CN101608230A - Kit for genetic detection of uterine cancer - Google Patents
Kit for genetic detection of uterine cancer Download PDFInfo
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- CN101608230A CN101608230A CNA2008100392727A CN200810039272A CN101608230A CN 101608230 A CN101608230 A CN 101608230A CN A2008100392727 A CNA2008100392727 A CN A2008100392727A CN 200810039272 A CN200810039272 A CN 200810039272A CN 101608230 A CN101608230 A CN 101608230A
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Abstract
The invention discloses a kind of test kit that detects the uterus carcinoma genetic risk.This test kit comprise the genotypic Auele Specific Primer of mononucleotide polymorphism site that detects on estrogen receptor alpha gene (ER-α), Cytochrome P450 17A1 gene (CYP17A1), Cytochrome P450 1A1 gene (CYPIA1), the Cytochrome P450 1B1 gene (CYP1B1) to and the specificity fluorescent probe to, quantification PCR normal assembly etc.Test kit of the present invention is assessed individual uterus carcinoma genetic risk by detection simultaneously and the pleomorphism site genotype on the closely-related gene of uterus carcinoma genetic risk.
Description
Technical field
The present invention relates to molecular biology and medical field, more specifically, the present invention relates to a kind of test kit that detects individual uterus carcinoma genetic predisposition, assess individual uterus carcinoma genetic predisposition by detection simultaneously and the mononucleotide polymorphism site genotype on the closely-related estrogen receptor alpha gene of uterus carcinoma (ER-α), Cytochrome P450 17A1 gene (CYP17A1), Cytochrome P450 1A1 gene (CYP1A1), the Cytochrome P450 1B1 gene (CYP1B1).
Background technology
Uterus carcinoma is a carcinoma of endometrium, is the malignant tumour that originates from endometrial gland, owing to be primary in the uterus body, so claim carcinoma of uterine body again.The women of breeding time, uterine endometrium all thickens every month, prepares to accept a zygote, if ovum is unfertilized, endometrial outer tissue and blood vessel will strip off, and discharge by menstruation then.If endometrial proliferation just might develop into uterus carcinoma.This disease is women's common malignancy, and is a kind of elderly woman tumour basically, and pilosity is born in 55-59 year women, below 40 years old more than 70 years old morbidity all less.
The estrogen receptor of ER coding is one of SHRS member, and this receptor is expressed in mammary gland, liver, bone, urogenital tract, arteries and central nervous system.ER (comprising ER-α and ER-β) belongs to glycoprotein, and high specificity, avidity height, binding capacity be low, it is apt to deteriorate to be heated, and is the member of nuclear receptor family.ER-α gene codon10 T/C site is a T/C polymorphic site that is positioned in No. 1 exon TAF-1 district of this gene, causes that Ser same sense mutation takes place the 10th amino acid of albumen of ER-α genes encoding.This loci gene type has TT, TC, CC, studies show that C allelotrope shields, and can reduce the ill risk of uterus carcinoma, and TT/TC is the risk genes type of uterus carcinoma.Studies show that, when individuality carries the risk genes type, changed and be positioned at ER-α N-terminal TAF-1 district, thereby influence is by the physiology and the pathologic process of estrogen-mediated the estrogenic level of replying, promote endometrial hyperplasia and vascular proliferation, cause the ill risk of uterus carcinoma to rise.
