CN106399304B - A kind of SNP marker relevant to breast cancer - Google Patents

A kind of SNP marker relevant to breast cancer Download PDF

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CN106399304B
CN106399304B CN201611027831.3A CN201611027831A CN106399304B CN 106399304 B CN106399304 B CN 106399304B CN 201611027831 A CN201611027831 A CN 201611027831A CN 106399304 B CN106399304 B CN 106399304B
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breast cancer
snp
diagnosis
site
snp marker
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CN106399304A (en
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何劲松
陈伟财
欧阳依雯
毛有胜
罗雪莹
潘悦
刘宝儿
李锋
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Shenzhen bike Biotechnology Co., Ltd.
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Shenzhen Second Peoples Hospital
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers

Abstract

The invention discloses a kind of biomarkers relevant to breast cancer, the biomarker includes positioned at the gene C 12orf45 nucleotide sequence SNP site that the 8187th bit base is G from 5 ' ends, and the site is C12orf45:NM_152318:exon4:c.C368G.Present invention research SNP illustrates influence of the SNP for breast cancer progression, discloses its diagnostic value in the application prospect of Computer-aided Diagnosis of Breast Cancer.Therefore, the present invention passes through the development and application of SNP biomarker and diagnostic kit, it may make the diagnosis of breast cancer more convenient and easy, conditions of patients is quick and precisely grasped for clinician, it lays the foundation for clinical therapeutic efficacy evaluation, and provide help to be found to have the new small molecule drug targets of potential treatment value.

Description

A kind of SNP marker relevant to breast cancer
Technical field
The present invention relates to field of biomedicine technology, and in particular to a kind of SNP marker relevant to breast cancer.
Background technique
Breast cancer is that a kind of systemic disease, its occurrence and development are one and are related to multifactor, too many levels complex process, The inactivation etc. of activation and tumor suppressor gene including oncogene.Therefore, gene mutation rises in the generation, development process of breast cancer Very important effect.
Breast cancer is a multifactor hereditary variability disease, is only due to caused by single-gene defect less than 10%. It is more and more to be found with breast cancer related gene with the development of high-throughput gene technology, potential heredity on these genes Variation (mononucleotide polymorphic and copy number variation) may cause the difference of breast cancer medicines therapeutic effect.Due to hereditary variation May be affected in the presence of the metabolic pathway and pharmaceutically-active target gene for making anti-tumor drug, so affect the treatment and Prognosis.
SNP (singlenucleotidepolymorphism, SNP, i.e. single nucleotide polymorphism) is 1996 by the U.S. The molecule genetic marker that the human genome research center scholar Lander of the Massachusetts Institute of Technology is proposed, is primarily referred to as gene The horizontal DNA sequence polymorphism caused by a single nucleotide variation of group.The polymorphism that SNP is shown relates only to individually The variation of base, performance are that have conversion, transversion, insertion and missing etc..Single nucleotide polymorphism is third generation genetic marker, human body Many phenotypic differences, all may be related with SNP to neurological susceptibility of drug or disease etc..It is pre- for different parting breast cancer at present Afterwards, the predictive research of curative effect is concentrated mainly on SNP level.
SNP assigns differential responses of the individual to environmental exposure, drug therapy etc., to generate different phenotypes, therefore SNP It may be the important hereditary basis for leading to individual disease development difference.It is diagnosed the illness, is had using the SNP spectrum of disease-susceptible humans Quickly, the features such as sensitive, accurate, thus have a extensive future.In recent years, it is had become using the occurrence and development that SNP diagnoses the illness Clinical and researcher research hotspot.
However, there is presently no the reports that SNP is applied to breast cancer diagnosis, if the SNP of breast cancer susceptibility can be filtered out As biomarker, and corresponding diagnostic kit is developed, will effectively push the status of China's early diagnosing mammary cancer, And new approach is opened up for its drug screening, evaluating drug effect and targeted therapy.
Summary of the invention
The purpose of the present invention is in view of the above technical problems, propose a kind of biological marker relevant to Computer-aided Diagnosis of Breast Cancer Object.
A second object of the present invention is to provide application of the biomarker in prediction breast cancer diagnosis reagent.
Third object of the present invention is to provide Computer-aided Diagnosis of Breast Cancer kits.
