CN106048060A - Breast cancer susceptibility gene detection kit - Google Patents
Breast cancer susceptibility gene detection kit Download PDFInfo
- Publication number
- CN106048060A CN106048060A CN201610651265.7A CN201610651265A CN106048060A CN 106048060 A CN106048060 A CN 106048060A CN 201610651265 A CN201610651265 A CN 201610651265A CN 106048060 A CN106048060 A CN 106048060A
- Authority
- CN
- China
- Prior art keywords
- breast cancer
- gene
- cancer susceptibility
- detection kit
- snp site
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a breast cancer susceptibility gene detection kit. The kit comprises endonuclease HaeIII used for detecting No. rs2294021 SNP site on Foxp3 gene and No. rs2569190 SNP site on CD14 gene. The assessment of the breast cancer susceptibility risk is realized through detecting the polymorphisms of the No. rs2294021 SNP site on the Foxp3 gene and the No. rs2569190 SNP site on the CD14 gene.
Description
Technical field
The present invention relates to molecular biology and medical domain, be specifically related to a kind of breast cancer susceptibility gene detection kit.
Background technology
Breast carcinoma is one of common malignant tumor of women, and China's female mammary gland cancer morbidity increases year by year, sickness rate
Accounting for the 7%～10% of malignant tumor, the whole year about 1,200,000 there is breast carcinoma in women, accounts for the 18% of female's surname tumor.
After deliberation, the sudden change of BRCA1 Yu BRCA2 gene largely adds women and suffers from the probability of breast carcinoma.
Under normal circumstances, both genes can transmit the information controlling cell proliferation.If they variation occur, cell will be without joint
The breeding of system ground causes formation of cancer.But BRCA1 and BRCA2 is unsatisfactory in prediction breast carcinoma familial risk,
Because the crowd carrying BRCA1 with BRCA2 gene mutation is relative little.
Human transcriptionfactor hTF forked head wing shape helix transcription factor (forkhead/winged
Helixtranscription factor Foxp3) gene mapping Xp11.2～Xq13.3, belong to transcription factor
Forkhead/winged-he2lix family.Foxp3 contains 11 exons and 10 introns, and cDNA total length is
1869bp, the albumen Foxp3/ Scurfin of its coding are made up of 431 aminoacid, and N end has zinc finger protein, leucine
Slide fastener and the region of a proline rich.Fork2hea domain is positioned at c-terminus (the 337 of amino acid residue～420),
Be combined with DNA specific site by this domain, the activation of regulation genes of interest and expression.It is proved to be CD4+ in 2003
The specificity marker of CD25+ regulatory T cells (regulatoryT cell, Treg).
People's CD14 gene is positioned at the autosomal long arm end 5q23-q31 of people No. 5, about contains 1338 nucleotide residual
Base.From the 76th of nucleotide the to 1200, encode one section of polypeptide chain having 375 amino acid residues.
CD14 has two forms, i.e. mCD14 and sCD14.MCD14 is the glycoprotein of a kind of 55kDa, by glycosyl phosphorus
Acyl inositol (GPI) is anchored in cell membrane.MCD14 protein portion is by the polypeptide including that 356 amino acid residues form
Chain and a terminal polypeptide chain being made up of 19 amino acid residues are constituted, and its terminal polypeptide is strong-hydrophobicity polypeptide chain.People
In CD14 aminoacid sequence, 39～44 sections are the necessary parts being combined with LPS.SCD14 be also a kind of glycoprotein its
Protein structure is essentially identical with the protein structure of mCD14.
MCD14 is by the mononuclear cell containing CD14 gene, macrophage, transcribes voluntarily, translated protein polypeptide
Chain, in Golgi complex after saccharifying, its c-terminus is combined with PI again, and by the phospholipid moiety of PI with cell membrane even
Connect.IL-1 β and TNF-α can regulate the expression of CD14 gene, promote transcribing of CD14mRNA；FMLP and GM-CSF can
Stimulate neutrophilic granulocyte so that it is the mCD14 of cell surface increases.SCD14 is then to be produced by mononuclear cell.
