CN112662766A

CN112662766A - Method for detecting familial thoracic aortic aneurysm and interlayer related mutant gene based on high-throughput sequencing technology

Info

Publication number: CN112662766A
Application number: CN202011297996.9A
Authority: CN
Inventors: 段艳宇; 刘子由
Original assignee: First Affiliated Hospital of Gannan Medical University
Current assignee: First Affiliated Hospital of Gannan Medical University
Priority date: 2020-11-19
Filing date: 2020-11-19
Publication date: 2021-04-16

Abstract

The invention discloses a method for detecting familial thoracic aortic aneurysm and interlayer related mutant gene based on high-throughput sequencing technology, which comprises the steps of (1) collecting samples; (2) designing a panel; (3) constructing a library; (4) sequencing on a computer; (5) and (5) data analysis annotation. The invention is based on the technical process of target area capture high-throughput sequencing, and the method uses the multiple PCR capture process to detect 12 pathogenic genes at one time by selecting 12 pathogenic gene sets related to FTAAD. Compared with the prior art, the method has the advantages of strong pertinence, low cost, fast flow and the like.

Description

Method for detecting familial thoracic aortic aneurysm and interlayer related mutant gene based on high-throughput sequencing technology

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a method for detecting genes related to familial thoracic aortic aneurysm and interlayer disease based on an illumina sequencing platform.

Background

Thoracic aortic aneurysms are a very aggressive cardiovascular crisis severe and often asymptomatic. However, if the aneurysm is not fixed, it becomes larger and larger over time, and may even tear the aorta. Rupture of the aorta can result in blood flow to the wrong part of the body, potentially life threatening. About one fifth of patients with thoracic aortic aneurysms, which are caused by parentally inherited defective genes, are known as familial thoracic aortic aneurysms. If one of the parents carries a defective gene associated with Familial Thoracic Aortic Aneurysm and Dissection (FTAAD), then each of their children has 50% of the opportunity to inherit the gene, and the siblings or parents of the person carrying the FTAAD risk gene also have 50% of the opportunity to inherit the gene. If genetic testing indicates that a person is at risk of developing FTAAD, then they should have a 50% -80% probability of having FTAAD that is 50-fold higher than a person who does not carry the defective gene. These patients should be periodically examined imagewise to monitor their own aorta. Currently, hypotensive drugs are commonly used in FTAAD patients to relieve aortic pressure; but if the aorta becomes too large, surgery is required. Several genes have been found to be associated with FTAAD, and alterations in some genes, including ACTA2, MYLK and MYH11, have been shown to result in FTAAD; other genes such as TGFBR1, TGFBR2, SMAD3, PRKG1, LOX, COL3A1, TGFB2, TGFB3, and FBN-1 are also associated with risk of developing FTAAD, and therefore we can identify people at risk of FTAAD through gene testing.

The Chinese first edition 'Single Gene genetic cardiovascular disease Gene diagnostic guidance' formulated by the cardiovascular disease society of the Chinese medical society, accurate cardiovascular group and the like suggests twelve gene-related results of ACTA2, FBN1, MYH11, MYLK, SMAD3, TGFBR1, TGFBR2, PRKG1, LOX, COL3A1, TGFB2, TGFB3 and the like, wherein the gene mutation of ACTA2 can explain 10-15% of familial TAAD. Patients who carry mutations in the causative gene but who are temporarily asymptomatic may undergo early disease prediction and intervention, such as periodic (1 time per year before aortic dilation and 1 time in the second half of the year) imaging examinations to discover and monitor early disease. Can be used for individual treatment according to genotype and phenotype, such as advanced operation (such as maximum internal diameter of thoracic aorta up to 4.0-5.0cm) to prevent dissection and rupture; the maximum inner diameter of thoracic aorta of TGFBR1 and TGFBR2 pathogenic gene mutation carriers is 4.2cm, and operation can be considered; for patients with ACTA2 gene mutation, the maximum internal diameter of the thoracic aorta is 4.5cm, which is considered for surgery. The risk of coronary artery disease in the early stroke of ACTA2 gene mutation patients is high, and attention should be paid. If the first-class relatives of 1 or more of the first-class relatives have the phenotype of thoracic aortic dilatation, tumor or dissection, the second-class relatives can carry out imaging examination and gene detection.

Currently, the genetic detection technologies commonly used in clinic include sequencing technologies such as Sanger sequencing and high-throughput sequencing, probe technologies such as fluorescence PCR, gene chips, suspension chips, mass spectrometry and ARMs, and mass spectrometry such as DHPLC, wherein the probe and mass spectrometry can also detect mutation, but can only detect known variation of single nucleotide such as SNP. The sequencing technology is a gold standard for gene mutation detection, is the most advanced level which can be achieved by the prior art and is closest to objective facts, or reflects the objective facts; as in pathological diagnosis. In sequencing, Sanger sequencing is widely applied technology and has the advantages of rapidness, accuracy, simplicity and convenience and the like, but has the problems of low flux, high relative cost, low automation degree and the like, and is difficult to meet the requirement of detecting multiple genes and multiple variations at one time. High throughput sequencing achieves the goal of simultaneously detecting unknown genetic mutations in multiple genes, divided into whole genome sequencing and targeted sequencing, which attempts to obtain different combinations from millions of sequences, and thus cannot leave an enrichment step, wherein the hybrid capture method uses probes complementary to the target of interest, the amplicon-based method relies on multiplex PCR reactions, and selective circularization relies on molecular inversion probes; whole genome sequencing can detect all types of variation on a genome at the same time, but has weak pertinence, large data volume and higher cost of each item. Clinical application usually pursues to be fast, economical and simple, and a group of effective gene sets are selected to perform high-throughput sequencing so as to reduce cost, so that the method becomes an effective method for detecting the FTAAD mutant genes.

Other names for FTAAD are: thoracic Aortic Aneurysm (TAA), Thoracic Aortic Aneurysm and Dissection (TAAD), Familial Aortic Aneurysm (FAA), and aortic annulectasia.

Disclosure of Invention

The invention aims to solve the technical problem that an effective and low-cost gene mutation method for detecting FTAAD is lacked at present, a multiple PCR (polymerase chain reaction) capture technology is adopted for the technical problem, a pathogenic gene set of the FTAAD is captured and enriched by using an amplification principle, then the detection is carried out through a high-throughput sequencing platform, and the gene mutation related to the FTAAD is detected and read by using a biological information analysis process and an ACMG genetic reading guide so as to assist the accurate diagnosis and treatment of the FTAAD in clinic. The method has high flux, can realize single-tube reaction detection of dozens of mutation sites, and has simple and convenient operation and low cost.

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

a method for detecting familial thoracic aortic aneurysm and dissecting associated mutant gene based on high throughput sequencing technology comprises the steps of,

(1) collecting samples:

selecting 12 pathogenic genes ACTA2, FBN1, MYH11, MYLK, SMAD3, TGFBR1, TGFBR2, PRKG1, LOX, COL3A1, TGFB2 and TGFB3 related to the familial thoracic aortic aneurysm and interlayer related genes;

(2) the panel design:

the kit consists of 7 tubes of reagents,

wherein 5 tubes of reagents are stored at-20 ℃ and comprise 1 tube of IGT-I7Index (10uM) reagent, 1 tube of IGT-I5 Index (10uM) reagent, 1 tube of Primer pool reagent, 1 tube of IGT-EM808 polymerase mix reagent and 1 tube of Enhancer buffer NB (1N) reagent;

the other 2 tubes of reagents are stored at 4 ℃ and comprise 1 tube of Enhancer buffer M reagent and 1 tube of YF buffer B reagent;

in the designed kit, the Primer pool reagent contains 367 amplicons, the sequence of each amplicon is 1-367, each amplicon comprises a forward Primer and a reverse Primer, and the sequences of the forward Primer and the reverse Primer are SEQ ID No.1-SEQ ID No. 734;

(3) library construction:

(3.1) 1 st round of multiplex PCR reaction

Preparing reaction liquid in a PCR tube according to the formula shown in the table below, and gently blowing, sucking and mixing the reaction liquid up and down by using a gun;

operating a PCR instrument, putting the PCR tube into the PCR tube, and carrying out reaction according to the following procedures: the cycle was performed after first 95 ℃ for 210 s: circulating for 18 times at 98 deg.C for 10s and 60 deg.C for 5min, and extending for 5min at 72 deg.C;

(3.2) purification of the product by magnetic beads

(3.2.1) adding 27ul of AMPure XP magnetic beads which are balanced at room temperature into 30ul of PCR products processed in the step (3.1), and sucking and uniformly mixing the products for 20 times by using a pipette;

(3.2.2) after incubating for 5min at room temperature, placing the PCR tube on a DynaMag-96Side magnetic frame for 3 min;

(3.2.3) completely removing the supernatant, taking down the PCR tube from the magnetic rack, adding 50ul YF buffer B into the tube, and sucking and mixing the mixture by a pipettor for 20 times;

(3.2.4) after incubating for 5min at room temperature, placing the PCR tube on a DynaMag-96Side magnetic frame for 3 min;

(3.2.5) removing the supernatant, placing the PCR tube on a magnetic frame, adding 180ul of 80% ethanol solution with volume percentage concentration into the PCR tube, and standing for 30 s;

(3.2.6) removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180ul of ethanol solution with the volume percentage concentration of 80%, standing for 30s, and completely removing the supernatant;

(3.2.7) standing at room temperature for 3min to completely volatilize residual ethanol;

(3.2.8) removing the PCR tube from the magnetic frame, adding 24. mu.l of nucleic-free water, pipetting to gently pipette the resuspended beads to avoid air bubbles, and standing at room temperature for 2 min;

(3.2.9) putting the PCR tube on the magnetic frame again, and standing for 3 min;

(3.2.10) pipetting 13.5. mu.l of the supernatant and transferring the supernatant into a new 200. mu.l PCR tube, wherein the supernatant transferred into the tube is a multiplex PCR product;

(3.3) round 2 adaptor sequence PCR reaction

The reaction solution was prepared in a PCR tube according to the following formulation, and gently mixed by up-and-down blowing and sucking with a gun. Wherein the PCR product mix is the purified multiplex PCR product obtained in the step (3.2.10);

operating the PCR instrument, putting the PCR tube into the PCR tube, and carrying out reaction according to the following program: the cycle was performed after first 95 ℃ for 210 s: circulating at 98 deg.C for 20s, 68 deg.C for 1min, and 72 deg.C for 30s for 9 times, and extending at 72 deg.C for 5 min;

