tggggaagga 120

<210> 107

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 107

cacgtcgtcc atgtcttctt catccatcag ggcacggtag aagttggagt ctgtaggact 60

tggcaaatgc attctttcat ccccctggtg cagagatttt gaggctatta gaatggcccg 120

<210> 108

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 108

cagcttgaga gagagagaga gacagagatt tcataccaga gagctcagga ggggagtccg 60

tgacgtggag gggctgctga agaagccctg ctgtgggatg aggtactcgt cggcatccac 120

<210> 109

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 109

gctgtaaatt ctggcttata aggtgttcat acatacccca tttctatcaa tgcaagccac 60

ggtggaattg ttgctggttg cactctgaaa aagaaacaga gaagttgccc atggaatgct 120

<210> 110

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 110

ctcaccaggc actgggagga aggtgtcgtc tatgctgtcc tcagtcaagg cgcctgtggg 60

gtctgagctg tatcgctgca agaagctgtc ttccttgatg ggacagcttt gcagctgcag 120

<210> 111

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 111

tgatagacag gattctgcac agagccagcg ggccttttgg gaacggactg gtttatgtat 60

tctgaaagtg gaaagaaatc agaggtgaag taatcagcaa agcaccatcc catgcaggat 120

<210> 112

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 112

aatgtgctgt tgacacaggt gggctggaca gtgttgagat actcggggtt gcccactgca 60

gtgctgtggg ggtcctggta gtgtgggtct ctgctgggcg cggggttcag aggctgattg 120

<210> 113

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 113

gtggagccct taaagatgcc atttggcttg gcttccttgg gaaagaagtc ctgctggtag 60

tcagggttgt ccaggctaat ttggtggctg cctttctggg cccagtgggc agggctgtcg 120

<210> 114

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 114

ggcttggtcc tgggtatcga aagagtctgg atttttaggg ctcatactat cctccgtggt 60

catgctccaa taaattcact gctttgtggc gcgaccctta ggtattctgc attttcagct 120

<210> 115

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 115

tcttcgggga gcagcgatgc gaccctccgg gacggccggg gcagcgctcc tggcgctgct 60

ggctgcgctc tgcccggcga gtcgggctct ggaggaaaag aaaggtaagg gcgtgtctcg 120

<210> 116

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 116

ctatcattcc tttgggccta ggattgcatt tatttccatg acaaaagggc ctgtctggtg 60

tttcagcaaa tgaaaacaaa aatataaagc ccatctcctt ttgaatgagc tctaaaacag 120

<210> 117

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 117

ttctccactg gacttcagaa caagagggag ctctgggctg ctggctggtt gtgcatttgc 60

tgtgggttcc ctccggcagg cgacctctcc gcgctgagaa ggttatccgg ataaccaagt 120

<210> 118

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 118

tgcatttctc agtatttcat gtgatatctg tctttttctt ccagtttgcc aaggcacgag 60

taacaagctc acgcagttgg gcacttttga agatcatttt ctcagcctcc agaggatgtt 120

<210> 119

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 119

caataactgt gaggtggtcc ttgggaattt ggaaattacc tatgtgcaga ggaattatga 60

tctttccttc ttaaaggttg gtgactttga ttttcctaca caaataaaat tggagaaaat 120

<210> 120

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 120

atcacgcatt tatgttttct cttcttagac catccaggag gtggctggtt atgtcctcat 60

tgccctcaac acagtggagc gaattccttt ggaaaacctg cagatcatca gaggaaatat 120

<210> 121

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 121

gtactacgaa aattcctatg ccttagcagt cttatctaac tatgatgcaa ataaaaccgg 60

actgaaggag ctgcccatga gaaatttaca gggtgagagg ctgggatgcc aaggctgggg 120

<210> 122

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 122

ggaatttaaa ggagctggaa agagtgctca ccgcagttcc attctcccgc agaaatcctg 60

catggcgccg tgcggttcag caacaaccct gccctgtgca acgtggagag catccagtgg 120

<210> 123

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 123

cgggacatag tcagcagtga ctttctcagc aacatgtcga tggacttcca gaaccacctg 60

ggcagctgta agtgtcgcat acacactatc tctgcctcca gctcctatgg gggacagctc 120

