CN105969889A - Use of SNP (single nucleotide polymorphism) locus of MUC3B gene - Google Patents
Use of SNP (single nucleotide polymorphism) locus of MUC3B gene Download PDFInfo
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- CN105969889A CN105969889A CN201610527752.2A CN201610527752A CN105969889A CN 105969889 A CN105969889 A CN 105969889A CN 201610527752 A CN201610527752 A CN 201610527752A CN 105969889 A CN105969889 A CN 105969889A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses use of an SNP (single nucleotide polymorphism) locus of an MUC3B gene. The SNP site is rs100389954. The SNP site rs100389954 of the MUC3B gene is found to be related with intracranial aneurysm for the first time, so that a kit for diagnosing risks of susceptibility to intracranial aneurysm by detecting a genotype of the SNP site is disclosed on such a basis. The kit is sensitive and specific, and can be used for screening susceptibility of a normal population to intracranial aneurysm to effectively achieve the effects of risk early warning and early diagnosis.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to purposes and the phase thereof of MUC3B gene SNP site
Close test kit.
Background technology
Intracranial aneurysm be entocranial artery be the pathological dilatation of pouch sample.Rupturing of intracranial aneurysm is
For one of serious nervous system disease.China belongs to the hotspot of intracranial aneurysm, and crowd's sickness rate reaches
0.1-4%.Although in last decade, the treatment of intracranial aneurysm has had significant progress, but the prognosis of patient is still
The most very poor, its fatality rate is up to 30-40%.
Research confirms, the formation of intracranial aneurysm, grows and rupture the pathophysiological change essentially consisting in its tumor wall.
Morphology based on blood vessel wall and histopathologic research disclose, and the formation of intracranial aneurysm is by outside different
Cause and the coefficient result of endogenous cause of ill.Intracranial aneurysm have in human body uniqueness histologic characteristics, they be by
Containing endotheliocyte and the theca interna of smooth muscle cell, containing interior elastic membrane and the middle film layer of smooth muscle cell and contain
The theca externa of loose connective tissue is formed.Intracranial aneurysm is usually located at the furcation of Wills arterial ring,
These crotches, there is interval in the continuity of middle film, and middle seam is broadening, and this is considered as aneurysmal sack
The susceptible position of bag development.The disappearance of outer elastic membrane, the organizational structure of extracellular matrix is disorderly, atheromatous plaque
Formed and the inflammatory infiltration of complement activation, the minimizing etc. of reticular fiber, add among stitch the impact of broadening, blood pressure and
Furcation answers shear force to have important effect in aneurysmal development.
Wear frequently with routine blood test, waist when checking intracranial aneurysm clinically, cerebrospinal fluid biochemical analysis, CT scan,
The means such as MRI scan, somatosensory evoked potential inspection, ultrasonic examination by Doppler's method, cerebral angiography, but adopt
When making a definite diagnosis by above-mentioned means, patient is often in the middle and advanced stage of intracranial aneurysm, and prognosis is poor, how to realize cranium
The early diagnosis of internal aneurysm has important function to the prognosis of patient.
Single nucleotide polymorphism (SNP) refers to cause in genomic level due to the variation of single core thuja acid
DNA sequence polymorphism, form includes the disappearance of single base, inserts, changes and transversion etc..It is said that in general,
In above-mentioned form, minimum a kind of allele frequency in colony is not less than 1%, but at certain in particular cases
(in cDNA) might be less that 1%.1 SNP has single core thuja acid on certain site of genome
Change, is mainly occurred by two kinds of forms: a kind of be single base conversion caused by, i.e. with a kind of cytosine
Another kind of pyrimidine or a kind of another kind of purine of purine displacement；Another form is exactly transversion, i.e. purine and pyrimidine
Exchange.Analyzing from principle, the base at sudden change can be C, G, A, T, and actually SNP is multiple
Raw between T and C.The essence performance in hereditary information of human inheritance's gene polynorphisms, 90% is above
Caused by SNP.Because its polypeptide contains much information, the most become the restriction fragment length polymorphism that continues
(RFLP) third generation genetic marker after mark and microsatellite i.e. Short tandem repeatSTR (STR) indicate.