Inventor is by separating and studying patient with breast cancer and compare in peripheral blood DNA with the healthy women of its age-matched Single nucleotide polymorphism, find the SNP of one group of high specific and sensibility highly relevant with breast cancer, and developing can be just In the Computer-aided Diagnosis of Breast Cancer kit of clinical application, data support is provided for the screening and diagnosis of breast cancer.
The purpose of the present invention is what is realized by following technical proposal:
A kind of SNP marker relevant to breast cancer, the biomarker include being located at gene C 12orf45 nucleotides sequence The SNP site mutation by C to G has occurred in column the 8187th bit base from 5 ' ends, and the SNP site sports C12orf45:NM_ 152318:exon4:c.C368G:p.S123W。
Wherein, gene C 12orf45 (chromosome 12open reading frame 45) is to be located at the mankind the 12nd An open reading frame on number chromosome, genome sequence are classified as NC_000012.12, total 8408bp;NM_152318 is A transcript of C12orf45, the SNP site mutation occur on the 4th exon of the transcript, and the 368th The missense mutation by C to G has occurred, which results in transformation of the coding albumen by serine S to tryptophan W.
Further, the application the present invention provides the biomarker in prediction breast cancer diagnosis reagent.
Preferably, the reagent includes the primer pair for expanding the SNP site, or including described for expanding The primer pair and restriction enzyme of SNP site.
Preferably, the primer pair for expanding the SNP site has nucleotide sequence shown in SEQIDNO:3-4.
Preferably, shown in the nucleotide sequence SEQIDNO:1 of the primer pair amplifies.
Closer, the present invention provides a kind of kits of Computer-aided Diagnosis of Breast Cancer comprising detection is located at gene The reagent of C12orf45 nucleotide sequence the 8187th SNP site genotype from 5 ' ends.
Preferably, the reagent includes the primer pair for expanding the SNP site, or including described for expanding The primer pair and restriction enzyme of SNP site.
Preferably, the primer pair for expanding the SNP site has nucleotide sequence shown in SEQIDNO:3-4.
Preferably, the kit further includes that PCR reacts common enzyme and reagent, such as dNTPs, Taq enzyme, Mg2+, PCR it is anti- Answer buffer etc.;Standard items and/or reference substance can also be contained.
The invention has the advantages that:
Present invention research SNP illustrates influence of the SNP for breast cancer progression in the application prospect of Computer-aided Diagnosis of Breast Cancer, Disclose its diagnostic value.Therefore, the present invention passes through the development and application of SNP biomarker and diagnostic kit, may make cream The diagnosis of gland cancer is more convenient and easy, quick and precisely grasps conditions of patients for clinician, establishes for clinical therapeutic efficacy evaluation Basis, and help is provided to be found to have the new small molecule drug targets of potential treatment value.
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.
Technical solution of the present invention specifically includes: acquiring standard compliant blood sample, system collects complete demography Data and clinical data;Genotype detection: selection breast cancer case is compareed with the healthy women of breast cancer case age-matched, It is sequenced using exon, finds out SNP relevant to breast cancer;To the positive association SNP filtered out, Genotyping is further used It is detected, verifies its repeatability for being applied to clinical diagnosis;The development of Computer-aided Diagnosis of Breast Cancer kit: according to breast cancer The genotype distribution frequency SNP that there were significant differences develops SNP auxiliary diagnostic box in case and healthy women control.