<210> 124

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 124

atgcatctat tacttttaca tttcaggcca aaagtgtgat ccaagctgtc ccaatgggag 60

ctgctggggt gcaggagagg agaactgcca gaaacgtaag tcagtgaaca gcctcagacc 120

<210> 125

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 125

tgaccaaaat catctgtgcc cagcagtgct ccgggcgctg ccgtggcaag tcccccagtg 60

actgctgcca caaccagtgt gctgcaggct gcacaggccc ccgggagagc gactgcctgg 120

<210> 126

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 126

gctgagtgta cttacctcac ttgcccagcg tgtcctctct cctccatagg tctgccgcaa 60

attccgagac gaagccacgt gcaaggacac ctgcccccca ctcatgctct acaaccccac 120

<210> 127

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 127

cacgtaccag atggatgtga accccgaggg caaatacagc tttggtgcca cctgcgtgaa 60

gaagtgtccc cgtgagtcct cctctgtggg ccctctaact ggtcaggcat ccttgtcccg 120

<210> 128

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 128

aggtaattat gtggtgacag atcacggctc gtgcgtccga gcctgtgggg ccgacagcta 60

tgagatggag gaagacggcg tccgcaagtg taagaagtgc gaagggcctt gccgcaaagg 120

<210> 129

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 129

agctattctt aatccaacaa atgtgaacgg aatacacgtc tctcttatct ctgcagtgtg 60

taacggaata ggtattggtg aatttaaaga ctcactctcc ataaatgcta cgaatattaa 120

<210> 130

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 130

acacttcaaa aactgcacct ccatcagtgg cgatctccac atcctgccgg tggcatttag 60

ggggtgagtc acaggttcag ttgcttgtat aaagaaaaac aaaatctgcc tttttaactg 120

<210> 131

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 131

taatcaccct gttgtttgtt tcagtgactc cttcacacat actcctcctc tggatccaca 60

ggaactggat attctgaaaa ccgtaaagga aatcacaggt ttgagctgaa ttatcacatg 120

<210> 132

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 132

aatataaatg ggaaatcagt gttttagaga gagaactttt cgacatattt cctgttccct 60

tggaataaaa acatttcttc tgaaatttta ccgttaatgg ctgatgtttt gatatttttc 120

<210> 133

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 133

catctgcctt acagggtttt tgctgattca ggcttggcct gaaaacagga cggacctcca 60

tgcctttgag aacctagaaa tcatacgcgg caggaccaag caacagtaag ttgaccacag 120

<210> 134

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 134

ttttcttctc tccaatgtag tggtcagttt tctcttgcag tcgtcagcct gaacataaca 60

tccttgggat tacgctccct caaggagata agtgatggag atgtgataat ttcaggaaac 120

<210> 135

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 135

aaaaatttgt gctatgcaaa tacaataaac tggaaaaaac tgtttgggac ctccggtcag 60

aaaaccaaaa ttataagcaa cagaggtgaa aacagctgca gtaagtcacc gctttctgtt 120

<210> 136

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 136

cccctcgggt ccctgctctg tcactgactg ctgtgaccca ctctgtctcc gcagaggcca 60

caggccaggt ctgccatgcc ttgtgctccc ccgagggctg ctggggcccg gagcccaggg 120

<210> 137

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 137

actgcgtctc ttgccggaat gtcagccgag gcagggaatg cgtggacaag tgcaaccttc 60

tggaggggta ggaggttatt tctttaatcc ccttgcgttg atcaaaaata aggctccagg 120

<210> 138

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 138

cttcctcctc tcagtgagcc aagggagttt gtggagaact ctgagtgcat acagtgccac 60

ccagagtgcc tgcctcaggc catgaacatc acctgcacag gacgggtaag agccccttgc 120

<210> 139

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 139

gaaagagaca tgcatgaaca tttttctcca ccttggtgca gggaccagac aactgtatcc 60

agtgtgccca ctacattgac ggcccccact gcgtcaagac ctgcccggca ggagtcatgg 120

<210> 140

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 140

gagaaaacaa caccctggtc tggaagtacg cagacgccgg ccatgtgtgc cacctgtgcc 60

atccaaactg cacctacggg tgagtggaaa gtgaaggaga acagaacatt tcctctcttg 120

