The application of the SNP site of CLIP3 gene
Technical field
The present invention relates to biomedicines to lead detection technique field, and in particular to CLIP3 relevant to dissection of aorta disease
The application of the SNP site of gene.
Background technique
Dissection of aorta (Aortic Dissection, AD) means aorta under a series of effect of external factors (such as height
The factors such as blood pressure, wound), based on aorta wall itself presence or absence of lesion, there is cut in aortic tunica intima, and blood is autonomous
Endarterium cut invades the middle layer of aorta wall, and medial constantly tears, is longitudinally separated, causes to show a kind of active
State vessel lumen true and false two chamber and deposited.Dissection of aorta onset is hurried, quickly grows, and prognosis is dangerous.Although current is various
Treatment means are continuously improved, but the morbidity and mortality of AD are still high, and if without appropriate and timely
Treatment, the AD death rate is high.It has been reported that there are about nearly 20% AD patients before reaching hospital is dead, and 30% patient
It is dead during hospitalization.Effective pharmaceutical intervention means clinically are lacked to AD at present, are mainly carried out using surgical operation
Vascular replacement, intervention operation carry out aorta intra-cavity stent implantation and the two connected applications, but operation risk is big, expense
Height, and recurrence rate and re-treatm ent rate at a specified future date is still higher.Along with the progress of Imaging Technology and Medical Devices condition, AD
Disease detection rate is in rising trend, and the research of generation and pathogenesis for AD is also deepening continuously.AD generting machanism and something lost
It is closely related to pass.AD correlation tumor susceptibility gene is current AD genetics research hot spot, relevant to AD easily from gene angle analysis
It is meaningful to feel gene.
The development of plan completion and high throughput sequencing technologies, the research work of medical domain are sequenced along with human genome
Person to human genome have deeper into understanding, function and regulated and control network to disease correlative heritability, related gene
Understanding is also gradually deepened, and high-flux sequence is used widely.Full sequencing of extron group (Whole Exome Sequencing,
WES) technology is to capture entire exon 1 domain dna using exon trapping kit, then carries out high-flux sequence, is current
Highly developed a part in genomics research.Over the past two years, started using the document report of WES technical research complex disease
Occur, shows the huge advantage of the technology.The scholars such as Ziganshin BA carry out 102 aneurysm of thoracic aorta and AD patient
WES analysis, 21.6% patient is there are the mutation of one or more other genes for discovery, 3.9% patient there are FBN1, COL5A1,
The detrimental mutation of MYLK, FLNA gene.Janice L.Farlow etc. has carried out WES, discovery 68 to 45 intracranial aneurysm patients
There is variation in a gene, the expression of TMEM132B gene is dramatically different in patient and collator (44VS 16).Show common
Rare variation can be identified in complex disease by high-flux sequence.Also, there is increasing evidence that rare variation is to normal
See disease susceptibility have in arrive intensity effects, significant portion of genetic defect can be explained.It is rare mutation (MAF < 1% or
5%) limitation, and expense, time cost are higher.Whole-genome association (genome wide association
Studies, GWAS) can only identify common variation (common variants, CV:MAF > 5%), have ignored it is certain it is unknown with
The relevant function candidate gene of AD morbidity.WES compared to genome sequencing (Whole Genome Sequencing, WGS),
GWAS, which has, is sequenced the high depth in genome encoding region, in addition to the repetition of the missing of large fragment and SNP, moreover it is possible to find that low frequency is prominent
Become, rare mutation, identification range is wider, has low cost, efficient advantage, is that searching diseases predisposing gene or site are special
It is one of the new tumor susceptibility gene of discovery or the most common method in site, can make up GWAS cannot screen the defect of unknown candidate gene.
AD vascular risk is high, it is not easy to which early detection, the genetic diagnosis positive rate based on known Disease-causing gene is limited, difficult
In doing early stage risk assessment, it would be highly desirable to find new molecular marker.
Summary of the invention
To solve the above-mentioned problems, the purpose of the present invention is to provide one kind is screened and master based on full exon sequencing technologies
The relevant SNP site of artery dissection disease, the SNP site include following dissection of aorta neurological susceptibility SNP site: CLIP3:
NM_001199570:exon1:c.32C>T:p.P11L。
Further, the present invention provides the SNP sites in the reagent of preparation detection dissection of aorta disease
Using.