Each numerical value is expressed as follows in data analysis:
1, ljb23_sift:SIFT score value (version 2.3) indicates influence of the variation to protein sequence, includes three A value, first is that SIFT initial value, second is that the value (1-SIFT) after conversion, third is that T or D.When the variation while influencing multiple When protein sequence, there is a SIFT value to every protein sequence, be minimized.SIFT score value is smaller more " nocuousness ", shows the SNP A possibility that causing protein structure or function to change, is big;D:Deleterious (sift≤0.05);T:tolerated(sift> 0.05));
2, ljb23_pp2hvar: predict the variation to protein sequence based on HumanVar database using PolyPhen2 It influences, is used for single gene inheritance disease.The column include two values, and first is PolyPhen2 score value, and the numerical value the big more " nocuousness ", Show that a possibility that SNP causes protein structure or function to change is big;Second is D or P or B (D:Probably damaging (>=0.909), P:possibly damaging (0.447≤pp2_hvar≤0.909);B:benign (pp2_hvar≤ 0.446));
3, ljb23_pp2hdiv: predict the variation to protein sequence based on HumanDiv database using PolyPhen2 It influences, is used for complex disease.The column include two values, and first is 2 score value of PolyPhen, and the numerical value the big more " nocuousness ", are shown A possibility that SNP causes protein structure or function to change is big;Second be D or P or B (D:Probably damaging (>= 0.957), (0.453≤pp2_hdiv≤0.956) P:possibly damaging;B:benign (pp2_hdiv≤ 0.452));
4, ljb23_mt:tionTaster score value (version 2.3) indicates influence of the variation to protein sequence, packet It is worth containing three, first is that Mutation Taster initial value, second is that the value after conversion, third is that A, D, N or P.Second value is got over Greatly more " nocuousness ", show that a possibility that SNP causes protein structure or function to change is big, wherein " A " (" disease_ causing_automatic");"D"("disease_causing");"N"("polymorphism");"P"(" polymorphism_automatic")。
The experimental method specifically studied mainly includes following components:
1. studying the selection of sample
(1) breast cancer case clarified a diagnosis through pathology 25 and healthy women 10 with breast cancer case age-matched Example wherein has 3 patients to have cancer family history as control in breast cancer case;
(2) radiotherapy or chemotherapy were not received, without the past tumour medical history before blood sampling;
(3) it is compareed with the healthy women of case age-matched
2. phenol-chloroform method extracts peripheral blood genomic DNA, operate according to a conventional method.Usually lead to 20-50ng/ μ LDNA, purity (ultraviolet 2600D:2800D) is in 1.6-2.0.
3. full Exon chip detection
(1) subject's complete genome DNA sample is taken;
(2) it is scanned in full Exon chip (Beijing source Nuo Hezhi Science and Technology Co., Ltd., similarly hereinafter);
(3) detection and difference difference of more each genotype in breast cancer case is compareed with healthy women.
4. the Genotyping of single SNP
(1) subject's DNA sample is taken;
(2) specificity amplification primer of single SNP is designed;
(3) PCR reaction is carried out, recovery product is sequenced;
(4) compare breast cancer case compareed with healthy women in different genotype distributional difference.
5. diagnostic reagent box preparation method
Full Exon chip be scanned with determine breast cancer case after single SNP detection with healthy women and compare in gene The type distribution frequency SNP that there were significant differences, the index as breast cancer diagnosis.The SNP related with pathogenesis of breast carcinoma filtered out Auxiliary diagnostic box comprising detection is located at gene C 12orf45 nucleotide sequence the 8187th SNP site base from 5 ' ends Because of the reagent of type, diagnostic kit can also include the reagents such as specificity amplification primer and Taq enzyme, the dNTPs of these SNP.
6. clinical application example
Using the present inventor preparation Computer-aided Diagnosis of Breast Cancer kit detection to screening patient with breast cancer and with reality Clinical detection compares so that the validity of Computer-aided Diagnosis of Breast Cancer kit has been determined.Specifically include measurement subject's blood specimen The specificity amplification primer of above-mentioned SNP and other detection reagents in cDNA, the disease of patient is quick and precisely grasped for clinician State and coincident with severity degree of condition take the control prece of more personalized to provide support in time.
The collection of 1 sample of embodiment and the arrangement of sample data
Inventor has collected a large amount of new hair-cream gland in Shenzhen City Second People's Hospital in January, 2010 in December, 2015 Cancer patient's blood specimen, by the arrangement to sample data, inventor has therefrom selected 25 samples for meeting following standard, simultaneously It selects 10 ages to compare in 25-55 years old healthy women and carries out full Exon chip detection, sample selection criteria is as follows:
1, the breast cancer case clarified a diagnosis through pathology, wherein having 3 patients that there is cancer family history and marking respectively For X1, X2, X3;
2, radiotherapy or chemotherapy were not received, without the past tumour medical history before blood sampling;
3, it is compareed with the healthy women of case age-matched
And situations such as demographic data and clinical data of system acquisition these samples.
The extraction and purifying of 2 peripheral blood DNA of embodiment
In above-mentioned qualified 25 patient with breast cancers and 10 healthy women controls, two groups of age equilibriums are comparable.