SNP marker as at present the most potential molecular marker, because its quantity in genome is many, point
Cloth is wide and need not divide band according to clip size by DNA during gene analysis, can realize extensive height
Automatization, thus be more suitable for the detection analysis of substantial amounts, be widely used in biology, agronomy, medical science,
The various fields such as biological evolution.The research of SNP is that the early diagnosis of gene diagnosis, especially disease provides more
Foundation.SNP is owing to its distribution is wide, density is high and is desirably in such as cancer, diabetes, hypertension, melancholy
Its important function in the complex diseases such as strongly fragrant disease and asthma, these complex diseases are multiple hereditary variation site and environment
The coefficient result of the factor, owing to pathogenic factor is complicated, the gene dosage related to is many, it has also become disease in the world
The emphasis of ospc gene group research.Test the judgement that SNP is applied to tumor prognosis and susceptibility at present,
There is individual variation in the carcinogenic susceptibility of such as pulmonary carcinoma, i.e. the Gene Susceptibility of pulmonary carcinoma, and studying more is generation
Thank to enzyme gene pleiomorphism, such as human-cytochrome P450-CYP450 and myeloperoxidase (MPer) MPO etc..By passing
The Candidate Gene Study method of system, by being measured the gene relevant to intracranial aneurysm generating function, also sends out
Showed many tumor susceptibility genes, as endoglin gene, matrix metalloproteinase gene, IR Collagen Type VI gene,
Otl-antitrypsin gene, elastin gene, angiotensin converting enzyme gene, Apolipoprotein genes etc.,
Although being found that above-mentioned related gene, but the research to gene the most only rests on the experimental stage, clinical at present
On still there is no the diagnosis for intracranial aneurysm of the effective tumor susceptibility gene.
Summary of the invention
In order to make up the deficiencies in the prior art, it is an object of the invention to provide one and can be used for diagnosis of intracranial tremulous pulse
The SNP site of tumor susceptibility.Carry out Diagnosis of intracranial aneurysms by detection SNP genotype and there is early warning.
To achieve these goals, present invention employs following technical scheme:
Diagnosis of intracranial aneurysms prepared by the reagent of the SNP site that the invention provides detection MUC3B gene
Purposes in the test kit of susceptibility.
Further, the SNP site of described MUC3B gene is rs100389954.Wherein, MUC3B
When the genotype of SNP site rs100389954 is T/G, the susceptibility of intracranial aneurysm increases.
Further, described test kit is include for drawing of expanding nucleotide sequence including rs100389954 site
Thing pair.
Further, described amplimer sequence forward primer sequence as shown in SEQ ID NO.1, reverse primer
Sequence is as shown in SEQ ID NO.2.
Those skilled in the art can detect MUC3B base by any method of detection SNP site or technology
SNP locus of gene.Such as, Taqman method, mass spectrography, DNA microarray method, sequencing, micrometering sequence,
Hybridization, restriction fragment analysis, oligonucleotide connect detection, ApoE gene-HRM or connection
Conjunction should be in aforementioned manners.
The invention provides the test kit of a kind of Diagnosis of intracranial aneurysms susceptibility, described test kit includes detection
The reagent of MUC3B gene SNP site rs100389954.Wherein, the SNP site of MUC3B
When the genotype of rs100389954 is T/G, the susceptibility of intracranial aneurysm increases.This test kit of the present invention
The reagent using any technology for detection SNP site genotype known in the art can be included, as long as it can
Rs3890213 loci gene type in detection sample.
Further, the reagent of described detection MUC3B gene SNP site rs100389954 includes amplification
Rs100389954 site is at the primer pair of interior nucleotide sequence.
In a specific embodiment of the present invention, the forward primer sequence such as SEQ ID of described primer pair
Shown in NO.1, reverse primer sequences is as shown in SEQ ID NO.2.Those skilled in the art it will be appreciated that this
The primer of invention is not limited to this to primer.
Further, described test kit also include genome DNA extraction reagent, PCR reaction system reagent and
DHPLC related reagent.
Further, described PCR reaction system reagent includes dNTP, MgCl2, Taq archaeal dna polymerase, PCR
Reaction buffer, deionized water.