Rs2569190 is positioned at 5 ' UTR on CD14 gene, polymorphic for A/G, and in asian population, this polymorphic frequency is divided
Cloth is A0.500, G0.500, genotype A/A0.205, A/G0.590, G/G0.205.Rs2569190 site is different
Genotype can be by affecting the transcriptional level of CD14 gene, thus the expression to CD14 plays regulatory role.SNP
(Single nucleotide polymorphism), i.e. single nucleotide polymorphism, is that a class is drawn based on single nucleotide variation
The DNA polymorphism risen, is referred to as third generation genetic marker by heredity circle.It is primarily referred to as by the variation in genome nucleotide level
The DNA sequence polymorphism caused, including single base transition, transversion, and the insertion/deletion etc. of single base.It is in genome
Be widely present the most a class polymorphism mark, account for about 90%.These Genomic changes can cause table between individuality
The difference of type and Different Individual are to the susceptibility of disease, particularly complex disease with to environmental factors, the difference of drug reaction.
Based on this, research and develop and design a kind of breast cancer susceptibility gene detection kit.
Summary of the invention
It is an object of the invention to provide a kind of breast cancer susceptibility gene detection kit, this test kit elaboration is high, steady
Qualitative good.
The present invention is achieved through the following technical solutions:
A kind of breast cancer susceptibility gene detection kit, described test kit includes detecting on Foxp3 gene No. rs2294021
Rs2569190 SNP site restriction endonuclease HaeIII on SNP site and CD14 gene.
Preferably, described test kit also includes the general components of PCR augmentation detection: Taq enzyme, dNTP mixed liquor, MgCl2
Solution, PCR reaction buffer, deionized water, enzyme action general components: reaction buffer, deionized water.
Preferably, each constituent content of described PCR augmentation detection general components is: every 10 μ LPCR amplification systems include: 1
μ L 10 × PCR reaction buffer, 0.5 μ L 25mM MgCl2Solution, 0.2 μ L 25mM dNTP mixed liquor, 1U Taq DNA
Polymerase, surplus is deionized water.
Preferably, in described test kit, component and the content of enzyme action general components are, every 20 μ L endonuclease reaction systems include
2 μ L 10 × restriction endonuclease reaction buffers.
Inventor is it has been investigated that on Foxp3 gene on rs2294021 SNP site and CD14 gene
The polymorphism in rs2569190 SNP site is relevant to breast cancer susceptibility to human body, can be used for assessment individual to breast carcinoma
The basis of susceptibility, and, when the rs2294021 SNP site genotype on the Foxp3 gene of detected DNA is miscellaneous
Close CT type, for breast cancer susceptibility type；Rs2569190 SNP site genotype on the CD14 gene of detected DNA carries
G type, for breast cancer susceptibility type；Simultaneously for Foxp3 gene rs2294021 SNP site be heterozygosis CT type, CD14 base
Because G type is carried in upper rs2569190 SNP site, the risk suffering from breast carcinoma is the highest.
The present invention compared with prior art, has such advantages as and beneficial effect:
The present invention is by rs2569190 on rs2294021 SNP site on detection Foxp3 gene and CD14 gene
The polymorphism in number SNP site achieves the assessment to breast cancer susceptibility gene risk.
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with embodiment, the present invention is made
Further describing in detail, the exemplary embodiment of the present invention and explanation thereof are only used for explaining the present invention, are not intended as this
The restriction of invention.
Embodiment 1:
One detects breast cancer susceptibility test kit, and its component and content include: 1 μ l 10 × PCR reaction buffer, 0.5 μ
L25mM MgCl2 solution, 0.2 μ l 25mM dNTP mixed liquor, 0.2 μ l (5Units/ μ l) Taq DNA polymerase, 10 μMs
(four) each 0.2ul, 0.5 μ l HaeIII restriction endonuclease (10U/ul), 2 μ l 10 × restriction endonuclease is reacted by specific primer
Buffer, deionized water 9 μ l.This test kit is for a person-portion detection application, and test kit storage temperature is-20 DEG C.Mentioned reagent box
Using method comprise the following steps:
1) extraction of DNA template
Poba gene group DNA extraction system is used to extract the genomic DNA of peripheral blood.