CYP17A1 is one of cytochrome P 450 enzymes family member, is positioned on the endocytoplasmic reticulum, at each histoorgan of whole body expression is arranged all, and wherein liver and kidney expression amount are more.CYP17A1 has 17 α-hydroxylase, 17 simultaneously, and the activity of 20-lyase plays a crucial role in the approach that steroid generates, and can synthesize Progesterone, mineralocorticoid, glucocorticosteroid, male sex hormone and oestrogenic hormon.CYP17A1 gene M spA1 I restriction enzyme site is the C/T polymorphic site (T27C) on 5 ' the UTR end, A1 (T), A2 (C) are two allelotrope in this site, the former can increase this gene transcription efficient, thereby the raising enzymic activity produces and more can be converted into estrogenic androgen precurosor thing.The genotype of this polymorphic site has three kinds of A1/A1, A1/A2, A2/A2, and wherein A1/A1 and A1/A2 are the risk genes types of uterus carcinoma.Domestic association study shows that the CYP17A1 gene pleiomorphism is relevant with the generation of uterus carcinoma.Carry this gene transcription efficient of wild-type (A1) allelotrope person and improve, cause the gross activity of enzyme to improve, produce and more can be converted into estrogenic androgen precurosor thing, body inner estrogen level raises, thereby has increased the ill risk of carcinoma of endometrium.
Cytochrome P450 1A1 is the carcinogenic main enzyme of activation polycyclic aromatic hydrocarbons.It is relevant with the bioactivation of some carcinogenic substances as a kind of little neurone somatocyte enzyme, for example benzopyrene [benzo (a) pyrene].Simultaneously, CYP1A1 is easy to by 2,3,7,8-tetrachloro hexichol-right-Dioxins (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) and polycyclic aromatics such as 3-MECA induce.CYP1A1 Msp I site is the T/C polymorphic site that is positioned at this gene 3 '-264bp place, end non-coding region poly A downstream.Have three kinds of genotype: TT (m1/m1), TC (m1/m2), CC (m2/m2).Wherein TC, CC are the risk genes type, carry being limited property of C allelotrope endonuclease Msp I identification.Studies show that, after CYP1A1 Msp I restriction enzyme site is undergone mutation, the induced activity of this enzyme strengthens, promptly be subjected to the influence of environmental carcinogen easily, produce more CYP1A1 thereby induce, make the activation of carcinogenic substance quicken, can not in time such substance metabolism be discharged if other separate toxenzyme, then can cause the distortion of dna damage and cell, increase cancered risk.Therefore when carrying risk genes type TC, CC, the uterus carcinoma onset risk rises.
CYP1B1 is unique member of present CYP1B subfamily.Activation, the metabolism of multiple procarcinogen and anticarcinogen in the CYP1B1 participation environment, and the C4 hydroxylation of mediation 17-estradiol (E2).Meta-bolites 4-hydroxyl estradiol carinogenicity in all estrogen metabolism products of CYP1B1 is the strongest, shows all in many animal models that 4-hydroxyl estradiol is relevant with the uterus carcinoma morbidity.4-hydroxyl estradiol can combine with DNA by its quinones meta-bolites, causes the DNA oxidative damage; Can also combine with estrogen receptor target cell is brought into play estrogenic effect.Therefore, CYP1B1 genovariation is relevant with the multiple pathogenesis of cancer that is caused by hyperestrogenism, such as uterus carcinoma.CYP1B1 gene Leu432Val site is the C/G polymorphic site that is positioned at this gene the 432nd codon place, causes Leu to become Val.This site has three kinds of genotype CC (Leu/Leu), CG (Leu/Val), GG (Val/Val), and wherein CG and GG are the tumor susceptibility gene types of uterus carcinoma.Studies show that, CYP1B1 gene Leu432 becomes Val432, make the catalytic activity of CYP1B1 gene encoding production increase 2-3 doubly, the estrogen metabolism product 4-hydroxyl estradiol level that causes having high carcinogenic raises, and 4-hydroxyl estradiol is the inducing DNA single-strand break directly, also can generate the benzoquinones metabolite by a series of redox reactions, such material is an electrophilic compound, can combine with dna molecular and form dna adduct, cause the damage of DNA, cause the ill risk of uterus carcinoma to increase.
Summary of the invention
Can be used to assess on the basis of individual uterus carcinoma genetic predisposition based on 4 SNP loci polymorphisms on estrogen receptor alpha gene (ER-α), Cytochrome P450 17A1 gene (CYP17A1), Cytochrome P450 1A1 gene (CYP1A1), the Cytochrome P450 1B1 gene (CYP1B1), the invention provides a kind of test kit that detects individual uterus carcinoma genetic predisposition.