(3.4) round 2 magnetic bead purification

(3.4.1) taking 30ul of the PCR reaction system treated in the step (3.3), adding 27ul of AMPure XP magnetic beads balanced at room temperature, and sucking and uniformly mixing the mixture for 20 times by using a pipette;

(3.4.2) after incubating at room temperature for 5min, placing the PCR tube on a DynaMag-96Side magnetic frame for 3 min;

(3.4.3) completely removing the supernatant, taking down the PCR tube from the magnetic rack, adding 50ul YF buffer B into the PCR tube, and sucking and mixing the mixture for 20 times by using a pipettor;

(3.4.4) after incubating for 5min at room temperature, the PCR tube was placed on a DynaMag-96Side magnetic frame for 3 min;

(3.4.5) removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180ul of 80% ethanol solution in volume percentage into the PCR tube, and standing for 30 s;

(3.4.6) removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180ul of 80% ethanol solution in volume percentage into the PCR tube, standing for 30s, and completely removing the supernatant;

(3.4.7) standing at room temperature for 3min to completely volatilize residual ethanol;

(3.4.8) removing the centrifuge tube from the magnetic frame, adding 24 μ l of nucleic-free water or 1 × TE buffer (pH8.0), pipetting and mixing for 20 times, resuspending the magnetic beads to avoid generating bubbles, and standing at room temperature for 2 min;

(3.4.9) putting the PCR tube on the magnetic frame again, and standing for 3 min;

(3.4.10) pipetting 20. mu.l of the supernatant and transferring the supernatant into a new 200. mu.l PCR tube, wherein the supernatant transferred into the tube is the prepared multiplex PCR library;

(3.5) library quantification

The multiplex PCR library obtained in 2ul step (3.4.10) was taken and used

3.0fluorometer (qubit dsDNA HS Assay kit) to perform library concentration determination and record library concentration; the concentration range of a normal library constructed by human genome DNA is 5-40 ng/ul;

(4) sequencing on machine

Sequencing the qualified multiplex PCR library obtained in the step (3.6) by using Hiseq Xten PE150 to obtain a gene sequence table, wherein the computer-operated data volume is 300 Mb;

(5) data analysis annotation: after the sequencing data is downloaded, performing bioinformatics analysis on the downloaded data, and detecting variation on genes;

(5.1) the quality control of the off-line data Fastq format: for each pair of original Fastq sequence raw reads generated by each sample library and lane, removing a sequencing joint and sequence reads containing a large number of 'N' bases by using software cutAdpt to obtain sequence Clean reads meeting quality control, and counting basic information of the sequence Clean reads by using software fastqc, wherein the basic information comprises the number of sequences and bases, GC content and distribution, sequencing error rate and distribution and base quality distribution;

(5.2) Alignment: using software bwa, aligning the sequence Clean reads with a human reference genome GRCh38 to obtain an alignment information file BAM of the sequence Clean reads on the reference genome;

(5.3) BAM preprocessing of the comparison information file: combining BAM files of comparison information files generated by different libraries and Lane of the same sample together by using picard and GATK software, sequencing according to genome coordinates, verifying the BAM files of the comparison information files, removing a repetitive sequence generated in the PCR process, correcting a base quality value, and obtaining a Clean BAM of the comparison file after pretreatment; counting and comparing the file Clean BAM by using the picard software to obtain quality control information; the quality control information comprises coverage, depth, capture efficiency and uniformity of a target region capture chip, the number of comparable sequences and bases, and the length distribution of insert fragments;

(5.4) mutation detection: detecting small fragment variation on the gene by using GATK software, wherein the small fragment variation comprises Single Nucleotide Polymorphism (SNP) and insertion and deletion indels; and (3) filtering the detected variation, wherein the SNP filtering conditions are as follows: QD <3.37| | FS >31.397| | SOR >10.419| | MQ <20.0| | MQRankSum < -12.49| | ReadPosRankSum < -3.721, and the Indel filtering conditions are as follows: QD <5.2| | FS >52.254| | SOR >9.044| | ReadPosRankSum < -5.504;

annotating the variations that meet the filtering conditions, and noting the corresponding genes, transcripts, variation types, functions, and frequencies in normal populations;

and (5) counting variation results to obtain variation quantity, length distribution, various type variation quantity and crowd variation frequency information.

For better technical effect, the concentration of gDNA to be detected is diluted to the same concentration before step (3.1), and then transferred to the PCR8 tube.

In order to obtain better technical effect, step (3.6) of library quality detection is carried out after step (3.5),

taking 1ul of the multiple PCR library obtained in the step (3.4), and measuring the length and purity of the library fragment by using a Qsep100 full-automatic nucleic acid protein analysis system, wherein the target fragment distribution interval of the normal library is between 300bp and 420 bp;

performing library detection on the multiplex PCR library obtained in the step (3.4) by using align 2100 and QPCR;

the result of the multiple PCR library Aligent 2100 fragment obtained in the step (3.4) is qualified when the result is between 250bp and 350bp, and the result of QPCR is qualified when the result is more than 10 nM;

and (4) sequencing the qualified multiplex PCR library in the step (4) to obtain a gene sequence table.

In order to obtain better technical effect, the method also comprises the step (6) of mutation interpretation: explaining the relationship between variant SNPs and variant indels and diseases;

variants were classified into 5 classes by using the software REO-HIT according to the ACMG/AMP guidelines: pathogenic, suspected pathogenic, unknown meaning, suspected benign, benign;

in order to obtain better technical effects, the method also comprises the step (7) of giving a report, verifying the sequencing results of the pathogenicity, suspected pathogenicity, unknown significance and suspected virtuosity one by one through the Sanger method sequencing, and giving the report.

The invention is based on a target area capturing high-throughput sequencing technical process, 12 pathogenic gene sets related to FTAAD are selected, a multiple PCR capturing process and the following implementation scheme are used for performing high-throughput capturing sequencing analysis, the gene variation situation related to the FTAAD is decoded by a gene detection technology, and the multi-gene variation analysis result refers to the guidance decoding variation published by American genetic society (ACMG), molecular pathology society (AMP) and China medical society cardiovascular disease society accurate cardiovascular disease group and the like.

Compared with the prior art, the method has the advantages of strong pertinence, low cost, fast flow and the like.

Reagent manufacturers: illumina Corp.

Drawings

FIG. 1 is a flowchart illustrating genetic variation analysis and interpretation according to an embodiment of the present invention;

FIG. 2 shows the results of quality control library of Qsep100 full-automatic nucleic acid protein analysis system according to the present invention;

FIG. 3 is a flowchart of a detection technique according to an embodiment of the present invention.

Detailed Description

The invention is further explained in detail below with reference to the drawings and the detailed description.

A method for detecting familial thoracic aortic aneurysm and dissecting associated gene based on high throughput sequencing technology comprises the steps of,

(1) collecting samples:

based on the above situation, the invention selects genes/variants of FTAAD with sufficient evidence including gene, protein function research, family inheritance research, population variant analysis, etc. by querying a large number of DISEASE databases and FTAAD research records in literature research, including OMIM, ClinVar, CGD, Orphanet, DISEASE, GeneReviews, and 'single-gene hereditary cardiovascular DISEASE gene detection guidelines'. Finally, 12 pathogenic genes of FTAAD are selected, and a gene list is shown in table 1;

TABLE 1

Finally, 12 pathogenic genes ACTA2, FBN1, MYH11, MYLK, SMAD3, TGFBR1, TGFBR2, PRKG1, LOX, COL3A1, TGFB2 and TGFB3 related to familial thoracic aortic aneurysm and interlayer are selected;

(2) the panel design:

the kit consists of 7 tubes of reagents,

designing the region as the exon region of each gene, and allowing the two sides of the exon region to extend 15bp respectively;

in the designed kit, the Primer pool reagent contains 367 amplicons, the sequence of each amplicon is 1-367, each amplicon comprises a forward Primer and a reverse Primer, the sequences of the forward Primer and the reverse Primer are SEQ ID No.1-SEQ ID No.734, and the detailed Primer sequences are shown in appendix 3;

the position information of the IGT-I7Index is disclosed in appendix 1, "96 IGT-I7Index position information";

the information of IGT-I5 Index is described in appendix 2;

reagent manufacturers: illumina Corp.;

the kit adopts two rounds of PCR reactions to construct the library, has the advantages of short library construction period, high contrast ratio, high capture rate, good uniformity, good repeatability, simple and convenient operation and the like, and supports various samples to extract human whole genome DNA for constructing the library, such as saliva, blood and the like;

(3) library construction:

(3.0) diluting the concentrations of all gDNAs to be detected to the same concentration, and transferring the gDNAs to a PCR8 union tube, wherein on one hand, the gDNA concentration is convenient for the gun arrangement operation, and on the other hand, the gDNA concentration difference of the final library is reduced for adding the same initial amount of gDNA, so that the library is convenient to mix; reaction numbering is carried out on the upper part of the PCR tube wall or the tube cover, so that the marks are prevented from disappearing due to high temperature or other reasons, and the cross contamination of samples caused by the misoperation of mixing subsequent products is avoided;

(3.1) 1 st round of multiplex PCR reaction

Preparing reaction liquid in a PCR tube (or a calandria or a PCR plate) according to the formula in the following table, and gently blowing and sucking up and down by using a gun to mix uniformly;

(3.2) magnetic bead purification of pooled products

(3.2.6) removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180ul of 80% ethanol solution with volume percentage concentration into the PCR tube, standing for 30s, completely removing the supernatant, and recommending to remove the residual ethanol solution at the bottom by using a 10 mu l pipette;

(3.3) round 2 adaptor sequence PCR reaction

Preparing reaction liquid in a PCR tube (or a calandria or a PCR plate) according to the formula shown in the table below, and gently blowing and sucking up and down by using a gun to mix uniformly, wherein the PCR product mixture is the purified multiplex PCR product obtained in the step (3.2.10);

(3.4) round 2 magnetic bead purification

(3.4.3) completely removing the supernatant, taking down the PCR tube from the magnetic rack, adding 50ul YF buffer B into the tube, and sucking and mixing the mixture by a pipettor for 20 times;

(3.4.6) removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180ul of 80% ethanol solution in volume percentage into the PCR tube, standing for 30s, completely removing the supernatant, and recommending to remove the residual ethanol solution at the bottom by using a 10-microliter pipettor;

(3.5) library quantification

The multiplex PCR library obtained in 2ul step (3.4.10) was taken and used

3.0fluorometer (qubit dsDNA HS Assay kit) to perform library concentration determination and record library concentration; the concentration range of a normal library formed by saliva gDNA or blood gDNA is 5-40 ng/ul;