Further, the present invention also provides the SNP sites in preparation prediction dissection of aorta disease early diagnosis
Application in kit.
Preferably, the kit includes detection dissection of aorta neurological susceptibility SNP site CLIP3:NM_001199570:
The reagent whether exon1:c.32C > T:p.P11L mutates.
Preferably, the kit can be to use any technology known in the art to detect the reagent of SNP, as long as its energy
Enough detecting susceptible SNP site CLIP3:NM_001199570:exon1:c.32C > T:p.P11L in sample whether there is mutation.Its
It include but is not limited to each embodiment being set forth below.
In the first embodiment, the kit is including the use of susceptible SNP site CLIP3:NM_ in PCR sequencing PCR detection sample
Reagent existing for the T allele of the site 001199570:exon1:c.32C > T:p.P11L.PCR sequencing PCR is known in the field
Technology, the reagents such as required primer, those of ordinary skill in the art can voluntarily select (referring to ABI, Beckman as needed
Equal companies' sequenator explanation used in connection with), details are not described herein.Using the kit, sample can directly be measured by PCR sequencing PCR
The sequence of susceptible SNP site CLIP3:NM_001199570:exon1:c.32C > T:p.P11L in this, to judge whether it takes
Variation with corresponding site allele, and then judge the neurological susceptibility of its dissection of aorta.
In this second embodiment, the kit is including the use of susceptible in Taqman probe SNP detection method detection sample
The reagent of SNP site CLIP3:NM_001199570:exon1:c.32C > T:p.P11L genotype.Wherein Taqman probe used
For the probe designed for susceptible SNP site CLIP3:NM_001199570:ex on1:c.32C > T:p.P11L, which can
With the probe for using Reagent Company to provide;Software designed, designed can also be used, such as the Beacon of PREMIER Biosoft company
Designer 7.5。
In the third embodiment, the kit is using susceptible in PCR- Single strand conformation polymorphism detection sample
The kit of SNP site CLIP3:NM_001199570:exon1:c.32C > T:p.P11L genotype.It include using in the kit
In the primer for expanding susceptible SNP site CLIP3:NM_001199570:exon1:c.32C > T:p.P11L, PCR reagent, control sample
Reagent needed for the electrophoresis of this and detection conformation.The preferred native polyacrylamide gel electrophoresis of electrophoresis.It is wrapped in check sample
Include the homozygous negative control sample of susceptible SNP site CLIP3:NM_001199570:exon1:c.32C > T:p.P11L CC and
At least one of homozygous positive control of site TT, additionally may include can not also include that heterozygote is accordingly miscellaneous
The check sample of zygote.It is preferred that simultaneously including above-mentioned three classes check sample.The amplification of sample to be tested amplified production and check sample
Product while electrophoresis, compare its electrophoresis result it can be concluded that whether carrying the detection knot of corresponding allelic variation in sample to be tested
Fruit.
Further, the present invention provides the SNP sites in preparation diagnosis dissection of aorta disease device
Using.
Preferably, the diagnostic device is sequence testing chip.
Still further, the present invention provides a kind of method for screening SNP site relevant to dissection of aorta disease, it is described
Method the following steps are included:
(1) DNA of the peripheral blood sample of dissection of aorta patient and normal control is extracted;
(2) DNA is subjected to ultrasonic fragmentation, interrupts fragment ends filling-in, 3 ' ends plus A, connects adaptor, select 350~
Segment between 400bp prepares full-length genome library;
(3) full exon detection is carried out using GenCap liquid phase capture target gene technology;It is carried out with high-flux sequence instrument
Both-end sequencing, reads a length of 100bp;
(4) after exon sequencing, conventional filtration analysis is carried out, is to be with reference to genome version with hg19, dbSNP (v147)
Screening and filtering standard;Pathogenic sites, normal person's frequency are left 5% hereinafter, and there are a same sense mutation pathogenic sites reported in the literature;
In order to more accurately screen disease related locus, it is complete to filter out SIFT, Polyphen2, MutationTaster, GERP++ prediction
For benign SNP;Filter out Mutcount > 5, MutRatio > 30%, the Indel of MAF > 1% in normal data library;
(5) the exclusion work in false positive site is carried out, first is that by IGV software to the bam after de-redundancy duplicates
File (rmdup.sorted.bam) carries out checking verifying, and catastrophe is incongruent to be considered as false positive site;Pass through simultaneously
Samtools software checks bam file.Then, respectively with the gene (a) of mutation, mutation site (b) as
Maker carries out rare mutation load (RVB) analysis, the sample number of the gene mutation and calculating in statistics case group and control group
OR value, OR>1, P<0.01.As a result, it has been found that thering are multiple mutational sites and AD to have statistical correlations;
(6) depth is carried out to sequencing result by gene function and the analysis of the annotation of related pathways and literature search data
Analysis, final inventor filter out dissection of aorta neurological susceptibility SNP site: CLIP3:NM_ from 48 mutational sites
001199570:exon1:c.32C>T:p.P11L。
The invention has the advantages that:
The present invention is pre- in dissection of aorta early stage by having found the susceptible SNP site of a dissection of aorta and having studied it
The application prospect of survey, the relevant gene loci of dissection of aorta provided by the invention may be the early stage biology mark of dissection of aorta
Will object, for the genetic molecule mechanism of further research dissection of aorta, the drug target for exploring dissection of aorta early prevention and treatment is mentioned
New direction is supplied.