<210> 141

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 141

ccaggtccta ataaatcttc actgtctgac tttagtctcc cactaaaact gcatttcctt 60

tctacaattt caatttctcc ctttgcttca aataaagtcc tgacactatt catttgacat 120

<210> 142

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 142

ttaggatcag attatagtgt tacaccaggg ctccccaggc ctctcacata ttgaaatgta 60

cttgtccatc tttctccagg ccaggaaatg agagtctcaa agccatgtta ttctgccttt 120

<210> 143

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 143

ttaaactatc atcctgtaat caaagtaatg atggcagcgt gtcccaccag agcgggagcc 60

cagctgctca ggagtcatgc ttaggatgga tcccttctct tctgccgtca gagtttcagc 120

<210> 144

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 144

tgggttgggg tggatgcagc cacctccatg cctggccttc tgcatctgtg atcatcacgg 60

cctcctcctg ccactgagcc tcatgccttc acgtgtctgt tccccccgct tttcctttct 120

<210> 145

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 145

gccacccctg cacgtgggcc gccaggttcc caagagtatc ctacccattt ccttccttcc 60

actccctttg ccagtgcctc tcaccccaac tagtagctaa ccatcacccc caggactgac 120

<210> 146

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 146

ctcttcctcc tcgctgccag atgattgttc aaagcacaga atttgtcaga aacctgcagg 60

gactccatgc tgccagcctt ctccgtaatt agcatggccc cagtccatgc ttctagcctt 120

<210> 147

<211> 119

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 147

ggttccttct gcccctctgt ttgaaattct agagccagct gtgggacaat tatctgtgtc 60

aaaagccaga tgtgaaaaca tctcaataac aaactggctg ctttgttcaa tgctagaac 119

<210> 148

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 148

aatgcataat aaataattaa ccaccaatcc aacatccaga cacatagtga ttttaattat 60

ttaagagtag tttagcatat attgctttat gatttaatta aaaatctcca aaatatatgc 120

<210> 149

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 149

agaatgagaa aaatgtatat ttctctttca cttcctacag atgcactggg ccaggtcttg 60

aaggctgtcc aacgaatggg taagtgttca cagctctgtg tcacatggac ctcgtcaaga 120

<210> 150

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 150

ttagaagcta catagtgtct cactttccaa gatcattcta caagatgtca gtgcactgaa 60

acatgcaggg gcgtgttgag tgccaaggcc atggaatctg tcagcaacct cacccttcct 120

<210> 151

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 151

ggaatctgtc agcaacctca cccttccttg ttcctccacc tcattccagg cctaagatcc 60

cgtccatcgc cactgggatg gtgggggccc tcctcttgct gctggtggtg gccctgggga 120

<210> 152

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 152

tcggcctctt catgcgaagg cgccacatcg ttcggaagcg cacgctgcgg aggctgctgc 60

aggagaggga ggtgagtgcc agtcctgggt gggctcagga gccctcgcac cccgacagga 120

<210> 153

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 153

ctttccagca tggtgagggc tgaggtgacc cttgtctctg tgttcttgtc ccccccagct 60

tgtggagcct cttacaccca gtggagaagc tcccaaccaa gctctcttga ggatcttgaa 120

<210> 154

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 154

ggaaactgaa ttcaaaaaga tcaaagtgct gggctccggt gcgttcggca cggtgtataa 60

ggtaaggtcc ctggcacagg cctctgggct gggccgcagg gcctctcatg gtctggtggg 120

<210> 155

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 155

tctgtcatag ggactctgga tcccagaagg tgagaaagtt aaaattcccg tcgctatcaa 60

ggaattaaga gaagcaacat ctccgaaagc caacaaggaa atcctcgatg tgagtttctg 120

<210> 156

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 156

cactgacgtg cctctccctc cctccaggaa gcctacgtga tggccagcgt ggacaacccc 60

cacgtgtgcc gcctgctggg catctgcctc acctccaccg tgcagctcat cacgcagctc 120

<210> 157

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 157

atgcccttcg gctgcctcct ggactatgtc cgggaacaca aagacaatat tggctcccag 60