(3.6) library quality testing

Taking 1ul of the multiple PCR library obtained in the step (3.4), and using a Qsep100 full-automatic nucleic acid protein analysis system to measure the length and purity of the library fragment, wherein the target fragment distribution interval of the normal library is between 300bp and 420bp, and the detection result is shown in figure 2;

(4) sequencing on machine

(5.3) BAM preprocessing of the comparison information file: the method comprises the steps of using picard and GATK software to combine BAM files of comparison information files generated by different libraries and Lane of the same sample, sequencing according to genome coordinates, verifying the BAM files of the comparison information files, removing repeated sequences generated in a PCR process, correcting a base quality value, and obtaining a Clean BAM of the preprocessed comparison file. Counting and comparing the file Clean BAM by using the picard software to obtain quality control information; the quality control information comprises coverage, depth, capture efficiency and uniformity of a target region capture chip, the number of comparable sequences and bases, and the length distribution of insert fragments;

(5.4) mutation detection: detecting small fragment variation on the gene by using GATK software, wherein the small fragment variation comprises Single Nucleotide Polymorphism (SNP) and insertion and deletion indels;

and (3) filtering the detected variation, wherein the SNP filtering conditions are as follows: QD <3.37| | FS >31.397| | SOR >10.419| | MQ <20.0| | MQRankSum < -12.49| | ReadPosRankSum < -3.721, and the Indel filtering conditions are as follows: QD <5.2| | FS >52.254| | SOR >9.044| | ReadPosRankSum < -5.504;

annotating the variations that meet the filtering conditions to obtain variation information, wherein the variation information comprises genes, transcripts, variation types, functions and frequencies in normal population;

counting variation results to obtain variation quantity, length distribution, various type variation quantity and proportion appearing in population;

(6) mutation interpretation: explaining the relationship between variant SNP and variant Indel and diseases,

the variants were classified into 5 classes by using the software REO-HIT according to the ACMG/AMP guidelines (Genet Med.2015May; 17 (5): 405-: pathogenic, suspected pathogenic, unknown meaning, suspected benign, benign;

the software REO-HIT process is shown in fig. 1.

The method comprises the specific steps of carrying out,

loading a database: loading ClinVar, CGD and OMIM databases;

collecting variation information: reading the variation information obtained in the step (5.4) one by one to obtain crowd variation frequency information, disease variation information and variation function prediction information;

processing interpretation evidence:

i) by database comparison with the collected mutation information, it was sequentially judged whether each mutation satisfied ACMG/AMP guidelines (Genet med.2015may; 17(5): 405-424.) said pathogenic evidence or benign evidence;

wherein the evidence of pathogenicity includes: strong pathogenic PVS, strong pathogenic PS, medium pathogenic PM and weak pathogenic PP;

benign evidence includes: independent benign BA, strong benign BS, weak benign BP;

ii) combining the evidence of variation, classifying each variation according to the ACMG/AMP combination rules as: pathogenic, suspected pathogenic, unknown meaning, suspected benign, benign;

(7) issue a report

And (3) verifying the sequencing results of the detected diseases, suspected diseases, unknown meanings and suspected benign one by one through Sanger sequencing, and giving a report.

Comparative example

Sanger sequencing is a gold standard for the detection of DNA mutations, but due to its limited sensitivity, it is not possible to simultaneously probe multiple targets in parallel.

When the Sanger sequencing is adopted to detect ACTA2, FBN1, MYH11, MYLK, SMAD3, TGFBR1, TGFBR2, PRKG1, LOX, COL3A1, TGFB2 and TGFB3 genes, the respective Sanger sequencing of each exon is required, and the cost is high.

The target region capture high-throughput sequencing used by the invention relates to the targeted enrichment of ACTA2, FBN1, MYH11, MYLK, SMAD3, TGFBR1, TGFBR2, PRKG1, LOX, COL3A1, TGFB2 and TGFB3 gene exons, and the detection of all the gene exons can be realized only once by parallel sequencing of Illumina XTen.

FTAAD is consistent with the clinical symptoms of sporadic TAAD and requires detection of FTAAD-associated gene mutations for definitive diagnosis. FTAAD is autosomal dominant hereditary disease, but the penetrance of different families is different, 50% -80% of carriers have TAAD, and the FTAAD has obvious genetic heterogeneity. 12 pathogenic genes such as ACTA2, FBN1, MYH11, MYLK, TGFBR1, TGFBR2 and COL3A1 have been reported. The traditional technology is difficult to examine the variation of the genes at one time, and a new generation of sequencing technology provides a new solution.

The invention provides an experimental process and a reading method which can detect 12 pathogenic genes related to FTAAD at one time, including screening of all FTAAD related genes and capturing of related genes by using a multiplex PCR technology. Accurately reading the relation between variation and diseases, and providing important basis for FTAAD screening, early diagnosis, differential diagnosis, individualized medication, birth guide and exercise suggestion.

The technology adopts a multiple PCR capture technology, captures a selected specific effective gene set by using an amplification principle, then detects the gene mutation of the FTAAD through a high-throughput sequencing platform, and detects the gene mutation of the FTAAD by using a self-established bioinformation analysis flow and an ACMG genetic interpretation guide so as to assist the clinical accurate diagnosis and treatment of diseases.

A method for detecting the pathogenic gene mutation of FTAAD based on high-throughput sequencing technology features that the total 12 pathogenic genes of FTAAD are enriched by multiple PCR targets. Secondly, 367 pairs of primers designed can enrich the 12 pathogenic gene exons and the intervals extending 15bp on both sides in a targeted manner from the human genome through one PCR reaction. ③ constructing the experimental process and technical parameters of the library by matching with the labeling technology used by MiniSeq, NextSeq, HiSeq 3000/4000, Hiseq X Ten, MiSeq, HiSeq 2000/2500 and NovaSeq sequencers published by Illumina company; the labeled joint is used in the process of capturing and building a library, so that the sequencing noise pollution is reduced, the PCR error is corrected, the accuracy of the result is improved, and particularly, the low-frequency mutation is easier to detect. The library constructed by the kit is used for sequencing technical parameters, data quality requirements after sequencing and the like of MiniSeq, NextSeq, HiSeq 3000/4000, Hiseq X Ten, MiSeq, HiSeq 2000/2500 and NovaSeq sequencers published by Illumina. The bioinformatics analysis method, parameters and flow of the sequencing sequence corresponding to the invention. Sixthly, sequencing variation result pathogenicity reading process and clinical explanation.

The method can be based on a high-throughput sequencing technology, and has a deformation scheme 1. a method for specifically enriching a pathogenic gene to be detected from a genome, such as changing from multiplex PCR to hybridization capture or selective cyclization, 2. a joint and a PCR amplification condition and an experimental flow are changed based on a probe used by a sequencing platform except Illumina, 3. a sequencing depth and a sequencing quality parameter and the like are changed based on a sequencing method used by the sequencing platform except Illumina, 4. a sequencing data interpretation method and a variation interpretation means corresponding to a bioinformatics algorithm and detection accuracy, and 5. a pathogenic interpretation method and a clinical application interpretation.

The invention detects gene sequences based on a high-throughput targeted sequencing technology, analyzes and reads gene variation and pathogenic probability thereof through bioinformatics, and is suitable for medical and third-party detection institutions with sequencers, large-scale servers, bioinformatics teams and the like. The possible deformation scheme of the clinical application of the invention can be based on probe technology such as fluorescence PCR, gene chip, suspension chip, mass spectrum technology and ARMs, and mass spectrum such as DHPLC directly detects the pathogenic hot point SNP and the corresponding result interpretation method.

Appendix 1:

position information for IGT-I7Index, using BOX1, 96-well plates,

1

2

3

4

5

6

7

8

9

10

11

12

A

A01

A02

A03

A04

A05

A06

A07

A08

A09

A10

A11

A12

B

B01

B02

B03

B04

B05

B06

B07

B08

B09

B10

B11

B12

C

C01

C02

C03

C04

C05

C06

C07

C08

C09

C10

C11

C12

D

D01

D02

D03

D04

D05

D06

D07

D08

D09

D10

D11

D12

E

E01

E02

E03

E04

E05

E06

E07

E08

E09

E10

E11

E12

F

F01

F02

F03

F04

F05

F06

F07

F08

F09

F10

F11

F12

G

G01

G02

G03

G04

G05

G06

G07

G08

G09

G10

G11

G12

H

H01

H02

H03

H04

H05

H06

H07

H08

H09

H10

H11

H12

。

appendix 2: IGT-I5 Index sequence information

The IGT-I5 Index end sample separation sequence is related to a sequencing platform:

MiniSeq, NextSeq, HiSeq 3000/4000, and Hiseq X Ten sequencing platforms, please use the sample sequences in the Index column;

MiSeq, HiSeq 2000/2500 and NovaSeq sequencing platforms, please use the subsampling sequence of the Inprimer column;

3. in the case of single-ended sequencing, the data are resolved only using the IGT-I7Index sample separation sequence, as shown in the following table,

Name	indcx sample separation sequence	Inprimcr partial sample sequence
			IGT-15-31#	GTAGAGGA	TCCTCTAC
IGT-15-32#	CCGCCTTA	TAAGGCGG
			IGT-15-33#	ATAGTACG	CGTACTAT
lGT-15-34#	TTCTGCCT	AGGCAGAA

。

Appendix 3:

primer pool reagent is provided comprising 367 amplicons, the sequence of the amplicons is 1-367, each amplicon comprises a forward Primer and a reverse Primer, the sequence of the forward Primer and the reverse Primer is SEQ ID No.1-SEQ ID No. 734; in particular, as shown in the following table,

SEQUENCE LISTING

<110> heart cerebrovascular prevention and cure education department key laboratory

FIRST AFFILIATED HOSPITAL OF GANNAN MEDICAL University

Paragraph, yanyu

Liu, Zi is composed of

<120> method for detecting familial thoracic aortic aneurysm and interlayer related mutant gene based on high-throughput sequencing technology

<130> 2020

<160> 734

<170> PatentIn version 3.5

<210> 1

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cagttgtgtg ctagagacag agag 24

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tgtcctaccg cactaacgac t 21

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ccaggaaaag ggcgtttgtt g 21

<210> 4

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cagaccctaa tgtttgccac aatg 24

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ttcaatccca tctctggcag tg 22

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtgtgctctc ctgaccattc tt 22

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cagcagtagt gtggtgttct gt 22

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cacaagtttt gggagatgct gac 23

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

attgccactg ggtctgtcac 20

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ctctccactt aaggaccatg gc 22

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tggtcctgga agttactgag ca 22

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

catggtgact tatcacgccc aa 22

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cttctctctg ctgtgctgca t 21

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ggaaatgcag gacctcatga ca 22

<210> 15

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

acagaggaac ctaatctgtg tcct 24

<210> 16

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cacaaatgcc caattacagc tgag 24