Detailed description of the invention
It includes two or more base in access that 48 site GO path analysis (MolecuLar Function) of Fig. 1, which are chosen,
Cause, p value are less than or equal to 0.05 access, to enrichment score mapping;
It includes two or more base in access that 48 site GO path analysis (biological process) of Fig. 2, which are chosen,
Cause, p value are less than or equal to 0.05 access, to enrichment score mapping.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means.
Term is explained as follows in the present invention:
GO (Gene Ontology): the comprehensive library of gene function is described, molecular function (Molecular can be divided into
Function, MF), bioprocess (biological process, BP) and cell composition (cellular component,
CC) three parts.GO enrichment is significant enrichment less than 0.05 with p.
1 sample collection of embodiment
It is made a definite diagnosis from January, 2017 in April, 2018 in Shenzhen's sun yat-sen angiocardiopathy hospital by aorta CTA
59 patients of Stanford A type dissection of aorta collect whole blood 2mL and 590 normal control samples and are sequenced from Mai Jinuo
The database of company's retrieval.The informed consent of patient has been obtained, and has been audited by Ethics Committee.
Sample process: EDTA anticoagulated whole blood is mixed in 1:1 ratio with Trizol, mixes well and is placed on 1.8mL cell
In cryopreservation tube, the refrigerator preservation that 30s is placed on -80 DEG C is cooled down rapidly in liquid nitrogen.
Embodiment 2 extracts blood sample DNA
(1) 1mL is passed through and is added in the blood of EDTA (0.01M, China, Huamei Bioengineer) anticoagulation
1mLCL cell pyrolysis liquid is gently mixed by inversion 6 times, is centrifuged 5min, reject supernatant with the revolving speed of 3600rpm;
(2) it as pouring into 1mLCL cell pyrolysis liquid in centrifuge tube again, is gently mixed by inversion 6 times, with turning for 3600rpm
Speed centrifugation 5min, reject supernatant;Under the premise of ensuring that precipitating is retained in pipe, centrifuge tube is upside down on clean blotting paper
Stand 2min;
(3) mixed liquor of Proteinase K and buffer FG is configured;
(4) mixed liquor of 500 μ L Proteinase Ks and buffer FG is added, is mixed immediately to solution without agglomerate;
(5) 65 DEG C of water-bath 30min, are during which mixed by inversion for several times;
(6) 1mL isopropanol is added, is mixed by inversion immediately to there is tufted or Filamentous genomic DNA;
(7) 8min, reject supernatant are centrifuged with the revolving speed of 3600rpm;It, will be under the premise of ensuring to precipitate and being retained in pipe
Heart pipe is upside down on clean blotting paper and stands 2min;
(8) 1mL70% ethyl alcohol is added, vibrates 5sec, is centrifuged 3min, reject supernatant with the revolving speed of 3600rpm;
(9) step (8) are repeated;
(10) under the premise of ensuring that precipitating is retained in pipe, centrifuge tube is upside down on clean blotting paper and stands 5min;
(11) under normal temperature state, be air-dried gene group DNA be precipitated to all liq volatilize completely (at least 5min) oscillation
5sec is centrifuged 3min, reject supernatant with the revolving speed of 3600rpm;
(12) 200 μ LTB buffers are added, low speed vortex vibrates 5sec, during which 65 DEG C of heating water bath 1h flick hydrotropy number
It is secondary;
(13) extracted genomic DNA concentration and purity are identified using NanoDrop ND8000 (THERMO, USA).It is dense
Spend > 30ng/ μ L, 1.8 < OD260/OD280< 2.0, and the genomic DNA sample of 3 μ g of total amount > is considered as qualification, is stored in -80
DEG C refrigerator it is spare;
(14) on the preliminary quantitative basis of DNA, further using agarose gel electrophoresis detection (gum concentration: 0.8%, electricity
Pressure: 120V, time: 20min) accurate quantification DNA.Occur illustrating that DNA may have there are miscellaneous band except weak band or master tape after electrophoresis
Signs of degradation or there are impurity illustrates that the DNA mass extracted is higher if occurring a strong band after electrophoresis.Full exon is surveyed
Sequence requires DNA concentration >=50ng/ μ L, and total amount is not less than 5 μ g, OD260/280Between 1.8~2.0, OD260/2302.0 or so, sample
This is polluted without RNA, no degradation or slight degradation.