tacctgctca actggtgtgt gcagatcgca aaggtaatca gggaagggag atacggggag 120

<210> 158

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 158

catgatgatc tgtccctcac agcagggtct tctctgtttc agggcatgaa ctacttggag 60

gaccgtcgct tggtgcaccg cgacctggca gccaggaacg tactggtgaa aacaccgcag 120

<210> 159

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 159

catgtcaaga tcacagattt tgggctggcc aaactgctgg gtgcggaaga gaaagaatac 60

catgcagaag gaggcaaagt aaggaggtgg ctttaggtca gccagcattt tcctgacacc 120

<210> 160

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 160

cctcatctct caccatccca aggtgcctat caagtggatg gcattggaat caattttaca 60

cagaatctat acccaccaga gtgatgtctg gagctacggt gagtcataat cctgatgcta 120

<210> 161

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 161

ttcattcatg atcccactgc cttcttttct tgcttcatcc tctcaggggt gactgtttgg 60

gagttgatga cctttggatc caagccatat gacggaatcc ctgccagcga gatctcctcc 120

<210> 162

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 162

atcctggaga aaggagaacg cctccctcag ccacccatat gtaccatcga tgtctacatg 60

atcatggtca agtgtgagtg actggtgggt ctgtccacac tgcctagctg agccttggtg 120

<210> 163

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 163

tccttcccca ggctggatga tagacgcaga tagtcgccca aagttccgtg agttgatcat 60

cgaattctcc aaaatggccc gagaccccca gcgctacctt gtcattcagg tacaaattgc 120

<210> 164

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 164

cgggccattc taatagcctc aaaatctctg caccaggggg atgaaagaat gcatttgcca 60

agtcctacag actccaactt ctaccgtgcc ctgatggatg aagaagacat ggacgacgtg 120

<210> 165

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 165

gtggatgccg acgagtacct catcccacag cagggcttct tcagcagccc ctccacgtca 60

cggactcccc tcctgagctc tctggtatga aatctctgtc tctctctctc tctcaagctg 120

<210> 166

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 166

agcattccat gggcaacttc tctgtttctt tttcagagtg caaccagcaa caattccacc 60

gtggcttgca ttgatagaaa tggggtatgt atgaacacct tataagccag aatttacagc 120

<210> 167

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 167

ctgcagctgc aaagctgtcc catcaaggaa gacagcttct tgcagcgata cagctcagac 60

cccacaggcg ccttgactga ggacagcata gacgacacct tcctcccagt gcctggtgag 120

<210> 168

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 168

atcctgcatg ggatggtgct ttgctgatta cttcacctct gatttctttc cactttcaga 60

atacataaac cagtccgttc ccaaaaggcc cgctggctct gtgcagaatc ctgtctatca 120

<210> 169

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 169

caatcagcct ctgaaccccg cgcccagcag agacccacac taccaggacc cccacagcac 60

tgcagtgggc aaccccgagt atctcaacac tgtccagccc acctgtgtca acagcacatt 120

<210> 179

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 179

cgacagccct gcccactggg cccagaaagg cagccaccaa attagcctgg acaaccctga 60

ctaccagcag gacttctttc ccaaggaagc caagccaaat ggcatcttta agggctccac 120

<210> 171

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 171

agctgaaaat gcagaatacc taagggtcgc gccacaaagc agtgaattta ttggagcatg 60

accacggagg atagtatgag ccctaaaaat ccagactctt tcgataccca ggaccaagcc 120

<210> 172

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 172

tcttcgggga gcagcgatgc gaccctccgg gacggccggg gcagcgctcc tggcgctgct 60

ggctgcgctc tgcccggcga gtcgggctct ggaggaaaag aaaggtaagg gcgtgtctcg 120

<210> 173

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 173

ctatcattcc tttgggccta ggattgcatt tatttccatg acaaaagggc ctgtctggtg 60

tttcagcaaa tgaaaacaaa aatataaagc ccatctcctt ttgaatgagc tctaaaacag 120

<210> 174

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 174

acttggttat ccggataacc ttctcagcgc ggagaggtcg cctgccggag ggaacccaca 60