Further, described test kit also includes enzyme action general components, including reaction buffer, deionized water.
Present invention also offers test kit recited above in the diagnosis preparing Diagnosis of intracranial aneurysms susceptibility examination
Application in product.
Wherein, described product can be chip, array or test kit.Described chip includes solid phase carrier；With
And it being fixed on the oligonucleotide probe on described solid phase carrier, described oligonucleotide probe includes specifically corresponding
SNP site in MUC3B gene recited above.Described chip can be used for detection and includes MUC3B base
The SNP site of cause is interior multiple SNP site (such as relevant to intracranial aneurysm susceptibility one or many
One or more SNP site of individual gene), by detecting the susceptible gene of multiple intracranial aneurysm simultaneously,
It is greatly improved the accuracy rate of intracranial aneurysm.
In the present invention, the oligonucleotide probe for the SNP site of MUC3B gene can be DNA,
RNA, DNA-RNA chimera, PNA or other derivant.The length of described probe does not limit, only
Specific hybrid to be completed is specific binding with purpose nucleotide sequence, and any length can.Described probe
Length can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of described probe can grow to
60,80,100,150,300 base pairs or longer, the most whole gene.Due to different probe length
Different impacts, the length of described probe is had to be typically at least 14 bases hybridization efficiency, signal specificity
Right, the longest it is usually no more than 30 base pairs, with the length of purpose nucleotide sequence complementary with 15-25 alkali
Base is to most preferably.Described probe self-complementary sequences is most preferably less than 4 base pairs, in order to avoid affecting hybridization efficiency.
In the present invention, described solid phase carrier includes plastic, microparticle, membrane carrier etc..Described plastics system
Product can be by non-covalent or physical absorption is machine-processed combines with antibody or proteantigen, and the most frequently used plastic is
Small test tube, globule and the micro-reaction plate that polystyrene is made；Described microparticle is to be aggregated into by high polymer monomer
Microsphere or granule, its diameter mostly is micron, due to can be with the functional group of protein bound, easily and antibody
(antigen) forms chemical coupling, and binding capacity is big；Described membrane carrier includes nitrocellulose filter, glass fibre
The element microporous filter membrane such as film and nylon membrane.
In some embodiments in the present invention, described detection kit carries out entocranial artery in human experimenter
Using in the in vitro method of the susceptibility evaluation of tumor and/or diagnosis and/or prediction, wherein said test kit comprises use
In the proper implements carrying out the following step:
(1) at least one and the MUC3B in the genome DNA sample of described human experimenter are evaluated
The type of gene SNP site and/or level；
(2) by biomarkcr data evaluated in (1) of described human experimenter with healthy and/or
The biomarkcr data of ill people compares, and makes the wind of the intracranial aneurysm of described human experimenter
Danger is evaluated and/or is diagnosed and/or prediction.
In the present invention, term " genotype " refers to the allelic homogeneity being present in individuality or sample.
Typically, it refers to the individual genotype relevant to specific gene interested；In polyploid individual, it refers to
Individuality carries which kind of combination of alleles.
In certain embodiments, described test kit identify in human experimenter with intracranial aneurysm susceptibility phase
Using in the in vitro method of the SNP closed, described test kit comprises the proper implements for carrying out the following step:
(1) at least one SNP of the MUC3B gene in the nucleic acid samples of the human experimenter described in detection,
At least one SNP wherein said imply that the susceptibility of intracranial aneurysm；
(2) the SNP haplotype of the human experimenter described in qualification, wherein said SNP haplotype comprises institute
State the SNP of detection in (1).
In the present invention, when referring to detection kit, term " proper implements " refers to any for reaching
Any technological means of specified purpose.As the non-limitative example of this proper implements, examination can be enumerated
Agent and/or material and/or flow process and/or description and/or software etc..All test kits of the present invention can comprise
Suitable packaging and description are for using in method disclosed herein.
In the present invention, " nucleic acid " means DNA and RNA, and both of which is deposited with any possible configuration
, i.e. with the form of double-strand (ds) nucleic acid, or the form with (ss) nucleic acid, or with a combination thereof form (portion
Divide ds or ss).Such nucleic acid is corresponding to optionally comprising at least two of at least one nucleotide modified even
Continuous deoxyribonucleotide or ribonucleotide.