The sequencing of the full exon of embodiment 3
1, DNA library constructs
3 μ g DNA are subjected to ultrasonic fragmentation, fragment ends filling-in, 3 ' ends plus A is interrupted, connects adaptor, select 350
Segment between~400bp prepares full-length genome library.Library sample uses Agilent2100 biological analyser (Agilent section
Skill company, the U.S.) carry out Quality Control.
2, target area capture sequencing
Full exon detection is carried out using GenCap liquid phase capture target gene technology (Beijing Mai Jinuo company).By 1 μ g
DNA library is mixed with BL buffer and probe, and 95 DEG C of heating 7min, the HY that 65 DEG C of 23 μ L of heating 2min addition are preheated to 65 DEG C are slow
Fliud flushing, 65 DEG C of hybridization 22h.Cleaning 50 μ L MyOne magnetic beads with 500 μ L 1X combination buffers, (U.S. Life Technology is public
Department) 3 times, it is resuspended in 80 μ L 1X combination buffers.64 μ L 2X combination buffers are added into hybridization mixture, be transferred to containing
In the test tube of 80 μ L MyOne magnetic beads.Rotation mixes 1h.Magnetic bead 15min is cleaned with WB1 buffer room temperature, with WB3 buffer 65
DEG C cleaning 3 times, each 15min.Then it is eluted with the combining DNA of elution buffer.The DNA of elution carries out PCR reaction,
Reaction condition is as follows: 98 DEG C of initial denaturation 30s;98 DEG C of denaturation 25s, 65 DEG C of annealing 30s, 72 DEG C of extension 30s carry out 15 altogether and follow
Ring;Last 72 DEG C of extensions 5min.PCR is purified with SPRI beads (U.S. Beckman Co μ Lter) according to product description to produce
Object.The library of enrichment carries out both-end sequencing with 2000 sequenator of illumina HiSeq, reads a length of 100bp.
3, data are analyzed
Obtained primitive sequencer sequence is sequenced, the inside, can be to follow-up point containing belt lacing, low-quality reads
Analysis interferes.In order to guarantee information analysis quality, initial data is filtered with Cutadapt software, connector sequence will be removed
Column and the sequencing data of low quality base are compared with Burrows-Wheeler Aligner (BWA) arrives human genome (version
GRCh37/hg19 on), and redundancy is removed with Picard tool.The depth of target area is calculated with coverageBed tool.It is logical
GATK software is crossed indel location proximate is compared again to improve the quality of sequence.GATK HaplotypeCaller inspection
SNP and indel abrupt information are surveyed, and mutation is annotated with ANNOVAR, annotations database includes dbSNP147,1000Genomes
project,Exome sequencing project(ESP6500),Inhouse database(MyGenostics),
gnomAD_genome_EAS.Online Mendelian Inheritance in Man(OMIM)and Human Gene
Mutation Database(HGMD Professional 2016.10).Pathogenic forecast database includes SIFT,
PolyPhen-2and MutationTaster, and divided by ACMG standard guide to pathogenic.
4, variation filtering
In order to find out potential disease cause mutation, need to be filtered data screening.Mainly for exon 1 and can
Become the mutation of share zone, step 1: leaving the disease cause mutation in pathogenic analysis (pathogenic_analysis)
(pathogenic) site.
