CN106636351B - One kind SNP marker relevant to breast cancer and its application - Google Patents
One kind SNP marker relevant to breast cancer and its application Download PDFInfo
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- CN106636351B CN106636351B CN201610991560.7A CN201610991560A CN106636351B CN 106636351 B CN106636351 B CN 106636351B CN 201610991560 A CN201610991560 A CN 201610991560A CN 106636351 B CN106636351 B CN 106636351B
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- 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 a kind of biomarkers relevant to breast cancer, the biomarker includes being mutated positioned at the 73420928th bit base of the 14th chromosome of the mankind by the SNP site of G to T, and the site mutation is DCAF4:NM_181341:exon8:c.G685T:p.A229S.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
Technical field
The present invention relates to field of biomedicine technology, and in particular to one kind SNP marker relevant to breast cancer and its application.
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 positioned at the 14th chromosome of the mankind the
73420928 bit bases are mutated by the SNP site of G to T, and the site mutation is DCAF4:NM_181341:exon8:
C.G685T:p.A229S.
Wherein, gene DCAF4 (DDB1 and CUL4 associated factor 4), the gene encode WD and repeat egg
White, which connect enzyme interacting with macromolecular complex cul4-ddb1 E3, and multiple montages are had found on this gene
Transcript variant.The WD repetitive proteins (WD-repeat protein) are the protein containing multiple conservative WD primitives, WD
Primitive is called Trp-ASP or WD40, is made of 40 or so amino acid residues, and WD is the abbreviation of two kinds of amino acid, and W is indicated
Tryptophan tryptophan D indicates Aspartic acid aspartic acid.
The NM_181341 is a transcript of DCAF4, and the SNP site is mutated the 8th occurred in the transcript
On exon, and the 685th missense mutation having occurred by G to T, the mutation result in coding albumen by alanine A to silk
The transformation of propylhomoserin S.
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 DCAF4
The reagent of the SNP site genotype of gene NM_181341:exon8:c.G685T.
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 (<=0.05 sift)；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 <=<=0.909 pp2_hvar)；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 is D or P or B (D:Probably damaging (>
=0.957), P:possibly damaging (0.453 <=<=0.956 pp2_hdiv)；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, (ultraviolet 2600D: 2800D) is in 1.6-2.0 for purity.
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 the SNP site genotype of DCAF4 gene NM_181341:exon8:c.G685T
Reagent, diagnostic kit can also specificity amplification primer and the reagents such as Taq enzyme, dNTPs including 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, (ultraviolet 2600D: 2800D) is in 1.8-2.0 for purity.
The full exon group of 3 SNP of embodiment 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, positioned at the SNP mutation of the 685th bit base G/T of DCAF4 gene NM_181341:exon8, the Mutation is to albumen influence value
It is as follows:
Ljb23_sift:0.03,0.97, D
Ljb23_pp2hvar:0.401, B
Ljb23_pp2hdiv:0.735, P
Ljb23_mt:1.000,1.000, D.
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 685th bit base G/T of DCAF4 gene NM_181341:exon8
SNP mutation, 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's is designed to DCAF4:NM_181341:exon8:c.G685T using 5 software of Primer Premier
Specificity amplification primer 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, 60 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 584bp is obtained as shown in SEQ ID NO.1, is GT, TT in the 62nd bit base；And 5 health
Female control is sequenced to obtain the nucleotide sequence of the segment of 584bp as shown in SEQ ID NO.2, is GG in the 62nd bit base；
Confirm the susceptible genotype for being judged as breast cancer when the site is GT, TT genotype, which is judged as when being GG genotype
The non-susceptible genotype of breast cancer, to further confirm that the SNP site of the DCAF4:NM_181341:exon8:c.G685T
It 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 (9)
1. a kind of biomarker relevant to breast cancer, which is characterized in that the biomarker includes being located at the mankind the 14th
The 73420928th bit base of chromosome is mutated by the SNP site of G to T, and the site mutation is DCAF4:NM_181341:
exon8:c.G685T:p.A229S。
2. application of the biomarker 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 drawing for the SNP site
Object pair, or include primer pair and restriction enzyme for expanding the SNP site.
4. application as claimed in claim 3, which is characterized in that expand the nucleotide sequence of the primer pair of the SNP site such as
Shown in SEQ ID NO:3-4.
5. application as claimed in claim 4, which is characterized in that the nucleotide sequence of the primer pair amplifies such as SEQ ID NO:
Shown in 1.
6. a kind of kit of Computer-aided Diagnosis of Breast Cancer, which is characterized in that it includes detection DCAF4 gene NM_181341:
The reagent of the SNP site genotype of exon8:c.G685T.
7. kit as claimed in claim 6, which is characterized in that including the primer pair for expanding the SNP site, or
Including the primer pair and restriction enzyme for expanding the SNP site.
8. kit as claimed in claim 7, which is characterized in that the nucleotide sequence of the primer pair such as SEQ ID NO:3-
Shown in 4.
9. kit as claimed in claim 7 or 8, which is characterized in that the kit further includes dNTPs, Taq enzyme, Mg2+With
PCR reaction buffer.
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