<210> 17

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gtttttcttt taattatttg gtctctggat gg 32

<210> 18

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

aagggatcag ctacctccac tt 22

<210> 19

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

agcaaagatg gctgtcttct ca 22

<210> 20

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tgtgtatgca gcataaggca ga 22

<210> 21

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ctccaaccat gaccaggaag ag 22

<210> 22

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

aacgaatgcc tcagcgctc 19

<210> 23

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

agccatgcat cttgagagtg ag 22

<210> 24

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

taggatgtgt aggggccaga tt 22

<210> 25

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ttcttatccc aacagcagag gaa 23

<210> 26

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

gttggcttga ctcaaatgcc tc 22

<210> 27

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ccacttgagg ataagccatc aga 23

<210> 28

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

acactgaagt gaccccctac ata 23

<210> 29

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

cacagcagca ttccgatttg g 21

<210> 30

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

gctttcccct cttgcttctt ct 22

<210> 31

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

gtcacttctg atgcactcaa agc 23

<210> 32

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

agtcaggtaa ttaaggcaga tatatgca 28

<210> 33

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

tgttcccagg atcagtacac gta 23

<210> 34

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

catcatgttt tggacacatt cctgg 25

<210> 35

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

aaagcctggg ccctaaacta c 21

<210> 36

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

gagtattgga ggggacagac atc 23

<210> 37

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

ttccctatga ggttcacgca ac 22

<210> 38

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

ccataacaca gagggaagtt accg 24

<210> 39

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

gccatcaaag cttcatggaa tcc 23

<210> 40

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

aaggctgtcc tgagactcat ttg 23

<210> 41

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

ggcaattggc catggaaaac g 21

<210> 42

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

atccgcctgg aaacctgctt 20

<210> 43

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

ccctatcgga catgctgaat tttg 24

<210> 44

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

agatatagat gaatgtgaag tgttccc 27

<210> 45

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

gacagcttta tccagtccga gt 22

<210> 46

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

tgtcagaact gcaaagtctg ga 22

<210> 47

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

ttcttttgca ggaaaagctg aca 23

<210> 48

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

taggcccaag actagatttt agcag 25

<210> 49

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

caggcaatgt ttcagaaaat gggta 25

<210> 50

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

cctaaggtca ttacatttat tgtagtgtta tatt 34

<210> 51

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

aagacctcaa tggtggcaga ag 22

<210> 52

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

gtgacagtgt gatgacagat gc 22

<210> 53

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

cataaggagg agaaaaggca cgt 23

<210> 54

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

ccttcgtaag cttactcttc tggtc 25

<210> 55

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

tatgtcccac attccacgtc ag 22

<210> 56

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

ggaacccaga aagtcttaga attatgag 28

<210> 57

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

acttgaacaa tgcaagaaaa ataactagat g 31

<210> 58

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

ctgttgtgtt ttgttttgtt gtgtttt 27

<210> 59

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

ggaactgact tacacaaacc atgc 24

<210> 60

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 60

ctgagtcctt ctactgacga atgg 24

<210> 61

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 61

gagaatggct ctccagagca a 21

<210> 62

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 62

acaggacagg cccatgtttt 20

<210> 63

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 63

ttcaggaagt agccatgcag ac 22

<210> 64

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 64

aagcgtctca gctctctcct ta 22

<210> 65

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 65

tgcaggaaag aggaaagcca a 21

<210> 66

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 66

catgtgctaa cagacctctg gt 22

<210> 67

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 67

cttgaaactt gggagaccca ca 22

<210> 68

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 68

caagaggcgg cgggag 16

<210> 69

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 69

aataggttcc agccactggc tt 22

<210> 70

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 70

cagaagccaa tgtgagtctt gc 22

<210> 71

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 71

agacatcagg agaaactaac ttctgac 27

<210> 72

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 72

ccttgtcttc ccattctaat gaaaaaca 28

<210> 73

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 73

cgcagagcca cattcattga tg 22

<210> 74

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 74

agcaagtggc cagatccaat g 21

<210> 75

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 75

acaaggattc accagctgga tc 22

<210> 76

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 76

ggaattttaa cccctctttg ccc 23

<210> 77

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 77

gtggagttct tacaggcaaa gga 23

<210> 78

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 78

gatgcacagt cacgctgtat ttc 23

<210> 79

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 79

agattccctg caagtatttt tgga 24

<210> 80

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 80

cagacaatcg ggaagggtac t 21

<210> 81

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 81

ttaggaaagt gcggtgccaa 20

<210> 82

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 82

aaggaaggag ctccatcctc ta 22

<210> 83

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 83

aagggaagct ttgagggaca tc 22

<210> 84

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 84

ggtaggttcc cttttgttgc tg 22

<210> 85

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 85

tggtacctat attcatggct atacagtg 28

<210> 86

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 86

cttcacgttt aaaaaatacc ttgttattca ct 32

<210> 87

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 87

cttacatcat ggccagtctg ca 22

<210> 88

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 88

aggaaagcaa ctgaagggtg tc 22

<210> 89

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 89

ttctgctaag tccagtggac ac 22

<210> 90

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 90

catgccagtg ggaacctctt 20

<210> 91

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 91

actgacttcc tttgctgatg ca 22

<210> 92

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 92

tgctctttag ccactgtaac cg 22

<210> 93

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 93

agacaaactc ttgggtaggc atg 23

<210> 94

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 94

tgagaggctt tgttgactgg ac 22

<210> 95

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 95

gaaagttctg acaatgccgt catg 24

<210> 96

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 96

ccaattattg ttctttgctg acccc 25

<210> 97

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 97

gaacttgtga gctctcttcc tct 23

<210> 98

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 98

tcaggccatt ccaaaatgtg aag 23

<210> 99

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 99

cccaaggaaa ttcaagttgt gtgt 24

<210> 100

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 100

cagacgggca gagtaacaac taa 23

<210> 101

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 101

acacagtatg cttgcttctc tga 23

<210> 102

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 102

ttgggccctg ttcttttatg gt 22

<210> 103

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 103

cctaatctca tcaagcccag ca 22

<210> 104

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 104

aaccgaggaa gagtaacgtg tg 22

<210> 105

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 105

gcagtccttg ataagcaacc tct 23

<210> 106

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 106

ccaaaagaca tttgtgctga gcc 23

<210> 107

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 107

cagtgcttat gactaacaag acaagatg 28

<210> 108

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 108

gactgcggtc agttaatgtt ttctc 25

<210> 109

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 109

cagagtacat agagtgtttt agggagag 28

<210> 110

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 110

gacagacatc aatgaatgtg agctg 25

<210> 111

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 111

gaccacaagt aaatggtgtg aaagtc 26

<210> 112

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 112

gtcaagatgg acacccagca at 22

<210> 113

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 113

tttaaaggac gtcccctctc ct 22

<210> 114

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 114

acctcctgac tgcttgctca ta 22

<210> 115

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 115

cactcctcgc attcctcagt ac 22

<210> 116

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 116

ggaagccgtg tggctctatt ta 22

<210> 117

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 117

cacttgaatg accccctagt gt 22

<210> 118

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 118

gttctggttg ctattcaggc ac 22

<210> 119

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 119

cattggagtg gtataggaac caca 24

<210> 120

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 120

tgctattttt gtctataatt ccaaggtgt 29

<210> 121

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 121

ggcattccaa aagatagcaa agtacac 27

<210> 122

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 122

gcaaagtaga tacaggcaaa gtttgg 26

<210> 123

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 123

catcccagat acatggcaca gt 22

<210> 124

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 124

aagggcagga tctacctgtt ct 22

<210> 125

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 125

cctctctcat aaggttagcc atgatg 26

<210> 126

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 126

tcctatcttc cccattttca aggg 24

<210> 127

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 127

actgcaatgg aaggagagga ct 22

<210> 128

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 128

tgagtttgca aatggaggga gg 22

<210> 129

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 129

acagaattac aacagaccct tggt 24

<210> 130

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 130

tgtcagatta agtactgatg aaagatacca 30

<210> 131

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 131

gctgggatgg gatattctgc a 21

<210> 132

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 132

tcagcgatgt gtgtgtgtgt 20

<210> 133

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 133

actacacccc ccaactgcaa 20

<210> 134

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 134

tggtaacata attgtggaca aattatcaca 30

<210> 135

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 135

ccgtgcggat atttggaatg aag 23

<210> 136

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 136

ttcctctgca tgatggttcc tg 22

<210> 137

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 137

cccaggtaat cgaagaaaat ccatc 25

<210> 138

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 138

ctgtgagctg ttgcaatcta tgc 23

<210> 139

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 139

ccccatgcaa ccaacacaac 20

<210> 140

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 140

gtctgccagg attcatcttg ct 22

<210> 141

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 141

gttttcttgc cgtggtgcaa 20

<210> 142

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 142

atcccaacca tgaagatttc tttgc 25

<210> 143

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 143

cagtgctttt ctctggcctg a 21

<210> 144

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 144

cctaaccacc cctccaactc t 21

<210> 145

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 145

ggttcggaac tccacaccc 19

<210> 146

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 146

ctcatggcct tcactctcct c 21

<210> 147

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 147

ataaaatggg ggtcgaggat gc 22

<210> 148

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 148

gtactttcaa gggtggctga gt 22

<210> 149

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 149

catgccccaa gctcctagtg 20

<210> 150

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 150

tgagccaaag gacctccca 19

<210> 151

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 151

cgaaacatgg acgagaaaaa cca 23

<210> 152

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 152

gttgtagttg ttagccgagg agaa 24

<210> 153

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 153

agcgtccatg gccagagt 18

<210> 154

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 154

ttgccagcac cgtggaag 18

<210> 155

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 155

gttgagagga cccatgaagg aag 23

<210> 156

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 156

ccttcctcgg ggcaggt 17

<210> 157

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 157

accaccgcgc cacct 15

<210> 158

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 158

ctgttatccc tcccctctgc t 21

<210> 159

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 159

gtgggattga tgggcccc 18

<210> 160

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 160

tctgctgggt ctctccct 18

<210> 161

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 161

ggcaacagag caagacctca 20

<210> 162

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 162

tcctaaagct atctctttct ctttccc 27

<210> 163

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 163

gggctgcccc tgtgac 16

<210> 164

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 164

ctccccacca cctgcg 16

<210> 165

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 165

cccaaatgtg aaggctccag a 21

<210> 166

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 166

agcagggtgt ggcttcatg 19

<210> 167

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 167