2): PCR reacts, the duplication of purpose fragment uses and can detect the PCR detection kit of breast cancer susceptibility wherein,
Containing following primer:
Sense primer 2: 5 '-ACACACAATCCATAAAGTCACC-3 '
Antisense primer 2: 5 '-ATCTCCATGCCCTAAGAAGGCCA--3 '
Sense primer 1: 5 '-CCCCAAGACCCTACACTCAC 3 '
Antisense primer 1: 5 '-AGGACACTGCCAGGAGACAC-3 '
Sense primer 1, it is many that antisense primer 1 specifically replicates rs2294021 SNP site on Foxp3 gene
The fragment of state property, sense primer 2, antisense primer 2 specifically replicates rs2569190 SNP site on CD14 gene
The fragment of polymorphism.PCR reaction system cumulative volume is 10 μ l, including 1ul 10 × PCR reaction buffer, 0.5 μ l
25mMMgCl2 solution, 0.2 μ l 25mM dNTP mixed liquor, 0.2 μ l (5Units/ μ l) Taq DNA polymerase, 10 μMs of spies
Specific primer 0.2 μ l each to (four), deionized water 7.3 μ l.Expand at Biometra TprofessionalPCR
Increase on instrument and react, reaction condition be 94 DEG C 5 minutes, carry out 35 circulations 94 DEG C 40 seconds, 56 DEG C 40 seconds,
72 DEG C 40 seconds, 72 DEG C 10 minutes.
3) digestion with restriction enzyme
Use can detect the PCR detection kit of breast cancer susceptibility.Add included in test kit in step 2 products therefrom
10 × restriction endonuclease reaction buffer 2 μ l, HaeIII restriction endonuclease 0.5 μ l (10U/ μ l), deionized water 7.5 μ l, 37
Water-bath 3 hours under the conditions of DEG C.
4) SNP gene type assay
3% agarose gel electrophoresis detecting step 3 gained digestion products.
According to the different band of gel imaging display, representative gene type assay is as follows:
Wherein, contain 3 sizes is 320bp simultaneously, the band of 220bp, 110bp, and the Foxp3 of detected DNA is described
Rs2294021 SNP site genotype on gene is heterozygosis CT type, for breast cancer susceptibility type；Containing 2 290bp,
The band of 60bp size, illustrates that the rs2569190 SNP site genotype on the CD14 gene of detected DNA carries
G type, for breast cancer susceptibility type.Combine this 3 gene analysis, be miscellaneous for Foxp3 gene rs2294021 SNP site simultaneously
Closing CT type, on CD14 gene, to carry in site the risk suffering from breast carcinoma of G type the highest for rs2569190 SNP.
Above-described detailed description of the invention, has been carried out the purpose of the present invention, technical scheme and beneficial effect further
Describe in detail, be it should be understood that the detailed description of the invention that the foregoing is only the present invention, be not intended to limit the present invention
Protection domain, all within the spirit and principles in the present invention, any modification, equivalent substitution and improvement etc. done, all should comprise
Within protection scope of the present invention.
Claims (4)
1. a breast cancer susceptibility gene detection kit, it is characterised in that described test kit includes detecting on Foxp3 gene
Rs2569190 SNP site restriction endonuclease HaeIII on rs2294021 SNP site and CD14 gene.
A kind of breast cancer susceptibility gene detection kit the most according to claim 1, it is characterised in that: described test kit is also
General components including PCR augmentation detection: Taq enzyme, dNTP mixed liquor, MgCl2Solution, PCR reaction buffer, deionization
Water, enzyme action general components: reaction buffer, deionized water.
A kind of breast cancer susceptibility gene detection kit the most according to claim 2, it is characterised in that described PCR expands
Each constituent content of detection general components is: every 10 μ LPCR amplification systems include: 1 μ L 10 × PCR reaction buffer,
0.5μL 25mM MgCl2Solution, 0.2 μ L 25mM dNTP mixed liquor, 1U Taq DNA polymerase, surplus is deionized water.