Nucleic acid can also be modified (such as D2EHDTPA, H-phosphate ester, alkyl in the level of the key of nucleotide part
Phosphate ester), the level of main chain is modified (such as α-oligonucleotide) or PNA or 2 '-O-alkylribose).
These modify in each of can combine generation, as long as there is at least one phosphate ester in nucleic acid.Described core
Acid can be natural or synthesis, oligonucleotide, polynucleotide, nucleic acid fragment, ribosomal RNA, letter
Make RNA, transfer RNA, the nucleic acid that obtained by enzymatic amplification technology.Described enzymatic amplification technology is e.g.
PCR (polymerase chain reaction) and RT-PCR derivative form, PCR (reparation chain reaction), 3SR are (certainly
Dynamic maintain sequence amplification), NASBA (relying on the amplification of nucleotide sequence), TMA (amplification of transcriptive intermediate).
In the present invention, term " primer " refers to naturally occurring oligonucleotide (such as restriction fragment) or closes
Becoming the oligonucleotide produced, it can act as the starting point of primer extension product synthesis, when being in suitable condition
(such as buffer, salinity and temperature and pH) and exist nucleotide and for nucleic acid polymerization reagent (such as
Rely on DNA or the polymerase of dependenc RNA) under conditions of, described primer extension product and nucleic acid chains (mould
Plate or target sequence) complementary.
Further, described detection rs100389954 site agents includes for SNP site rs100389954
Primer and/or probe.Described reagent also include in prior art it has been reported that Diagnosis of intracranial aneurysms easy
The primer of predisposing genes SNP site and/or probe.Detection by multiple SNP site of one or more genes
Primer and/or probe are placed in same reagent box by detecting multiple SNP site index Combining diagnosis intracranial dynamic
Within the situation of arteries and veins tumor susceptibility is also contained in protection scope of the present invention.
In the present invention, " array " or " microarray " is that hybridised arrays original paper is ordered in substrate, institute
State hybridised arrays original paper such as polynucleotide probe (such as oligonucleotide) or bonding agent (such as antibody).Institute
Stating substrate can be solid matrix, such as, and glass or silicon dioxide slide, pearl, fibre optics binding agent or half
Solid state substrate, such as nitrocellulose filter.Nucleotide sequence can be any row of DNA, RNA or therein
Row.Microarray can be from the oligonucleotide probe system being produced from the gene of known intracranial aneurysm susceptibility-special
Standby.Array can be containing the different oligonucleotide probe of each gene SNP site, and array also can contain comparison,
The one or more suitable comparison of Non-specific hybridization may also comprise on microchip.
Advantages of the present invention and beneficial effect:
Present invention firstly discovers that MUC3B gene SNP site rs100389954 is relevant with intracranial aneurysm.
A kind of test kit by detecting intracranial aneurysm susceptibility risk is proposed the most on this basis.The present invention's
Detection kit has good susceptiveness, stability and specificity.Normal population is carried out by the test kit of the present invention
The examination of intracranial aneurysm susceptibility, can effectively play the effect of Risk-warning, early diagnosis.
Detailed description of the invention
Further illustrating the present invention below in conjunction with specific embodiment, embodiments of the invention are only used for explaining this
Bright, it is not intended to limit protection scope of the present invention.
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1 and the screening in intracranial aneurysm susceptibility related gene SNPs site
1, sample and the collection of clinical data and storage
Collect patient 10 example of intracranial aneurysm SAH, the diagnosis basis American Stroke of the SAH of aneurism
The standard of the aneurism SAH treatment guidelines that association (ASA) American Heart Association (AHA) formulates is entered
OK, and make a definite diagnosis through head CT inspection, get rid of hypertensive cerebral hemorrhage, cerebrovascular malformation patient, and get rid of
Because of SAH caused by other reasons such as brainpan cerebral trauma, intracranial infection, tumor apoplexy or intracranial metastatic tumor.Intracranial moves
Arteries and veins tumor is all diagnosed as entocranial artery through clinical symptoms and full brain Digital subtraction and/or CT blood vessel imaging
Tumor.