Second step (filter screen criteria): screening mutating alkali yl sequencing number be greater than 5, the frequency of mutation be more than or equal to 30% with
And retain in five normal person's mutation databases of 1000g2015apr, ESP6500si, Inhouse, ExAC_ALL, ExAC_EAS
Do not occur or the site less than 5%;The same sense mutation site in data set is removed, in addition has mutational site reported in the literature to stay
Under.
5, it statisticallys analyze
Rare mutation load Analysis is carried out to above-mentioned filtered mutation result, selects 590 number of cases of Mai Jinuo according to as right
In the same old way this, using the Metabin analysis OR value and 95% confidence interval in R language, and with Mantel-Haenszel ' s method into
Row statistical check, p value think significant less than 0.01.
6, result
After exon sequencing, conventional filtration analysis is carried out, screening and filtering standard: being with reference to base with hg19, dbSNP (v147)
Because of a group version.The pathogenic site (pathogenic), normal person's frequency are left 5% hereinafter, and having same sense mutation reported in the literature
Pathogenic sites obtain 59351 point mutation.In order to more accurately screen disease related locus, further filter out SIFT,
Polyphen2, MutationTaster, GERP++ prediction are all benign SNP;Filter out Mutcount > 5, MutRatio >
30%, the Indel of MAF > 1% in normal data library;29979 point mutation are obtained through above-mentioned screening.
Later, inventor has carried out the exclusion work in false positive site, first is that by IGV software to de-redundancy
Bam file (rmdup.sorted.bam) after duplicates carries out checking verifying, and catastrophe is incongruent to be considered as false sun
Property site;Bam file is checked by samtools software simultaneously.Then, respectively with the gene (a) of mutation, mutation
Site (b) carries out rare mutation load (RVB) analysis as maker, the sample of the gene mutation in statistics case group and control group
This number simultaneously calculates OR value, OR>1, P<0.01.As a result, it has been found that thering are 48 mutational sites and AD to have statistical correlations.
Further, inventor carries out the annotation of gene function and related pathways to 48 mutational sites filtered out.
The mapping that count is more than or equal to 2 is chosen in the access annotation of gene where 48 mutational sites, as shown in Figure 1, 2,
In BP analysis, gene significant enrichment is in redox bioprocess where these sites.
It is found in the selection result in 48 mutational sites:
CLIP3:NM_001199570:exon1:c.32C > T:p.P11L may exist with AD to be contacted.
Inventor combines gene function and the analysis of the annotation of related pathways and literature search data to carry out sequencing result
Depth analysis, final inventor filter out the gene C LIP3, the mutation SNP that the gene carries to mutate in case group
Site is as shown in table 1.The mutational site CLIP3:NM_001199570:exon1:c.32C > T:p.P11L may be the pass for leading to AD
Keyness molecule can be used as AD new biomarkers, to instruct its clinical early intervention and targeted therapy to provide important reason
By foundation.
1 dissection of aorta SNP site information of table
Note: Gene, Gene Name;Chr, chromosome;The position Pos, SNP;AA_change, amino acid change.
The identification of embodiment 4 and dissection of aorta neurological susceptibility related gene SNP site
1. research object
Dissection of aorta group: selected object is being made a definite diagnosis by aorta CTA for Shenzhen's sun yat-sen angiocardiopathy hospital
25 patients of Stanford A type dissection of aorta collect blood sample.
Control group: control group subject is Shenzhen's sun yat-sen angiocardiopathy hospital medical center without dissection of aorta and disease
The crowd of history, totally 30 subjects, collect blood sample.
2. pathology and clinical data
Group disease is recorded into because, pathogenic process and treatment pass through, and to general clinical data such as age, gender, medical history
Database is established etc. registration is carried out.
Two groups are affinity-less relation, area and the matched Chinese population of gender, this process, which follows the procedure, meets human body examination
The ethical standard of committee's formulation is tested, and obtains the informed consent of subject.Subject takes blood 10ml whole blood by ulnar vein
Or remaining blood sample after clinical examination is collected, sodium citrate is anticoagulant, extracts the detection with serological index for complete genome DNA.