gcaaatgcac aaccagccag cagcccagag ctccctcttg ttctgaagtc cagtggagaa 120

<210> 175

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 175

tgcatttctc agtatttcat gtgatatctg tctttttctt ccagtttgcc aaggcacgag 60

taacaagctc acgcagttgg gcacttttga agatcatttt ctcagcctcc agaggatgtt 120

<210> 176

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 176

attttctcca attttatttg tgtaggaaaa tcaaagtcac caacctttaa gaaggaaaga 60

tcataattcc tctgcacata ggtaatttcc aaattcccaa ggaccacctc acagttattg 120

<210> 177

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 177

atcacgcatt tatgttttct cttcttagac catccaggag gtggctggtt atgtcctcat 60

tgccctcaac acagtggagc gaattccttt ggaaaacctg cagatcatca gaggaaatat 120

<210> 178

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 178

ccccagcctt ggcatcccag cctctcaccc tgtaaatttc tcatgggcag ctccttcagt 60

ccggttttat ttgcatcata gttagataag actgctaagg cataggaatt ttcgtagtac 120

<210> 179

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 179

ggaatttaaa ggagctggaa agagtgctca ccgcagttcc attctcccgc agaaatcctg 60

catggcgccg tgcggttcag caacaaccct gccctgtgca acgtggagag catccagtgg 120

<210> 180

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 180

gagctgtccc ccataggagc tggaggcaga gatagtgtgt atgcgacact tacagctgcc 60

caggtggttc tggaagtcca tcgacatgtt gctgagaaag tcactgctga ctatgtcccg 120

<210> 181

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 181

atgcatctat tacttttaca tttcaggcca aaagtgtgat ccaagctgtc ccaatgggag 60

ctgctggggt gcaggagagg agaactgcca gaaacgtaag tcagtgaaca gcctcagacc 120

<210> 182

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 182

tgaccaaaat catctgtgcc cagcagtgct ccgggcgctg ccgtggcaag tcccccagtg 60

actgctgcca caaccagtgt gctgcaggct gcacaggccc ccgggagagc gactgcctgg 120

<210> 183

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 183

gctgagtgta cttacctcac ttgcccagcg tgtcctctct cctccatagg tctgccgcaa 60

attccgagac gaagccacgt gcaaggacac ctgcccccca ctcatgctct acaaccccac 120

<210> 184

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 184

cgggacaagg atgcctgacc agttagaggg cccacagagg aggactcacg gggacacttc 60

ttcacgcagg tggcaccaaa gctgtatttg ccctcggggt tcacatccat ctggtacgtg 120

<210> 185

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 185

aggtaattat gtggtgacag atcacggctc gtgcgtccga gcctgtgggg ccgacagcta 60

tgagatggag gaagacggcg tccgcaagtg taagaagtgc gaagggcctt gccgcaaagg 120

<210> 186

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 186

agctattctt aatccaacaa atgtgaacgg aatacacgtc tctcttatct ctgcagtgtg 60

taacggaata ggtattggtg aatttaaaga ctcactctcc ataaatgcta cgaatattaa 120

<210> 187

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 187

cagttaaaaa ggcagatttt gtttttcttt atacaagcaa ctgaacctgt gactcacccc 60

ctaaatgcca ccggcaggat gtggagatcg ccactgatgg aggtgcagtt tttgaagtgt 120

<210> 188

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 188

taatcaccct gttgtttgtt tcagtgactc cttcacacat actcctcctc tggatccaca 60

ggaactggat attctgaaaa ccgtaaagga aatcacaggt ttgagctgaa ttatcacatg 120

<210> 189

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 189

gaaaaatatc aaaacatcag ccattaacgg taaaatttca gaagaaatgt ttttattcca 60

agggaacagg aaatatgtcg aaaagttctc tctctaaaac actgatttcc catttatatt 120

<210> 190

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 190

catctgcctt acagggtttt tgctgattca ggcttggcct gaaaacagga cggacctcca 60

tgcctttgag aacctagaaa tcatacgcgg caggaccaag caacagtaag ttgaccacag 120

<210> 191

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 191

ttttcttctc tccaatgtag tggtcagttt tctcttgcag tcgtcagcct gaacataaca 60