gtaagaacgg tcccaccaag ag 22

<210> 168

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 168

caacggtaag tcccaaggtc tg 22

<210> 169

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 169

tgggagcccc agggtct 17

<210> 170

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 170

agaggctgat gccaccctg 19

<210> 171

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 171

gggtcccctg agacagagtc 20

<210> 172

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 172

ctgattcaag ccctacttgt ctcc 24

<210> 173

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 173

aagcccatct cagacaacca ag 22

<210> 174

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 174

tggagcccat tgagaataac cg 22

<210> 175

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 175

ccaaccccaa agtcattggt ca 22

<210> 176

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 176

taggctccaa aactccaggg ta 22

<210> 177

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 177

atcccttaac cccggattct ga 22

<210> 178

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 178

gtgaattggg tgtgacaggg at 22

<210> 179

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 179

gcaggagatg acaccaaagc tt 22

<210> 180

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 180

ctgtgggtta agggcagagt tt 22

<210> 181

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 181

ggttggctaa atcatggcct ct 22

<210> 182

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 182

tcaccatgga acgtgtgcat ag 22

<210> 183

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 183

gaactgcaga caagcaggac a 21

<210> 184

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 184

aggtctggaa gtaagctggg t 21

<210> 185

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 185

gtgaccttct tgccattctc ca 22

<210> 186

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 186

ctgttacctt attccacagg ggac 24

<210> 187

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 187

ccaagcctcc agattttgca ag 22

<210> 188

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 188

aaccgggctc cttcttgttt 20

<210> 189

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 189

agagttggag gggtggttag g 21

<210> 190

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 190

gtgacacacc agctacaagg g 21

<210> 191

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 191

agagacagta ggcagcgtga 20

<210> 192

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 192

ccaaggaggg ggcatctttt aa 22

<210> 193

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 193

gaaatctggg aatgcacaga ctg 23

<210> 194

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 194

aaggacatct gagcttgtcc tc 22

<210> 195

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 195

tggcctcccc ggcag 15

<210> 196

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 196

gctgcccgct ctgcc 15

<210> 197

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 197

cctctcatcc gcgtatttgg aa 22

<210> 198

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 198

aacctacact gcttacacct gc 22

<210> 199

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 199

tcgatctcct tctggaacct ct 22

<210> 200

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 200

agacatggtc ttcgcttctc ag 22

<210> 201

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 201

cctgcaaaag ggatgcaaag ag 22

<210> 202

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 202

aggatcccct tctagaggct tt 22

<210> 203

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 203

ctttatgcag accctgacct gt 22

<210> 204

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 204

ccattcactg tgttcctccc a 21

<210> 205

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 205

gggatacatg gacacacagc aa 22

<210> 206

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 206

cttcctgaag agcaccttgg tt 22

<210> 207

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 207

gcctcaagtg atccacatac ct 22

<210> 208

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 208

ccaaagctgt tactcttttc cctc 24

<210> 209

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 209

caagtcctgt tccccagcaa 20

<210> 210

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 210

tgacaacata tccctttgcc ac 22

<210> 211

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 211

cgcgtttgag gtattaggat gc 22

<210> 212

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 212

agtggaaatc aggagctctt cc 22

<210> 213

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 213

ggtacctcca gcatccattc c 21

<210> 214

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 214

gatccactgc cctctttgac c 21

<210> 215

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 215

tccagctcaa agcagaacat gg 22

<210> 216

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 216

cagaagatct ggaaggacaa tggg 24

<210> 217

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 217

acacacgtgt acaaggtgtg ac 22

<210> 218

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 218

gatgctcacg ccccgc 16

<210> 219

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 219

gggtgcatgg atggataagt ga 22

<210> 220

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 220

gagaacccgc tgtattcaca ga 22

<210> 221

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 221

ctaccctcca gactcaaggt gt 22

<210> 222

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 222

tgaccagatc tcggtctcat gt 22

<210> 223

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 223

ggaaagatag aggtggctct tgg 23

<210> 224

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 224

catggggtta acggctgaag t 21

<210> 225

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 225

accccatttc taccaccagc ta 22

<210> 226

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 226

ctgtttgggg ggatcaaagt ca 22

<210> 227

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 227

aagagaggca cttggaacca tg 22

<210> 228

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 228

agacccttag ctcagagctc at 22

<210> 229

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 229

tccttatgag gccaggagta gg 22

<210> 230

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 230

cttcgaggca gccagcatta 20

<210> 231

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 231

ccttttaata tgacttagaa actgcttttc tc 32

<210> 232

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 232

gatgaccagt caatcaggga gtc 23

<210> 233

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 233

atccagccac accctattca tg 22

<210> 234

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 234

caacgagtga agaaatgacc tgg 23

<210> 235

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 235

ctgggaatga aagtggcttg aac 23

<210> 236

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 236

aggtctcctc ctgctcaaaa atc 23

<210> 237

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 237

tggtccacca gtccaccaa 19

<210> 238

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 238

acacacagat tcgggcagc 19

<210> 239

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 239

tgcttgtgga tgtactccac tc 22

<210> 240

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 240

ggattcagga attgcccatc ac 22

<210> 241

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 241

agctgaggca ctgaatctaa ctg 23

<210> 242

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 242

tctgagactc acaccaaccc t 21

<210> 243

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 243

cgcacatttg tttcaagcca ct 22

<210> 244

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 244

acagtcctac agcatcgaga tct 23

<210> 245

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 245

ttgccccaag ctccatgc 18

<210> 246

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 246

ttcctttcct agcccagggg 20

<210> 247

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 247

atacccaggt gtcagtctag ca 22

<210> 248

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 248

agtggacaag actgcatctt cc 22

<210> 249

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 249

cagtgttgga ggcagttctg t 21

<210> 250

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 250

cacacctggt tcactccaca a 21

<210> 251

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 251

tcttgcagct tctgcttgaa gg 22

<210> 252

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 252

tcagggccct tgaaacccg 19

<210> 253

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 253

aaaatccggg gtggcaggtt 20

<210> 254

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 254

tcctggggaa gaaggtgagt aca 23

<210> 255

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 255

acccatagcg gtcactacca 20

<210> 256

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 256

ggatttgctc tcccctgaag tt 22

<210> 257

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 257

ttaagggagg ctggctggac 20

<210> 258

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 258

gttcatcagt aagcctcgct cag 23

<210> 259

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 259

actcgctctg agtgggtca 19

<210> 260

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 260

aaccaatccc ccttcccct 19

<210> 261

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 261

ctacaaatgt gcccttcctc ca 22

<210> 262

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 262

ctgagccttc ctgtgcttcc 20

<210> 263

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 263

ccatgaatgg ttgtcccaca ga 22

<210> 264

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 264

ttccgggatt ccaaagcctg 20

<210> 265

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 265

gcaatcggtg accctaatta cct 23

<210> 266

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 266

tcaccttctg gagaagagag gaa 23

<210> 267

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 267

cagggatcgt tctgcactag c 21

<210> 268

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 268

tgaccaagtt accccattgc c 21

<210> 269

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 269

caagtcagca ggaaagcaat cc 22

<210> 270

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 270

gatcaccatc actatcccac cc 22

<210> 271

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 271

tcgggcctcc tgggac 16

<210> 272

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 272

aggctcttac cctttcttct ttttct 26

<210> 273

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 273

ggcttctcca agactgttga ca 22

<210> 274

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 274

caacttgtgt tctctccctg tga 23

<210> 275

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 275

cctcaaaatg cccctttgtt cc 22

<210> 276

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 276

tgagtccaac cctcgtgtct at 22

<210> 277

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 277

agggagtagg gaagcaaaga ct 22

<210> 278

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 278

tagccacacc tgctgtcct 19

<210> 279

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 279

acactggtgt ccatgggaga 20

<210> 280

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 280

tcaggagggg agctgtttga 20

<210> 281

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 281

acgctgctgg cagtgg 16

<210> 282

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 282

gcttttctct cctccccctt tc 22

<210> 283

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 283

tgtcactgtc cggtaatcaa cc 22

<210> 284

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 284

tgtcactgtc cggtaatcaa cc 22

<210> 285

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 285

agttccttcc acgtcttgtt gg 22

<210> 286

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 286

cttcctttgc ctgcaattct gg 22

<210> 287

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 287

gctgtacgga ttattcccag ca 22

<210> 288

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 288

gacaatggcg tcgaatgtga tc 22

<210> 289

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 289

ggattcaggc aagagtgagt ga 22

<210> 290

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 290

ctcatttcct cagcagctgc ta 22

<210> 291

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 291

aggccccttc ttccccaa 18

<210> 292

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 292

actgcaagag aggccatgc 19

<210> 293

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 293

ttcagggtct cagcaggctt 20

<210> 294

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 294

ccaagaaggg gacttccaag ac 22

<210> 295

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 295

cttcaggtcg tcttccgata gg 22

<210> 296

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 296

acctctgtgg tggaggagtt 20

<210> 297

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 297

ctgcaggaca caaacaatct agg 23

<210> 298

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 298

ctgtgatcaa gtccacactg aac 23

<210> 299

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 299

agggctgcac cttctttaga ac 22

<210> 300

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 300

agctctgccc tttgccaatt 20

<210> 301

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 301

gagagcaata cccattgcaa cc 22

<210> 302

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 302

catgaatgtt caccccacgt tc 22

<210> 303

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 303

tggctcagag ggataagtga ga 22

<210> 304

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 304

gatctgagct gacctgtcct tt 22

<210> 305

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 305

tttcagcagg cagaggtaat gg 22

<210> 306

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 306

acctacctca caatcacagg gt 22

<210> 307

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 307

acaacatctt ggctccccag 20

<210> 308

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 308

ctgcaaacag ccagcctc 18

<210> 309

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 309

agggcatttc tcggtaacat cc 22

<210> 310

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 310

aaggccaaag acgttcagca 20

<210> 311

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 311

gagaacagac taatacaggc tggatc 26

<210> 312

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 312

gactggattc tctgccaatt tcct 24

<210> 313

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 313

cctccccatc cagccaca 18

<210> 314

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 314

agacagggct gtgctttgtc 20

<210> 315

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 315

gcatgactcc agcttggact c 21

<210> 316

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 316

gatcattcta ttctcccctc tcctca 26