3. test sample is extracted with embodiment 2
4. the PCR comprising SNP site reacts
4.1 design primer
Using Primer Premier5.0 software design synthetic primer, primer sequence is as follows:
Forward primer: 5 '-TAAGACAGATCCTGCCCCGA-3 ' (SEQ ID NO.1)
Reverse primer: 5 '-TCACGATCGTTCACATGGCA-3 ' (SEQ ID NO.2)
Amplified production length: 336bp (SEQ ID NO.3)
4.2PCR reaction
PCR is shown in reaction system such as table 2.PCR amplification program are as follows: 95 DEG C of initial denaturation 10min, 94 DEG C of denaturation 15s, 61 DEG C are moved back
Fiery 15s, 72 DEG C of extension 30s carry out 30 circulations, and last 72 DEG C of extensions 30min is saved in 4 DEG C, need -20 DEG C of placement cold overnight
Freeze.
2 reaction system of table
5. sequencing
After PCR amplification, 5 μ L amplified productions, 1% agarose gel electrophoresis are taken, electrophoresis 30min dyes 20min, so
Gel piece is placed in gel imager afterwards and is observed, according to the clip size situation for comparing Marker, tentatively judges amplified fragments
It is whether correct.And then satisfactory amplified production is purified: using Mag-BindOligonucleotidePurific
AtionKit kit, and operated by kit requirement.Loading sequencing: ABI company is used
BigDye3.1SequencingKit kit, and operated by kit requirement;It is carried out with 3730 type sequenator of ABI company
Sequencing.
6. interpretation of result
By Chromas sequence analysis software, sequencing result is compared with standard sequence, finds the site S NP, led to
Cross the type of base at analysis SNP site, so that it may obtain the genotype of SNP site.As the result is shown: in discovery CLIP3 gene
The 27th of the sequence as shown in SEQ ID NO.3 has found a SNP site, and there are allele Cs and allele in the site
T constitutes three kinds of genotype, C T, CC and TT.It is sequenced to obtain the nucleotide of the segment of 336bp in 25 dissection of aorta patients
It is CT, TT in the 27th genotype in sequence (as shown in SEQ ID NO.3);And it compares and surveys in 30 health volunteers
Sequence obtains in the nucleotide sequence (as shown in SEQ ID NO.3) of the segment of 336bp, in the gene of the 27th allele
Type is CC;Confirm the susceptible genotype for being judged as dissection of aorta when the site is CT, T T genotype, which is CC base
Because being judged as the non-susceptible genotype of dissection of aorta when type, to further confirm that the CLIP3:NM_001199570:ex
The SNP site of on1:c.32C > T:p.P11L can be used for the auxiliary such as detection, treatment, diagnosis, the prognosis evaluation of dissection of aorta and examine
It is disconnected.
Embodiment 5 is used to predict the production of dissection of aorta disease early diagnosis kit
Assemble the kit of the present invention for dissection of aorta:
The kit includes whether the detection site CLIP3:NM_001199570:exon1:c.32C > T:p.P11L occurs
The reagent of mutation.
The reagent includes for expanding the special of the site CLIP3:NM_001199570:exon1:c.32C > T:p.P11L
Property primer, which can be designed using software, such as use Primer5, Oligo6;The reagent can also have accordingly
Common agents needed for round pcr, such as: dNTPs, MgCl2, distilled water, Taq enzyme etc., these common agents are all this field skills
Known to art personnel, in addition it can have standard items and control (such as determining standard items and the blank control of genotype).This examination
The value of agent box is only to need peripheral blood without other tissue samples, by most simplifying and the detection of special primer pair
SNP, then auxiliary judgment dissection of aorta is composed by SNP, it is not only stable, easy to detect and accurate, greatly improve medical diagnosis on disease
Sensibility and specificity, therefore this kit is put into and is practiced, it can help to instruct diagnosis and more effective individualized treatment.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Pu Zuo
<120>application of the SNP site of CLIP3 gene
<130> P18073
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
taagacagat cctgccccga 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tcacgatcgt tcacatggca 20
<210> 3
<211> 336
<212> DNA
<213> Homo sapiens
<400> 3
taagacagat cctgccccga tggccccgcc accccgagga gaggaggaag aagaggagga 60
ggaggatgaa cccgtccccg aggcccccag ccccacccag gagcgccggc agaagcctgt 120
tgtgcacccc tcggcacctg cccccctccc taaggactac gctttcacct tcttcgatcc 180
caatgacccg gcgtgccagg agatcctgtt tgaccctcag accaccatcc ccgagctgtt 240
tgccattgtg cgccagtggg tgccccaagt ccagcacaag atagacgtca tcggcaatga 300
gattctgcgc cgaggctgcc atgtgaacga tcgtga 336