tccttgggat tacgctccct caaggagata agtgatggag atgtgataat ttcaggaaac 120

<210> 192

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 192

aacagaaagc ggtgacttac tgcagctgtt ttcacctctg ttgcttataa ttttggtttt 60

ctgaccggag gtcccaaaca gttttttcca gtttattgta tttgcatagc acaaattttt 120

<210> 193

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 193

cccctcgggt ccctgctctg tcactgactg ctgtgaccca ctctgtctcc gcagaggcca 60

caggccaggt ctgccatgcc ttgtgctccc ccgagggctg ctggggcccg gagcccaggg 120

<210> 194

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 194

cctggagcct tatttttgat caacgcaagg ggattaaaga aataacctcc tacccctcca 60

gaaggttgca cttgtccacg cattccctgc ctcggctgac attccggcaa gagacgcagt 120

<210> 195

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 195

cttcctcctc tcagtgagcc aagggagttt gtggagaact ctgagtgcat acagtgccac 60

ccagagtgcc tgcctcaggc catgaacatc acctgcacag gacgggtaag agccccttgc 120

<210> 196

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 196

gaaagagaca tgcatgaaca tttttctcca ccttggtgca gggaccagac aactgtatcc 60

agtgtgccca ctacattgac ggcccccact gcgtcaagac ctgcccggca ggagtcatgg 120

<210> 197

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 197

caagagagga aatgttctgt tctccttcac tttccactca cccgtaggtg cagtttggat 60

ggcacaggtg gcacacatgg ccggcgtctg cgtacttcca gaccagggtg ttgttttctc 120

<210> 198

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 198

ccaggtccta ataaatcttc actgtctgac tttagtctcc cactaaaact gcatttcctt 60

tctacaattt caatttctcc ctttgcttca aataaagtcc tgacactatt catttgacat 120

<210> 199

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 199

ttaggatcag attatagtgt tacaccaggg ctccccaggc ctctcacata ttgaaatgta 60

cttgtccatc tttctccagg ccaggaaatg agagtctcaa agccatgtta ttctgccttt 120

<210> 200

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 200

gctgaaactc tgacggcaga agagaaggga tccatcctaa gcatgactcc tgagcagctg 60

ggctcccgct ctggtgggac acgctgccat cattactttg attacaggat gatagtttaa 120

<210> 201

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 201

tgggttgggg tggatgcagc cacctccatg cctggccttc tgcatctgtg atcatcacgg 60

cctcctcctg ccactgagcc tcatgccttc acgtgtctgt tccccccgct tttcctttct 120

<210> 202

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 202

gtcagtcctg ggggtgatgg ttagctacta gttggggtga gaggcactgg caaagggagt 60

ggaaggaagg aaatgggtag gatactcttg ggaacctggc ggcccacgtg caggggtggc 120

<210> 203

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 203

ctcttcctcc tcgctgccag atgattgttc aaagcacaga atttgtcaga aacctgcagg 60

gactccatgc tgccagcctt ctccgtaatt agcatggccc cagtccatgc ttctagcctt 120

<210> 204

<211> 119

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 204

gttctagcat tgaacaaagc agccagtttg ttattgagat gttttcacat ctggcttttg 60

acacagataa ttgtcccaca gctggctcta gaatttcaaa cagaggggca gaaggaacc 119

<210> 205

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 205

aatgcataat aaataattaa ccaccaatcc aacatccaga cacatagtga ttttaattat 60

ttaagagtag tttagcatat attgctttat gatttaatta aaaatctcca aaatatatgc 120

<210> 206

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 206

agaatgagaa aaatgtatat ttctctttca cttcctacag atgcactggg ccaggtcttg 60

aaggctgtcc aacgaatggg taagtgttca cagctctgtg tcacatggac ctcgtcaaga 120

<210> 207

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 207

ttagaagcta catagtgtct cactttccaa gatcattcta caagatgtca gtgcactgaa 60

acatgcaggg gcgtgttgag tgccaaggcc atggaatctg tcagcaacct cacccttcct 120

<210> 208

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 208

ggaatctgtc agcaacctca cccttccttg ttcctccacc tcattccagg cctaagatcc 60