<210> 317

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 317

ggaggagaaa tggtgcgaga ag 22

<210> 318

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 318

tctctccctc ttcccatctc ca 22

<210> 319

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 319

cctctctttc tgcccctccc 20

<210> 320

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 320

tgctgtcccc acaggcag 18

<210> 321

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 321

catggtgtgc atgtgtgatg tc 22

<210> 322

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 322

aggaagggat ggaagggatg aa 22

<210> 323

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 323

agtagcccac cctgtgtcc 19

<210> 324

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 324

gcggggaatg gagccac 17

<210> 325

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 325

ggtccaggac ttgctttatc ca 22

<210> 326

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 326

ctgatgtagg cagcacccat aa 22

<210> 327

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 327

tctaggaagg gctgtattgt cct 23

<210> 328

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 328

gagaaactct gacagatctc tggc 24

<210> 329

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 329

actgagccac ctctgctct 19

<210> 330

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 330

agagattggg gccacagga 19

<210> 331

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 331

tctttctgct gtgttgggct ac 22

<210> 332

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 332

ttccagttgt gtgcacaagg ag 22

<210> 333

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 333

cctgtccagt ctaacctgaa tctc 24

<210> 334

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 334

tgagaaaggt ggactctctc ca 22

<210> 335

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 335

acttgtgtaa ccccctggag a 21

<210> 336

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 336

tccaattttc tccacctcct gc 22

<210> 337

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 337

ccgtcgagcc cagcc 15

<210> 338

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 338

ggatggtgat gcacttggtg 20

<210> 339

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 339

cgaggtttgc tggggtgag 19

<210> 340

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 340

gagaaagagc aggagcgagc 20

<210> 341

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 341

tcgagatagg ccgtttgtat gtg 23

<210> 342

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 342

tggacaagtc acttcttgcc tc 22

<210> 343

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 343

tgatggtcta tatctgccac aacc 24

<210> 344

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 344

tctcacattc tagcaagttg gctta 25

<210> 345

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 345

ctctagagaa gaacgttcgt ggtt 24

<210> 346

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 346

atgggtctaa tctacatgag agacatc 27

<210> 347

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 347

tgattggtat taccttttaa gcagtcatg 29

<210> 348

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 348

aagggaaaaa aggtgatttc agaagatatt aa 32

<210> 349

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 349

gatcttttaa tgccttggca ttagct 26

<210> 350

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 350

tgcttactaa gcagaagcag tttaga 26

<210> 351

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 351

aaccttgaga ttttttctaa gaatctttct ct 32

<210> 352

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 352

tggtcctgat tgcagcaata tgt 23

<210> 353

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 353

gtttttgtcg ttgttgatgt ttatttcact 30

<210> 354

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 354

gttgtcatat cataaattaa gtctttcaac gtag 34

<210> 355

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 355

gaaacatgta atattgttga ttgtgttgag t 31

<210> 356

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 356

aggatgtttt catgacgtaa cattacag 28

<210> 357

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 357

gcccaaccga aatgttaatt ctgt 24

<210> 358

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 358

tgaaaattta aagcttaaat aatagaactg ctt 33

<210> 359

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 359

tgtctgaaag gaggttcatc ca 22

<210> 360

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 360

ctcatgacaa actactgggg ga 22

<210> 361

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 361

tcttatccag accaatggaa aatggt 26

<210> 362

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 362

agcagatctg aagaaaaaag gagagt 26

<210> 363

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 363

tggcagttgg ataatcattt aatatatctt tctc 34

<210> 364

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 364

tgtaaaaagg ggaaaagaaa gaataacttc t 31

<210> 365

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 365

acctaccaca tccaactcct tc 22

<210> 366

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 366

cttccaggat gatggcacag t 21

<210> 367

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 367

gctgaggtct ataaggccaa gc 22

<210> 368

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 368

gtactcctgt aggttgccct tg 22

<210> 369

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 369

tcacctccac agtgatcaca ct 22

<210> 370

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 370

ggtaaagggg atctagcact agct 24

<210> 371

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 371

ctgcacatgc cattctcagt ga 22

<210> 372

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 372

tctgccacct aagaggcaac tt 22

<210> 373

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 373

gccaggggtc cgggaa 16

<210> 374

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 374

ctgtcaagcg cagcgga 17

<210> 375

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 375

tcaaaacagt ttcactttcc tgtcatc 27

<210> 376

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 376

agtgagggag catgactaaa aatagaa 27

<210> 377

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 377

ccctcgcttc caatgaatct ct 22

<210> 378

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 378

aggtcccaca cccttaagag aa 22

<210> 379

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 379

gaagctgagt tcaacctggg aa 22

<210> 380

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 380

gatcttgact gccactgtct ca 22

<210> 381

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 381

gggaaacaat actggctgat cac 23

<210> 382

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 382

aggttaggtc gttcttcacg ag 22

<210> 383

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 383

aaatgatggg cctcactgtc tg 22

<210> 384

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 384

cactacacaa tgatgctggt cca 23

<210> 385

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 385

ggtgcccttt ggatctcttt cc 22

<210> 386

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 386

tgagaggggc agcctcttt 19

<210> 387

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 387

acaaatgcca gtcggtgct 19

<210> 388

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 388

agagccccca gcctctg 17

<210> 389

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 389

cagaacgagc tggacaagta cc 22

<210> 390

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 390

gacagacaat acagagaccc gc 22

<210> 391

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 391

gagcctaaca ctcctcttca ca 22

<210> 392

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 392

aaggcctagg gaaaagacat tga 23

<210> 393

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 393

tgtggttcat gtgttcagca ct 22

<210> 394

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 394

catcacacag ctgtgaaaga agc 23

<210> 395

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 395

cctttgtcca ttgtgggtaa gca 23

<210> 396

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 396

aagcctattg tgctaggagc tca 23

<210> 397

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 397

tcactgacta ggttgccatt gc 22

<210> 398

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 398

gtgttgggat gtgtcaagtc ca 22

<210> 399

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 399

gcactcttac aagtctatgg gctt 24

<210> 400

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 400

ggatttgtca cttggagttg ggta 24

<210> 401

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 401

aattttttat acaattttca ttccatttct gca 33

<210> 402

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 402

agcaaatgaa cacagtttta taaacaaata ttt 33

<210> 403

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 403

aagtaatatc tcttgtcgtg tctctca 27

<210> 404

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 404

ctgatggatt gtccctaaaa ataaaataat gg 32

<210> 405

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 405

cctaactgct tcccattagc cat 23

<210> 406

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 406

gatcattgtt gactgacata aacacca 27

<210> 407

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 407

ccgcccgaga aaaagtttcg 20

<210> 408

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 408

acctgtacgt ctgcggct 18

<210> 409

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 409

atcggagagg ggaggaagc 19

<210> 410

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 410

tctgcttctg cgcctgc 17

<210> 411

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 411

gccagtaaat cttccctctc ttgc 24

<210> 412

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 412

tgtgcagctg ataagatgaa aaaagaa 27

<210> 413

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 413

ctcctttgca ggatgttgtt cac 23

<210> 414

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 414

tcagggtgct gcatttcaga t 21

<210> 415

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 415

gaagaggctg agctgtttgg ta 22

<210> 416

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 416

actctgcatg tgttcctact gg 22

<210> 417

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 417

cctggaggtg gataaatcag aagag 25

<210> 418

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 418

ccaagaactc atgtgtgttg ctg 23

<210> 419

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 419

aaccctgttc cacagtcatg tc 22

<210> 420

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 420

aggaagcaga ggcgatcaaa ag 22

<210> 421

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 421

aactataatc tgggcccccc aa 22

<210> 422

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 422

agaccaacat gggcaactgt t 21

<210> 423

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 423

aggagcttaa gtatcttgtt tttctctct 29

<210> 424

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 424

gtgtagctgc atgctggtct at 22

<210> 425

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 425

agggggattg acatgataga atttcc 26

<210> 426

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 426

acgtgttcag aactctgcag aaa 23

<210> 427

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 427

tgcacttaaa aattccagct tggaa 25

<210> 428

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 428

ctgtgtctca gaaaaagctg tcttc 25

<210> 429

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 429

ccgcccgaga aaaagtttcg 20

<210> 430

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 430

acctgtacgt ctgcggct 18

<210> 431

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 431

atcggagagg ggaggaagc 19

<210> 432

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 432

tctgcttctg cgcctgc 17

<210> 433

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 433

gccagtaaat cttccctctc ttgc 24

<210> 434

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 434

tgtgcagctg ataagatgaa aaaagaa 27

<210> 435

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 435

ctcctttgca ggatgttgtt cac 23

<210> 436

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 436

tcagggtgct gcatttcaga t 21

<210> 437

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 437

gaagaggctg agctgtttgg ta 22

<210> 438

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 438

actctgcatg tgttcctact gg 22

<210> 439

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 439

cctggaggtg gataaatcag aagag 25

<210> 440

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 440

ccaagaactc atgtgtgttg ctg 23

<210> 441

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 441

aaccctgttc cacagtcatg tc 22

<210> 442

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 442

aggaagcaga ggcgatcaaa ag 22

<210> 443

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 443

aactataatc tgggcccccc aa 22

<210> 444

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 444

agaccaacat gggcaactgt t 21

<210> 445

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 445

aggagcttaa gtatcttgtt tttctctct 29

<210> 446

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 446

gtgtagctgc atgctggtct at 22

<210> 447

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 447

agggggattg acatgataga atttcc 26

<210> 448

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 448

acgtgttcag aactctgcag aaa 23

<210> 449

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 449

tgcacttaaa aattccagct tggaa 25

<210> 450

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 450

ctgtgtctca gaaaaagctg tcttc 25

<210> 451

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 451

agctgctgta gtagcagaaa cag 23

<210> 452

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 452

ggaaaaagaa atactgagga actctttgt 29

<210> 453

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 453

gtttctctct gaggcttgag gtt 23

<210> 454

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 454

ctctgtatgt aactgaccac catct 25

<210> 455

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 455

cagtcagaac caggcacca 19

<210> 456

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 456

acctagaggg gtagggagtt g 21

<210> 457

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 457

ttgtaggggt tgtaagggtc gt 22

<210> 458

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 458

atcctgctga tccgcgacaa 20

<210> 459

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 459

cccgttgttc tcccattgga t 21

<210> 460

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 460

ttccctcacg tgatttgagc c 21

<210> 461

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 461

ccaggcactg atttatccat tgg 23

<210> 462

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 462

catcaggcat ctctgctgtt ga 22

<210> 463

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 463