A kind of breast cancer susceptibility gene detection kit the most according to claim 3, it is characterised in that: in described test kit
The component of enzyme action general components and content are that every 20 μ L endonuclease reaction systems include 2 μ L 10 × restriction endonuclease reaction bufferings
Liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610651265.7A CN106048060A (en) | 2016-08-10 | 2016-08-10 | Breast cancer susceptibility gene detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610651265.7A CN106048060A (en) | 2016-08-10 | 2016-08-10 | Breast cancer susceptibility gene detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106048060A true CN106048060A (en) | 2016-10-26 |
Family
ID=57480390
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610651265.7A Pending CN106048060A (en) | 2016-08-10 | 2016-08-10 | Breast cancer susceptibility gene detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106048060A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399304A (en) * | 2016-11-18 | 2017-02-15 | 深圳市第二人民医院 | Breast cancer related SNP marker |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101985662A (en) * | 2010-12-10 | 2011-03-16 | 苏州大学 | Kit for detecting breast cancer susceptibility |
-
2016
- 2016-08-10 CN CN201610651265.7A patent/CN106048060A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101985662A (en) * | 2010-12-10 | 2011-03-16 | 苏州大学 | Kit for detecting breast cancer susceptibility |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399304A (en) * | 2016-11-18 | 2017-02-15 | 深圳市第二人民医院 | Breast cancer related SNP marker |
| CN106399304B (en) * | 2016-11-18 | 2019-02-01 | 深圳市第二人民医院 | A kind of SNP marker relevant to breast cancer |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Casey et al. | Mutations in the protein kinase A R1α regulatory subunit cause familial cardiac myxomas and Carney complex | |
| Flørenes et al. | Levels of nm23 messenger RNA in metastatic malignant melanomas: inverse correlation to disease progression | |
| Zhang et al. | Identification of the cylindromatosis tumor-suppressor gene responsible for multiple familial trichoepithelioma | |
| Davies et al. | Inherited variants in the MC1R gene and survival from cutaneous melanoma: a BioGenoMEL study | |
| Koda et al. | The polymorphisms of fucosyltransferases | |
| SG178811A1 (en) | Genetic susceptibility variants associated with cardiovascular disease | |
| Nishimura et al. | Tissue-specific mRNA expression profiles of human solute carrier 35 transporters | |
| Castelli et al. | HLA‐G polymorphism and transitional cell carcinoma of the bladder in a Brazilian population | |
| Soejima et al. | Molecular mechanisms of Lewis antigen expression | |
| Chinen et al. | The leucine twenty homeobox (LEUTX) gene, which lacks a histone acetyltransferase domain, is fused to KAT6A in therapy‐related acute myeloid leukemia with t (8; 19)(p11; q13) | |
| CN106434982B (en) | The relevant molecular marked compound of cerebral arterial thrombosis and its application | |
| CN101985662B (en) | Kit for detecting breast cancer susceptibility | |
| CN103667444A (en) | Tumor markers associated with pancreatic cancer and application thereof | |
| CN106048060A (en) | Breast cancer susceptibility gene detection kit | |
| Govatati et al. | Mitochondrial NADH: ubiquinone oxidoreductase alterations are associated with endometriosis | |
| Haddadi et al. | Toward systems pathology for PTEN diagnostics | |
| Schreiber et al. | Chromosomal assignment and expression pattern of the murine Lasp-1 gene | |
| CN101200766B (en) | Multiple PCR reagent kit detecting breast cancer susceptibility gene mutation and preparation method thereof | |
| WO2016019633A1 (en) | Gene polymorphism variation site diagnostic reagent kit for early evaluation of breast cancer risk | |
| Iismaa et al. | Structural organization and chromosomal localization of three human galanin receptor genes | |
| Beder et al. | T‐lymphocyte maturation‐associated protein gene as a candidate metastasis suppressor for head and neck squamous cell carcinomas | |
| Kim et al. | Inter-individual variability in OCT1 expression and its relationship with OCT1 genotype in liver samples from a Korean population | |
| Smeitink et al. | Molecular characterization and mutational analysis of the human B17 subunit of the mitochondrial respiratory chain complex I | |
| Okolicsanyi et al. | Association of the SNP rs2623047 in the HSPG modification enzyme SULF1 with an Australian Caucasian breast cancer cohort | |
| Loginov et al. | Human chromosome 3P regions of putative tumor-suppressor genes in renal, breast, and ovarian carcinomas |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20161026 |
|
| RJ01 | Rejection of invention patent application after publication |