Itemized record enter to organize the cause of disease of patient, pathogenic process and treatment process, and to its general clinical data such as year
Age, sex, the data information such as hypertension, diabetes, atherosclerosis, smoking history and family history is stepped on
Note, and set up the disease data data base of this research project.
2, the extraction of DNA
All enter to organize patient and gather the anticoagulation cirumferential blood specimen of 2ml, use phenol-chloroform method to extract genomic DNA.
DNA specimen is isolatable from peripheral blood leucocyte.
(1) the anticoagulation specimen of collection is placed on room temperature from-80 DEG C of taking-ups, after dissolving, blood is proceeded to another
Clean centrifuge tube, is centrifuged 15min in 5000g after covering tightly；
(2) the blood upper strata after being centrifuged discards, and stagnant blood cell adds appropriate lysis buffer (containing 10mM pH
It is Tris-HCl, 0.1M EDTA, 20g/ml Pancreatic RNase, the 0.5%SDS of 8.0), mixing, in 37 DEG C
Temperature bath 1h, adds E.C. 3.4.21.64 (100g/ml) and mixes rear 37 DEG C of temperature baths overnight；
(3) after temperature bath terminates, addition equal-volume fully mixes through the balance phenol (pH7.4) of the saturated mistake of Tris-HCl
After be centrifuged 15min in 8000g；
(4) upper water is moved to mutually another centrifuge tube and adds equal-volume phenol: chloroform (1:1), fully after mixing
8000g is centrifuged 15min；
(5) upper water is moved in another centrifuge tube mutually, adds the ammonium acetate solution (10M) of 10% volume,
Add the dehydrated alcohol of 2 times of volumes after mixing, after mixing, put into-20 DEG C.The DNA being settled out is through 75% ethanol
After washing twice, it is dissolved in appropriate TE buffer.Use spectrophotometric determination DNA concentration.
3, genome dna electrophoresis is identified
Reclaim the DNA fragmentation obtained and use whether agarose gel electrophoresis qualification degrades.
4, the mensuration of DNA concentration
The DNA extracted measures its concentration and purity by ultraviolet spectrophotometry.DNA should reach following
Standard: concentration is at more than 10ng/ul；OD260/280=1.8～2.0.DNA sample is placed in 1.5ml EP pipe
In be stored in-80 DEG C of ultra cold storage freezers.When carrying out the detection of SNP typing, sample is sub-packed in 96 after being encoded
In orifice plate.
5, the mensuration of intracranial aneurysm full-length genome
The nucleic acid samples obtained is delivered to Hua Da gene and carries out genome sequencing.
6, data analysis
(1) initial data is carried out Quality Control, remove belt lacing (adapter) reads pair；Remove single-ended order-checking
When the ratio of N (N represents cannot determine base information) is more than 10% in read reads pair；Remove single-ended
Low quality (less than 5) the base number contained in order-checking read exceedes reads during the 50% of this read length ratio
Right.
(2) by Quality Control comparing to the mankind with reference to gene, in group sequences h g18, gone out by algorithm predicts
SNP site.
(3) to chromosome position, ref base, the sudden change result of alt base carries out annotating and understanding and screen,
Remove the site of dbsnp, retain the site of missense and splicesite；Change by chromosome base
Obtain amino acid whose change, and mutational site is navigated on gene, carry out SIFT prediction and marking,
Description information etc. to gene location.
(4) sort according to gene redundancy number, choose the gene of at least 3 gene redundancy numbers, add up each
The number of times that gene occurs in different samples, builds thermal map, finds the site relevant to disease.
7, result
By data analysis, the site relevant to the susceptibility of intracranial aneurysm, the SNP of MUC3B are filtered out
Site, finds that rs100389954 site is sported G by T.