gaagagggcc tattgatctg caa 23

<210> 464

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 464

gcatattatt tatcatagtc tgaccacata cc 32

<210> 465

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 465

gaaatgaaaa gcaacccaaa agtacc 26

<210> 466

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 466

tggatgtttt atttgtagtt ttgtctttag tt 32

<210> 467

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 467

acgtcgagaa gccacatagc 20

<210> 468

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 468

gagaagttcc tgcgctcagt 20

<210> 469

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 469

cagcggtgac tccagatgag 20

<210> 470

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 470

acagcagccc ccgcg 15

<210> 471

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 471

tcacctttca gcaaaaccta agg 23

<210> 472

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 472

tcacagcagt tggaggctg 19

<210> 473

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 473

ctgtgtcttg tttaacttgt ttcttttcc 29

<210> 474

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 474

tctgttgttt ggtgccatta ttcaaa 26

<210> 475

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 475

gatgttgcat gcacttctaa atgc 24

<210> 476

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 476

actgaattcc tagaagagca acca 24

<210> 477

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 477

cacaatcctg atttcttact gtcaagatg 29

<210> 478

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 478

ggtaggttcc atatggaatt atcgactta 29

<210> 479

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 479

tgagcaccta cgtattcttt atttctct 28

<210> 480

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 480

tggacctttg ataccctaaa aataatgaat g 31

<210> 481

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 481

gtgttatgaa gaccaattag aaaaatacca tg 32

<210> 482

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 482

aggtaaattt cagaaaacaa gttaaggct 29

<210> 483

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 483

cccaactgtt cttacacatg tcaag 25

<210> 484

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 484

tgtcactttg ggttgtaatt gtgtg 25

<210> 485

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 485

tcagctttca tttagttgaa aaagagct 28

<210> 486

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 486

gcaatctaat gcttttctct tgcct 25

<210> 487

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 487

tctctacaaa gcataacact catcga 26

<210> 488

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 488

aagccagtaa tgtgtggatg agataa 26

<210> 489

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 489

gaatcctccc tgtgtttcaa cca 23

<210> 490

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 490

agctcctgga acacctggaa ta 22

<210> 491

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 491

cttcagggtg agacagccaa 20

<210> 492

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 492

actgcccatt tgtggtgcta g 21

<210> 493

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 493

gatgtgcaaa tctgaggctt cac 23

<210> 494

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 494

cacacactca aggataggcc aa 22

<210> 495

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 495

tgaggattag taaataccga ccacttc 27

<210> 496

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 496

agagagaagt cagaaggttg gaga 24

<210> 497

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 497

caggtcctcc cttttcttca ca 22

<210> 498

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 498

gacagtcaga tatgccgtga ca 22

<210> 499

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 499

aatgcactct gatatgggcc ta 22

<210> 500

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 500

accaaacatt tgcaatgatg ctc 23

<210> 501

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 501

ggtttaaaaa atgcatttga tttccttctg 30

<210> 502

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 502

cgattacttt ccaggagtgt tgc 23

<210> 503

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 503

gagaaaagca tagcattcaa gccat 25

<210> 504

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 504

tgcatccaaa agagagagac attttc 26

<210> 505

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 505

cctaaaggag atgacgcaca ct 22

<210> 506

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 506

agcaagatag agatcctggg ct 22

<210> 507

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 507

gagttcctgg ttttcaaagg ctt 23

<210> 508

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 508

ggaactagtg tctcaaatgc agc 23

<210> 509

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 509

tctctagtaa gtctcaaata aaattatttg aagt 34

<210> 510

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 510

cctgggcacc aggcgat 17

<210> 511

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 511

cactgtcaca taaagatgag ctaagtc 27

<210> 512

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 512

tggttaagtg atctctgaag gctac 25

<210> 513

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 513

cctgctgaga atgcatggat g 21

<210> 514

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 514

gagagccatt ttcaccacga tc 22

<210> 515

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 515

aactagttcc gtgtatgtct tctca 25

<210> 516

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 516

ttcctgaaca cgctggaaaa taaa 24

<210> 517

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 517

cccagtgctt tttaaggcct tc 22

<210> 518

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 518

ggatgtccac tggttccatc tt 22

<210> 519

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 519

caatgtattc ttagagtggc gactg 25

<210> 520

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 520

cttgagtgaa gtcataatct catcgg 26

<210> 521

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 521

actttattac tggattttat aaccaattcc cat 33

<210> 522

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 522

cctttcctac ctgaaaacca cca 23

<210> 523

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 523

gaaatgtttg atctgtttta tttgttccct a 31

<210> 524

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 524

ctgtgtaggt gaatttgcta tttcctt 27

<210> 525

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 525

gtaatgtcat gatcatgtac attttgtcct 30

<210> 526

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 526

gaacaagagg aacacatatg gagttaaatt 30

<210> 527

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 527

tttttcctca accagatcaa tgtagatc 28

<210> 528

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 528

cctaatacag ccattactca gatgact 27

<210> 529

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 529

tcaaaataaa catggtaaaa taagcctggt 30

<210> 530

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 530

actgcaacat ggaaacagga gaa 23

<210> 531

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 531

gttttgctca aatgggctgc t 21

<210> 532

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 532

gatgaaagca tgccagatcc ac 22

<210> 533

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 533

gcatttcttg catgtaaacc tttgtg 26

<210> 534

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 534

cactgttaca gctccatatg taaaatagtt c 31

<210> 535

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 535

actacagggc agaataatac ttaaaggaa 29

<210> 536

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 536

gtagcaagta attttcatgc catatctct 29

<210> 537

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 537

gcatatgggt gtgcaaaact gt 22

<210> 538

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 538

catgaacata tggaatattg ttgttttaga at 32

<210> 539

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 539

tccatgacac cagataacaa gagaag 26

<210> 540

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 540

tagcctttat gggaactagc gttc 24

<210> 541

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 541

tctcagttga aggtggtctg ga 22

<210> 542

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 542

cttccatttt gccctccttg gt 22

<210> 543

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 543

acatgaccag gaagcactag gt 22

<210> 544

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 544

cttgagtcaa gtgccccatg tt 22

<210> 545

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 545

ccagaatacg acttcaaaac atatgaca 28

<210> 546

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 546

actgttcctg tttaggataa tgtgtga 27

<210> 547

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 547

gaaagagcct gctattcagg act 23

<210> 548

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 548

gctaagcatg aagatttctg tgca 24

<210> 549

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 549

gaatgactag tgactcagga tggtag 26

<210> 550

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 550

ttggtatggt cacttaatgt tggttc 26

<210> 551

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 551

actgaagcca gtgggaaaca ac 22

<210> 552

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 552

gcttatgcag ccttctaatg gc 22

<210> 553

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 553

tggaagatga aatggaaaac cgaa 24

<210> 554

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 554

ctagtggttc acgtagagag gaag 24

<210> 555

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 555

catcttttcc aggagctccc tag 23

<210> 556

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 556

ttggccacct catcatgatc c 21

<210> 557

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 557

gcttccagaa gttggtacaa atatgtc 27

<210> 558

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 558

caactgcata tcagcaacaa actga 25

<210> 559

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 559

atgcaggcca tagctagatc ac 22

<210> 560

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 560

gttggcttga tgatggctcc ta 22

<210> 561

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 561

cccttgagca ttttgagctg tac 23

<210> 562

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 562

ctactgatgc aaaaccgact cag 23

<210> 563

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 563

gtacctggag agccctatta aacag 25

<210> 564

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 564

gatggctgga catctgtata ctagc 25

<210> 565

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 565

tgtcattctt cactttggga ctca 24

<210> 566

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 566

tgacgccaaa ctctaaaaaa tgacc 25

<210> 567

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 567

cacatctcag cagtataagt aaaagca 27

<210> 568

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 568

gagtggggta gtcagactac tcta 24

<210> 569

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 569

catttgtttg tagccctctc tcct 24

<210> 570

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 570

tggttcttat gcctttgagg attga 25

<210> 571

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 571

actctggcaa aacgtcctgt 20

<210> 572

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 572

cacttttgcc ttcccaatct cc 22

<210> 573

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 573

ggaggacctg ggaagataaa taactc 26

<210> 574

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 574

atggcagagc caactgtcta aac 23

<210> 575

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 575

gtacgactgt gtttaagaag atgcat 26

<210> 576

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 576

aggacgttga tgtatgtctt ttactgt 27

<210> 577

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 577

gcttcttgct gcattcagag aac 23

<210> 578

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 578

gtcttcaact gaactgaatt aagtgtgg 28

<210> 579

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 579

ctaatctgtt ctggtttgct tgtgt 25

<210> 580

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 580

tgacatgttt ttgcccagca ag 22

<210> 581

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 581

ggatcaatta tgagttggcc ctaaac 26

<210> 582

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 582

gaaggatatg gtgaagaaat agcctg 26

<210> 583

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 583

tcctcttcac gctcttcctg a 21

<210> 584

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 584

tctcaggaga acccacatgt ct 22

<210> 585

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 585

cagggaacaa cttgatggtg cta 23

<210> 586

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 586

actgattttc ccctttacac ccc 23

<210> 587

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 587

gatgtctgga agccagaacc at 22

<210> 588

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 588

gactttcatt cccttttagg ctcc 24

<210> 589

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 589

tctgataatg attgtgaatc accagga 27

<210> 590

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 590

gtgagacgta atatcgagta aagaaagaag 30

<210> 591

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 591

gcgagtgtca ttgctttgaa gc 22

<210> 592

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 592

ggaagaaaac accaccaatg gc 22

<210> 593

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 593

ggttccttcc aggaatacat acact 25

<210> 594

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 594

cttctgggat tctgacataa agcat 25

<210> 595

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 595

acctccagtg agtcttcagc at 22

<210> 596

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 596

cagtggaata ccttgtaaac aacttagaa 29

<210> 597

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 597

tctttttcta ggctctataa acttttcca 29

<210> 598

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 598

cttgacatgt gtaagaacag ttggg 25

<210> 599

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 599

actcatgacc agccattcag a 21

<210> 600

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 600

tggtcaacag agatgctaat gtg 23

<210> 601

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 601

gaagggtgaa gtggctaagt ga 22

<210> 602

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 602

gaagcacatg ctttcatgga gt 22

<210> 603

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 603

ctggcttctt ttgcattttg catg 24

<210> 604

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 604

cagtcaacag agaaaaaaca aggca 25

<210> 605

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 605

cagatctggt tatttcttga aaaaatgca 29

<210> 606

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 606

tgctgctttt cttttagatg ccc 23

<210> 607

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 607

gttgctatct aggttagtga aggcta 26

<210> 608