cgtccatcgc cactgggatg gtgggggccc tcctcttgct gctggtggtg gccctgggga 120

<210> 209

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 209

tcctgtcggg gtgcgagggc tcctgagccc acccaggact ggcactcacc tccctctcct 60

gcagcagcct ccgcagcgtg cgcttccgaa cgatgtggcg ccttcgcatg aagaggccga 120

<210> 210

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 210

ctttccagca tggtgagggc tgaggtgacc cttgtctctg tgttcttgtc ccccccagct 60

tgtggagcct cttacaccca gtggagaagc tcccaaccaa gctctcttga ggatcttgaa 120

<210> 211

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 211

cccaccagac catgagaggc cctgcggccc agcccagagg cctgtgccag ggaccttacc 60

ttatacaccg tgccgaacgc accggagccc agcactttga tctttttgaa ttcagtttcc 120

<210> 212

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 212

tctgtcatag ggactctgga tcccagaagg tgagaaagtt aaaattcccg tcgctatcaa 60

ggaattaaga gaagcaacat ctccgaaagc caacaaggaa atcctcgatg tgagtttctg 120

<210> 213

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 213

cactgacgtg cctctccctc cctccaggaa gcctacgtga tggccagcgt ggacaacccc 60

cacgtgtgcc gcctgctggg catctgcctc acctccaccg tgcagctcat cacgcagctc 120

<210> 214

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 214

ctccccgtat ctcccttccc tgattacctt tgcgatctgc acacaccagt tgagcaggta 60

ctgggagcca atattgtctt tgtgttcccg gacatagtcc aggaggcagc cgaagggcat 120

<210> 215

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 215

catgatgatc tgtccctcac agcagggtct tctctgtttc agggcatgaa ctacttggag 60

gaccgtcgct tggtgcaccg cgacctggca gccaggaacg tactggtgaa aacaccgcag 120

<210> 216

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 216

ggtgtcagga aaatgctggc tgacctaaag ccacctcctt actttgcctc cttctgcatg 60

gtattctttc tcttccgcac ccagcagttt ggccagccca aaatctgtga tcttgacatg 120

<210> 217

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 217

cctcatctct caccatccca aggtgcctat caagtggatg gcattggaat caattttaca 60

cagaatctat acccaccaga gtgatgtctg gagctacggt gagtcataat cctgatgcta 120

<210> 218

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 218

ttcattcatg atcccactgc cttcttttct tgcttcatcc tctcaggggt gactgtttgg 60

gagttgatga cctttggatc caagccatat gacggaatcc ctgccagcga gatctcctcc 120

<210> 219

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 219

caccaaggct cagctaggca gtgtggacag acccaccagt cactcacact tgaccatgat 60

catgtagaca tcgatggtac atatgggtgg ctgagggagg cgttctcctt tctccaggat 120

<210> 220

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 220

tccttcccca ggctggatga tagacgcaga tagtcgccca aagttccgtg agttgatcat 60

cgaattctcc aaaatggccc gagaccccca gcgctacctt gtcattcagg tacaaattgc 120

<210> 221

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 221

cgggccattc taatagcctc aaaatctctg caccaggggg atgaaagaat gcatttgcca 60

agtcctacag actccaactt ctaccgtgcc ctgatggatg aagaagacat ggacgacgtg 120

<210> 222

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 222

cagcttgaga gagagagaga gacagagatt tcataccaga gagctcagga ggggagtccg 60

tgacgtggag gggctgctga agaagccctg ctgtgggatg aggtactcgt cggcatccac 120

<210> 223

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 223

agcattccat gggcaacttc tctgtttctt tttcagagtg caaccagcaa caattccacc 60

gtggcttgca ttgatagaaa tggggtatgt atgaacacct tataagccag aatttacagc 120

<210> 224

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 224

ctgcagctgc aaagctgtcc catcaaggaa gacagcttct tgcagcgata cagctcagac 60

cccacaggcg ccttgactga ggacagcata gacgacacct tcctcccagt gcctggtgag 120

<210> 225

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 225

atcctgcatg ggatggtgct ttgctgatta cttcacctct gatttctttc cactttcaga 60