Embodiment 2 and the qualification in intracranial aneurysm susceptibility related gene SNPs site
1, clinical data
1.1 object of study
Intracranial aneurysm group: selected object of study is the trouble of intracranial aneurysm SAH that neurosurgery is collected
Person.Diagnosis basis American Stroke association (ASA) American Heart Association (AHA) of the SAH of aneurism
The standard of the aneurism SAH treatment guidelines formulated is carried out, and makes a definite diagnosis through head CT inspection, row
Except hypertensive cerebral hemorrhage, cerebrovascular malformation patient, and get rid of because of brainpan cerebral trauma, intracranial infection, tumor apoplexy or
SAH caused by other reason such as intracranial metastatic tumor.Intracranial aneurysm is all through clinical symptoms and full brain Digital Subtraction blood
Pipe imaging and/or CT blood vessel imaging are diagnosed as intracranial aneurysm.
Matched group: matched group experimenter is the crowd of the medical history without intracranial aneurysm of health check-up section of the court health check-up.All
Object of study all has available blood preparation.
1.2 pathology and clinical data
Itemized record enter to organize the cause of disease of patient, pathogenic process and treatment process, and to its general clinical data such as year
Age, sex, the data information such as hypertension, diabetes, atherosclerosis, smoking history and family history is stepped on
Note, and set up the disease data data base of this research project.
Two groups of Chinese populations being affinity-less relation, area and gender matched.This process follows the procedure and meets people
Body tests the ethical standard that committee formulates, and obtains the informed consent of experimenter.Experimenter is all quiet through elbow
Arteries and veins takes 10ml whole blood or collects remaining blood sample after clinical examination, and sodium citrate anticoagulant, for complete genome DNA
Extract and the detection of serological index.
Enter group standard:
(1) intracranial aneurysm patients；(2) there is complete clinical case history data, make a definite diagnosis including intracranial aneurysm
Age, sex, race, aneurysm size, whether rupture, Hunt-Hess classification, GCS classification and
Previously Drug therapy situation；(3) all patients all can obtain complete Follow-up Data.
The most totally 148 example intracranial aneurysm patients include intracranial aneurysm group, 126 example non-intracranial aneurysm crowd in
Include matched group in.Wherein, case group man's 70 examples, female 78 example, the age is 20～78 years old, average 48 years old.
Male 60 examples in matched group, female 66 example, age 11～79 years old, average 43.5 years old.
2, DNA extraction step is with embodiment 1
3, the reaction of the PCR in the region of SNP mesh
3.1 design of primers and synthesis
According to determining the SNP site of MUC3B, centered by SNP site, intercept more than 100bp length
Gene order, utilizes Primer Premier5.0 software design synthetic primer, and primer sequence is as follows:
Forward primer: 5'-TCTTCTGCCAGCATAACT-3'(SEQ ID NO.1)
Reverse primer: 5'-GGACAGGTGACCATTTCG-3'(SEQ ID NO.2)
3.2PCR reaction
Configure 25 μ l PCR reaction systems, contain 1.0mM MgCl respectively2, each 120 μMs of 4 kinds of dNTP,
Various primers are respectively 0.12 μM, TaqDNA po1ymerase 1.6U, template DNA 20ng.PCR bar
Part is: denaturation 94 DEG C, 5min, (95 DEG C of 30s, 54-55 DEG C of 45s, 72 DEG C of 45s) × 35 circulations,
72 DEG C, 5min.
The PCR primer of amplification uses gel imaging system after detecting with 1.5% agarose gel electrophoresis (GelRed)
Carry out preliminary identification.
4, statistical procedures and analysis
During case-control is analyzed, each loci polymorphism and intracranial aneurysm are susceptible to suffer from risk correlations with or without significance
Difference is via χ2Detection judges.P ＜ 0.05 thinks there is significant difference.
5, experimental result
Result is as shown in table 1, in matched group rs100389954 loci gene type frequency in intracranial aneurysm case group and
There is between matched group significant difference (P < 0.05), and then analysis draws rs100389954 loci gene type
T/G is relevant to the susceptibility of intracranial aneurysm, and when this SNP site is sported G by T, patient has and suffers from cranium
The risk of internal aneurysm.