Specific steps are as follows:
1, to the peripheral blood addition hemolyzing reagent being stored in 2mL cryopreservation tube, (i.e. lysate, 40 deal configuration methods are such as Under: sucrose 219.72g, magnesium chloride 2.02g and triton x-100 (amresco0694) 20mL mixing after, with TrisHcl solution It is settled to 2000mL, similarly hereinafter), it is transferred to completely after being mixed by inversion.
2, it removing red blood cell: 5mL centrifuge tube being mended to 4mL with hemolyzing reagent, is mixed by inversion, 4000rpm is centrifuged 10 minutes, Abandon supernatant.4mL hemolyzing reagent is added into precipitating, is mixed by inversion cleaning again once, 4000rpm is centrifuged 10 minutes, abandons supernatant.
3, extract DNA: into precipitating plus 1mL extract (contains 122.5mL0.2M sodium chloride, 14.4mL in every 300mL 0.5M ethylenediamine tetra-acetic acid, 15mL10% lauryl sodium sulfate, 148.1mL distilled water, similarly hereinafter) and 8 μ L Proteinase Ks, oscillation Sufficiently oscillation mixes on device, and 37 DEG C of water-baths are stayed overnight.
4, it removes isolating protein: 1mL saturated phenol being added to mix well (hand jog 15 minutes), 4000rpm is centrifuged 10 minutes, is taken It is transferred to clearly in new 5mL centrifuge tube.Be added in supernatant isometric chloroform and isoamyl alcohol mixed liquor (chloroform: isoamyl alcohol=24: 1, v/v, similarly hereinafter), after mixing well (hand 15 minutes), 4000rpm is centrifuged 10 minutes, take supernatant (be divided into two 1.5mL from Heart pipe).
5, DNA is precipitated: the 60 μ L of sodium acetate of 3M being added in supernatant, adds the anhydrous second of the ice isometric with supernatant Alcohol, upper and lower jog, it is seen that white flock precipitate object, then 10min is centrifuged with 12000rpm.
6, DNA is washed: ice dehydrated alcohol 1mL being added in precipitating, 12000rpm is centrifuged 10min, and vacuum is taken out after abandoning supernatant It does or is placed in and be evaporated in cleaning dry environment.
7, it measures concentration: usually leading to 20-50ng/ μ LDNA, purity (ultraviolet 2600D:2800D) is in 1.8-2.0.
The full exon group of embodiment 3SNP detects
Two groups of crowds in embodiment 2 are detected through full Exon chip and obtain correlated results.
1, library construction
Beijing source Nuo Hezhi Science and Technology Co., Ltd. uses the liquid-phase chip capture systems of Agilent, to the complete outer of people Aobvious subregion DNA carries out efficiently concentrating, and high-throughput, high depth sequencing is then carried out on Illumina Hiseq platform.Build library Agilent SureSelect Human All ExonV5 kit is used with capture experiment, what stringent operation instructions were recommended Reagent and consumptive material, and operated referring to the newest experiment flow by optimization.
Experiment basic procedure: genomic DNA is crushed instrument through Covaris and is broken into the piece that length is 180-280bp at random Section is separately connected top connection preparation DNA library at segment both ends after repairing through end and adding A tail.Library with special index With up to 543 after pooling, the probe of 872 biotin labelings carries out solution hybridization, and reusing the magnetic bead with streptomysin will The 334 of 20,965 genes, 378 exon trappings get off, and through the laggard style of writing library quality inspection of PCR linear amplification, qualification can be into Machine is sequenced on row.
2, library is examined
After the completion of library construction, tentatively quantitative, dilution library to 1ng/ μ L is first carried out using Qubit2.0, then use Agilent 2100 detects the insert size in library, after insert size meets expection, uses Q-PCR method pair The effective concentration in library carries out accurate quantitative analysis (library effective concentration > 2nM), to guarantee Library Quality.
3, upper machine sequencing
Library inspection is qualified, carries out the sequencing of Illumina Hiseq platform according to the effective concentration in library and data output demand.
4, data analysis and processing
It is final to determine " breast cancer case " group and " be good for by data screening, deep processing and bioinformatics sequence alignment The genotype distribution frequency found in health female control " group 53 SNP sites that there were significant differences are preferred sensitivity level site.Its In, it is located at the gene C 12orf45 nucleotide sequence SNP site that the 8187th bit base is G from 5 ' ends, the site is C12orf45:NM_152318:exon4:c.C368G, the Mutation are as follows to albumen influence value:
Ljb23_sift:0,1.00, D
Ljb23_pp2hvar:0.951, D
Ljb23_pp2hdiv:0.999, D
Ljb23_mt:0.996,0.004, N.