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 608

ctgtcttcat ttgtcttgcc tttct 25

<210> 609

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 609

gcatgcttta atcttctctt tatcaaacc 29

<210> 610

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 610

gaagaggaga ctgagttttg acca 24

<210> 611

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 611

gcgttcatct ccaaccttct ga 22

<210> 612

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 612

aagcattctt cctgccccac 20

<210> 613

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 613

gcttttctat aagccatgtt tgaggt 26

<210> 614

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 614

acagtgtgta ttctttttgt ttctgagt 28

<210> 615

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 615

gtggcctgat tcaaaatgat gca 23

<210> 616

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 616

ctaggtgaat ggaatgctgt gga 23

<210> 617

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 617

cctgagagtt actcctcttc ttgg 24

<210> 618

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 618

gaagtgcagg actgtcccat at 22

<210> 619

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 619

tggatgcaag acagtgacat gg 22

<210> 620

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 620

ccaaattgag gctcagggaa ct 22

<210> 621

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 621

cagctgagag attgctgttg ttg 23

<210> 622

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 622

gagagagagg gagagataga cagataa 27

<210> 623

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 623

tccaagggga aacacaaacc at 22

<210> 624

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 624

catgaatgct ctttctgaag tgtatga 27

<210> 625

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 625

caaaaggtga tgctggccaa c 21

<210> 626

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 626

cagtgatccc agcaatccca a 21

<210> 627

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 627

acctgaaaat aaagatatct gataacacca 30

<210> 628

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 628

ctacctggct ggttgaataa aaact 25

<210> 629

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 629

cctggtggca aggtataata aacac 25

<210> 630

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 630

agacagatgc tgcttttggg aa 22

<210> 631

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 631

gggggatttt ttagctgata gaaagc 26

<210> 632

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 632

gaagggattt tccaacaaaa tgaatgc 27

<210> 633

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 633

caggtccaca tgtttcagat gtct 24

<210> 634

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 634

atgcgctgtg tttctgaaaa ttgg 24

<210> 635

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 635

tgccccgtat tatggagatg aac 23

<210> 636

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 636

cgttcccatt ctaatctaag cagc 24

<210> 637

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 637

cacgtttggt ttggagagtc ca 22

<210> 638

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 638

gcatgccatc actttcagta gc 22

<210> 639

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 639

aaagaaggcc ctgaagctga tg 22

<210> 640

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 640

ttccctgcaa tgaatcagta gatgc 25

<210> 641

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 641

ggtgctcatt gtcgatgtgt ct 22

<210> 642

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 642

tgtctgggaa ggttgctgtc ta 22

<210> 643

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 643

gtcatttgcc cctttgagaa cc 22

<210> 644

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 644

aacccttcag tcttgtgtat gaca 24

<210> 645

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 645

caggttttac gtggtacact gga 23

<210> 646

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 646

cgtaaggggc actctcactt aaa 23

<210> 647

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 647

ccgcctgatc ttcagtaaac tca 23

<210> 648

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 648

gagtgtctca ttggaactcc ctatg 25

<210> 649

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 649

gaagatgagc caactccagc tt 22

<210> 650

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 650

gagaatggca caatgcagga tc 22

<210> 651

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 651

gttcaagctt gaggaatttc atatttttca g 31

<210> 652

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 652

acgtattgtt gacatcctgt atctatgtaa 30

<210> 653

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 653

tcagaagtgt attggaacta tcaggaa 27

<210> 654

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 654

cacaggtgta aaacatgctg tcat 24

<210> 655

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 655

gagataaaat attgaccgat gcagcta 27

<210> 656

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 656

actgaaaaac ctgtaaagct gtacc 25

<210> 657

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 657

tcagtgactt gtctcactaa gttgac 26

<210> 658

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 658

aaggcttcag tcttcctgtg aaaa 24

<210> 659

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 659

agcaaaagag gctgaatgta cact 24

<210> 660

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 660

tgtctataag tcttataatc aattgcttgt ttgt 34

<210> 661

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 661

gctcaatctc acactaaaga acacca 26

<210> 662

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 662

tctcctccta cacagtgacc aat 23

<210> 663

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 663

tcctagccca gctagaaaag ga 22

<210> 664

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 664

gagaaaatgc acagtgtgag gc 22

<210> 665

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 665

cattctcaca ccatcatgcg tt 22

<210> 666

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 666

cagtagaagc catttctcag aaaca 25

<210> 667

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 667

gagccagcaa tgtatagtgt tgc 23

<210> 668

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 668

agtcttggag accccatgaa ttg 23

<210> 669

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 669

acctcctcca aatgaggatc atg 23

<210> 670

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 670

gtgacacttt gggtgatgct atg 23

<210> 671

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 671

tcaggagcac ttctcccctt 20

<210> 672

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 672

catgcatctg ctttgcctcg 20

<210> 673

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 673

agggaacctg caagcatttc tc 22

<210> 674

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 674

gatgggtctt tctggtaact gtcc 24

<210> 675

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 675

catgttatta ctcttggccc tcgt 24

<210> 676

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 676

agactgctac ctgtgtctct actt 24

<210> 677

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 677

tctgggctgg ttcttaaata tgct 24

<210> 678

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 678

ttggcctctt tcagtatttg ggt 23

<210> 679

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 679

gtccatcaac tttttattcc aagtgtct 28

<210> 680

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 680

gtctaaaatg taatcttatc ctgtcttgtc tc 32

<210> 681

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 681

ctgtgcttgt ctatcagctg gt 22

<210> 682

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 682

gtgatgtatc ggttgtaggc ttct 24

<210> 683

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 683

agagcctgtt cactaaacca cag 23

<210> 684

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 684

gttgctggaa aactgtgtac tagc 24

<210> 685

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 685

acctgaatgg aaaatgagga aaggta 26

<210> 686

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 686

gtctcaataa tgttttgaat tgtattactg gc 32

<210> 687

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 687

gcagtcatgt cacagagacc tt 22

<210> 688

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 688

tggagaatgt catcatcccc ct 22

<210> 689

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 689

cctgtacgtg acatcaaaca atgc 24

<210> 690

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 690

acactccctt gccattaaga agtc 24

<210> 691

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 691

tgctgcaaag caatgtctaa cttg 24

<210> 692

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 692

tgacctctct gaatatctgc agaaac 26

<210> 693

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 693

ctttcaaacg tttcagagca cagt 24

<210> 694

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 694

ccttttgaat tctgcttact gctga 25

<210> 695

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 695

gcaggtttcc aaatgtccct gt 22

<210> 696

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 696

gtgtttggct ccttggaagg tt 22

<210> 697

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 697

cctgaaactt acccataagt cttgaac 27

<210> 698

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 698

actcacctca caaatatctg tgtgt 25

<210> 699

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 699

atggaccagt tcatgcgcaa 20

<210> 700

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 700

gaagaagggc ggcatgtcta tt 22

<210> 701

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 701

atagttctgt gccaggcatc tc 22

<210> 702

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 702

caagttcccc acaggagaca aa 22

<210> 703

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 703

gaatgccaac tcagcctttt ctc 23

<210> 704

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 704

catcttggga ggaaaagaga gagtg 25

<210> 705

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 705

gtgaagctaa atgtttatta cccaaatgc 29

<210> 706

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 706

tgcaattact tggttttact tttctttcc 29

<210> 707

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 707

tgttgtctct cctctcctgt gt 22

<210> 708

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 708

catcgttgtc gtcgtcatca tc 22

<210> 709

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 709

caaacaactc tccttgatct atactttgag 30

<210> 710

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 710

actggtgagc ttcagcttgc 20

<210> 711

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 711

aagagtacta cgccaaggag gt 22

<210> 712

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 712

aaggacagga gaacgggaaa ag 22

<210> 713

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 713

agtcttctct gaaagcacaa tgga 24

<210> 714

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 714

caacaacaac aacaacaaca acaaatg 27

<210> 715

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 715

cctcccaaga tgttcagtat cccta 25

<210> 716

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 716

ttccctcccc cacctcatat g 21

<210> 717

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 717

gtgagggtgg tgaatcagct t 21

<210> 718

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 718

atggtaaccc aagaacagag gc 22

<210> 719

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 719

ccagtgcctc agatggcat 19

<210> 720

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 720

gagagcattc atgaagtttc ttttattgg 29

<210> 721

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 721

tcttgaggtc tggtaagggt ct 22

<210> 722

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 722

ccatcacaca gaggtgcttt tc 22

<210> 723

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 723

tggtttctgc tctgagagag ga 22

<210> 724

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 724

agcacactgt tcctgcatgt 20

<210> 725

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 725

tgatagggga cgtgggtcat c 21

<210> 726

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 726

tctcttcctc tccaggcctt g 21

<210> 727

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 727

aggcagtggt ggttctctct 20

<210> 728

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 728

ccaacctctc actgacatgt cc 22

<210> 729

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 729

agtaggcagg cagtagatgt tg 22

<210> 730

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 730

aggagattgt cactttcctt ccc 23

<210> 731

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 731

agggacctga ggttcattct ga 22

<210> 732

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 732

ctgtgctctg cattgtgaca tc 22

<210> 733

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 733

acaccccagc gagaatttgg 20

<210> 734

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 734

tggaagccat taggggacag a 21

Claims

1. A method for detecting familial thoracic aortic aneurysm and dissecting associated mutant gene based on high throughput sequencing technology comprises the steps of,

(1) collecting samples:

(2) the panel design:

the kit consists of 7 tubes of reagents,

(3) library construction:

(3.1) 1 st round of multiplex PCR reaction

(3.2) purification of the product by magnetic beads

(3.3) round 2 adaptor sequence PCR reaction

(3.4) round 2 magnetic bead purification

(3.5) library quantification

The multiplex PCR library obtained in 2ul step (3.4.10) was taken and used

(4) sequencing on machine

2. The method for detecting familial thoracic aortic aneurysm and dissection related mutant gene as claimed in claim 1, wherein the concentration of gDNA to be detected is diluted to the same concentration before step (3.1), and then transferred to PCR8 tube.

3. The method for detecting familial thoracic aortic aneurysm and dissection related mutant gene as claimed in claim 1 or 2, further comprising the step (3.6) of library quality detection after the step (3.5),

4. The method for detecting familial thoracic aortic aneurysm and dissection associated mutant gene of claim 1 or 2, further comprising the step of (6) mutational interpretation: explaining the relationship between variant SNPs and variant indels and diseases;

variants were classified into 5 classes by using the software REO-HIT according to the ACMG/AMP guidelines: pathogenic, suspected pathogenic, unknown meaning, suspected benign, benign.

5. The method for detecting familial thoracic aortic aneurysm and dissection related mutant genes as claimed in claim 4, further comprising the step of (7) providing a report, verifying the sequencing results of the above-detected pathopoiesia, suspected pathopoiesia, unknown significance and suspected benign by Sanger sequencing one by one, and providing the report.