The X 2 test of table 1rs100389954 loci gene type
Embodiment 3 intracranial aneurysm diagnosis of susceptibility test kit
According to the dependency of MUCB3 gene SNP site rs100389954 Yu intracranial aneurysm susceptibility,
Can be carried out the susceptibility of Diagnosis of intracranial aneurysms by the genotype detecting this SNP site, the present invention carries accordingly
Supply a kind of test kit based on diagnosis osteosarcoma susceptibility, this diagnostic kit has also included DNA extracting examination
Agent and PCR reaction system reagent, wherein expand MUCB3 gene SNP site in PCR reaction system
The primer sequence of rs100389954, as shown in SEQ ID NO.1 and SEQ ID NO.2, carries out PCR reaction
Reagent also include such as dNTP, MgCl2, Taq archaeal dna polymerase, PCR reaction buffer, deionized water.
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It is right to it should be pointed out that,
For those of ordinary skill in the art, under the premise without departing from the principles of the invention, it is also possible to the present invention
Carrying out some improvement and modification, these improve and modify also by the protection domain falling into the claims in the present invention.
Claims (10)
1. the reagent of the SNP site of detection MUC3B gene is in the examination preparing Diagnosis of intracranial aneurysms susceptibility
Purposes in agent box.
Purposes the most according to claim 1, it is characterised in that the SNP position of described MUC3B gene
Point is rs100389954.
Purposes the most according to claim 2, it is characterised in that described test kit includes for expanding
Rs100389954 site is at the primer pair of interior nucleotide sequence.
Apply the most according to claim 3, it is characterised in that described amplimer sequence forward primer sequence
Row are as shown in SEQ ID NO.1, and reverse primer sequences is as shown in SEQ ID NO.2.
5. the test kit of a Diagnosis of intracranial aneurysms susceptibility, it is characterised in that described test kit includes inspection
Survey the reagent of MUC3B gene SNP site rs100389954.
Test kit the most according to claim 5, it is characterised in that described detection MUC3B gene SNP
The primer pair of the reagent of site rs100389954 nucleotide sequence including amplification rs100389954 site.
Test kit the most according to claim 6, it is characterised in that the forward primer sequence of described primer pair
Row are as shown in SEQ ID NO.1, and reverse primer sequences is as shown in SEQ ID NO.2.
Test kit the most according to claim 5, it is characterised in that described test kit also includes genome
DNA extraction agent, PCR reaction system reagent.
Test kit the most according to claim 8, it is characterised in that described PCR reaction system reagent bag
Include dNTP, MgCl2, Taq archaeal dna polymerase, PCR reaction buffer, deionized water.
10. the test kit described in any one of claim 5-9 is preparing examining of Diagnosis of intracranial aneurysms susceptibility examination
Application in pregnancy ceased product.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949965A (en) * | 2018-08-22 | 2018-12-07 | 中国人民解放军军事科学院军事医学研究院 | Application of the SNP marker in type-2 diabetes mellitus diagnosis |
| KR102158714B1 (en) * | 2019-04-03 | 2020-09-22 | 한림대학교 산학협력단 | SNP marker for diagnosis of intracranial aneurysm comprising SNP of TCF24 gene |
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|---|---|---|---|---|
| WO2014193999A2 (en) * | 2013-05-28 | 2014-12-04 | Caris Science, Inc. | Biomarker methods and compositions |
-
2016
- 2016-07-06 CN CN201610527752.2A patent/CN105969889B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014193999A2 (en) * | 2013-05-28 | 2014-12-04 | Caris Science, Inc. | Biomarker methods and compositions |
Non-Patent Citations (2)
| Title |
|---|
| ANONYMOUS: "NC_018918.2", 《GENBANK》 * |
| PRATT WS ET AL: "Multiple transcripts of MUC3：evidence for two genes,MUC3A and MUC3B", 《BIOCHEM BIOPHYS RES COMMUN》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949965A (en) * | 2018-08-22 | 2018-12-07 | 中国人民解放军军事科学院军事医学研究院 | Application of the SNP marker in type-2 diabetes mellitus diagnosis |
| CN108949965B (en) * | 2018-08-22 | 2019-09-03 | 中国人民解放军军事科学院军事医学研究院 | Application of the SNP marker in type-2 diabetes mellitus diagnosis |
| KR102158714B1 (en) * | 2019-04-03 | 2020-09-22 | 한림대학교 산학협력단 | SNP marker for diagnosis of intracranial aneurysm comprising SNP of TCF24 gene |
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