Bioinformatic analysis is passed through in the site, can be confirmed as breast cancer candidate markers.
Embodiment 4 further analyzes the onset risk of SNP and breast cancer using risk score method
The present inventor passes through to 2 groups of samples (" breast cancer case group " and " healthy women control group ") genotype distribution frequency Comparison, select the SNP of positive association, using SNP regression coefficient single in full exon scanned samples as weight, further acquire Dangerous score value draws ROC to evaluate the sensitivity and specificity of diagnosis, and then diagnoses judgement of these SNP to pathogenesis of breast carcinoma Ability.Conjoint Analysis discovery to all SNP markers, is located at the 368th alkali of gene C 12orf45:NM_152318:exon4 Base is the mutation of G/C, and sensitivity and specificity all reach 60% or more.
Therefore, inventors demonstrated that the site marker can be well by healthy women control and patient with breast cancer area Point.
The Genotyping of the single SNP of embodiment 5
1, take 5 patient with breast cancers and 5 healthy women comparison DNA samples with embodiment 2;
2, PCR amplification
Single SNP is designed to C12orf45:NM_152318:exon4:c.C368G using 5 software of Primer Premier Specificity amplification primer it is as shown in table 1.
1 primer sequence of table
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
Component Additional amount
2×mix 25μL
Upstream primer (10uM) 3.0μL
Downstream primer (10uM) 3.0μL
Template 5μL
Sterile purified water is added To 50 μ L
3, it is sequenced
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.
4, interpretation of result
By Chromas sequence analysis software, sequencing result is compared with standard sequence, finds SNP site, pass through Analyze the type of base at SNP site, so that it may obtain the genotype of SNP site.As the result is shown: 5 patient with breast cancer's sequencings The nucleotide sequence of the segment of 392bp is obtained as shown in SEQ ID NO.1, is CG, GG in the 239th bit base;And 5 health Female control is sequenced to obtain the nucleotide sequence of the segment of 392bp as shown in SEQ ID NO.2, is CC in the 239th bit base; Confirm the susceptible genotype for being judged as breast cancer when the site is CG, GG genotype, which is judged as when being CC genotype The non-susceptible genotype of breast cancer, to further confirm that the SNP of the C12orf45:NM_152318:exon4:c.C368G Site can be used for the auxiliary diagnosis such as detection, treatment, diagnosis, the prognosis evaluation of breast cancer.
Embodiment 6 is used for the production of Computer-aided Diagnosis of Breast Cancer SNP kit
Based on the primer sets that embodiment 5 obtains, the kit of the present invention for breast cancer, the kit are assembled Primer pair including specific amplified nucleotide sequence as shown in SEQ ID NO.1 such as SEQ ID NO:3 and SEQ ID NO:4 institute Show.Common agents needed for the kit can also have corresponding round pcr, such as: dNTPs, MgCl2, distilled water, Taq enzyme etc., These common agents be all it is well known to those skilled in the art, in addition it can have standard items and control (as determination genotype Standard items and blank control etc.).The value of this kit is only to need peripheral blood without other tissue samples, by most It simplifies and detects SNP with special primer pair, then auxiliary judgment breast cancer is composed by SNP, it is not only stable, easy to detect and accurate, It greatly improves the sensibility and specificity of medical diagnosis on disease, therefore this kit is put into and is practiced, 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.

Claims (5)

1. a kind of SNP marker relevant to breast cancer, which is characterized in that the SNP marker includes that site is located at SEQ 239th SNP of nucleotide sequence shown in ID NO:1, can be G or C.
2. application of the SNP marker described in claim 1 in preparation prediction breast cancer diagnosis reagent.
3. application as claimed in claim 2, which is characterized in that the reagent includes for expanding the SNP marker Primer pair.
4. application as claimed in claim 2, which is characterized in that the reagent includes for expanding the SNP marker Primer pair and restriction enzyme.
5. application as described in claim 3 or 4, which is characterized in that the primer pair for expanding the SNP marker is SEQ ID Nucleotide sequence shown in NO:3-4.
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CN106868191B (en) * 2017-04-01 2019-10-18 深圳大学 Application of the eukaryotic translation elongation factors in detection breast cancer reagent
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