atacataaac cagtccgttc ccaaaaggcc cgctggctct gtgcagaatc ctgtctatca 120

<210> 226

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 226

aatgtgctgt tgacacaggt gggctggaca gtgttgagat actcggggtt gcccactgca 60

gtgctgtggg ggtcctggta gtgtgggtct ctgctgggcg cggggttcag aggctgattg 120

<210> 227

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 227

cgacagccct gcccactggg cccagaaagg cagccaccaa attagcctgg acaaccctga 60

ctaccagcag gacttctttc ccaaggaagc caagccaaat ggcatcttta agggctccac 120

<210> 228

<211> 120

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 228

ggcttggtcc tgggtatcga aagagtctgg atttttaggg ctcatactat cctccgtggt 60

catgctccaa taaattcact gctttgtggc gcgaccctta ggtattctgc attttcagct 120

Claims (10)

1. a kind of capture probe for high-flux sequence detection gene mutation, which is characterized in that the capture probe is single-stranded Probe mixture, the single-stranded probe mixture include being directed to the positive-sense strand of DNA to be measured respectively since one end of DNA to be measured With the nucleotide sequence of antisense strand, the probe for positive-sense strand and the probe for antisense strand successively designed；Every two is adjacent Single-stranded probe in, one for for positive-sense strand nucleotide sequence design single-stranded probe, another be for antisense strand The single-stranded probe of nucleotide sequence design；The targeted DNA to be measured of every two adjacent single-stranded probe is not overlapped；It is described single-stranded Probe mixture can cover all DNA to be measured.

2. capture probe according to claim 1, which is characterized in that the length of the single-stranded probe is 90~120nt；Institute Each single-stranded probe in single-stranded probe mixture is stated to mix with identical molar ratio；

Preferably, the length of the single-stranded probe is 110~120nt.

3. capture probe of any of claims 1 or 2 detects gene mutation in gene order-checking library construction or high-flux sequence In application.

4. a kind of construction method of gene mutation sequencing library, which is characterized in that interrupt genomic DNA, using claim 1 Or capture probe described in 2 constructs sequencing library.

5. construction method according to claim 4, which comprises the steps of:

(1) genomic DNA is extracted, is interrupted as small fragment；

(2) library preparation is carried out to the small fragment DNA that step (1) obtains；

(3) the small fragment DNA library that step (2) obtains is hybridized with capture probe of any of claims 1 or 2, is captured Target fragment；

(4) segment of step (3) capture is expanded, machine on amplified production is sequenced.

6. a kind of specific capture probe of EGFR gene, which is characterized in that including such as SEQ ID NO.172~SEQ ID Single-stranded probe shown in NO.228.

7. a kind of kit for detecting EGFR genetic mutation, which is characterized in that it includes capture probe as claimed in claim 6.

8. kit according to claim 7, which is characterized in that the capture probe is by such as SEQ ID NO.172~SEQ The mixing composition of single-stranded probe equimolar ratio shown in ID NO.228.

9. kit according to claim 7 or 8, which is characterized in that the working concentration of the capture probe be 0.1pM~ 0.75pM。

10. capture probe as claimed in claim 6 or the described in any item kits of claim 7~9 are examined in high-flux sequence Survey the application in the sequencing library of EGFR genetic mutation or building detection EGFR genetic mutation.