This test kit comprises:
The genotypic Auele Specific Primer of codon10 T/C SNP polymorphism is right to reaching the specificity fluorescent probe on the detection ER-α gene;
The genotypic Auele Specific Primer of MspA1 I SNP polymorphism is right to reaching the specificity fluorescent probe on the detection CYP17A1 gene;
The genotypic Auele Specific Primer of Msp I SNP polymorphism is right to reaching the specificity fluorescent probe on the detection CYP1A1 gene;
The genotypic Auele Specific Primer of Leu432Val SNP polymorphism is right to reaching the specificity fluorescent probe on the detection CYP1B1 gene;
The quantitative fluorescent PCR reaction component (comprises Taq enzyme, dNTP mixed solution, MgCl
2Solution, reaction buffer, deionized water etc.).
Primer described in this test kit is to being that those skilled in the art can finish design easily, and the synthetic technology of available routine is synthesized.
Specificity fluorescent probe described in this test kit is to being that those skilled in the art can finish design easily, and the specificity fluorescent probe synthesizes the probe synthetic technology of available routine.
The component and the content of test kit of the present invention comprise:
10X quantitative fluorescent PCR reaction buffer 4 μ l,
25mM dNTP mixed solution 0.4 μ l,
25mM MgCl2 solution 2.4 μ l,
5units/ μ l Taq archaeal dna polymerase 0.01 μ l,
20 μ M Auele Specific Primers are to each 0.225 μ l of every primer,
10 μ M specificity fluorescent probes are to each 0.25 μ l of every probe,
Deionized water 21.3 μ l.
This test kit detects for a person-portion and uses, and the storage temperature of test kit is-20 ℃.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
The use of embodiment 1. detection kit
The extraction of step 1:DNA template
Genomic dna with silica gel adsorption extracting mouth epithelial cells.
Step 2: quantitative fluorescent PCR reaction
Use the quantitative fluorescent PCR suit in the detection kit, carry out 4 independently quantitative fluorescent PCR reactions respectively, the system of each reaction is cumulative volume 10 μ l, and comprising concentration is dna profiling 2 μ l, 1 μ l, 10 X quantitative fluorescent PCR reaction buffers, 0.1 μ l 25mMdNTP mixed solution, the 0.6 μ l 25mM MgCl of 20ng/ μ l
2The band VIC fluorescent probe that adopted primer and antisense primer each 0.225 μ l, 10 μ M are arranged of solution, 0.025 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M and each 0.25 μ l of band FAM fluorescent probe, deionized water 5.325 μ l.
React on the pcr amplification instrument, reaction conditions is 50 ℃, 2 minutes, 95 ℃, 10 minutes, carries out 95 ℃ of 60 round-robin, 30 seconds, 60 ℃, 1 minute.Reaction finishes the back and read the fluorescent amount on quantitative real time PCR Instrument.
Step 4: gene type assay
According to the gene type diagram that the test kit working instructions indicate gene type assay is carried out in the SNP site.
The those skilled in the art that are familiar with fluorescent quantitative PCR technique can be by the final sample fluorescence volume that shows on the identification quantitative real time PCR Instrument, can determine the genotype in the SNP site detected according to the power of different sequence probe VIC and FAM fluorescent signal.
Embodiment 2. applying detection test kits carry out the service that individual uterus carcinoma genetic predisposition detects
Step 1:DNA extracts
Instructing the examinee to use the oral cavity sampling to wipe away by the hospital laboratory doctor carries out the mouth epithelial cells sampling, adopts silica gel adsorption to carry out the DNA extracting of mouth epithelial cells.
Step 2: genotype tests
Use test kit provided by the invention, the estrogen receptor alpha gene (ER-α) of detected person's genomic dna is gone up codon10 T/C SNP site, Cytochrome P450 17A1 gene (CYP17A1), and upward MspA1 I SNP site, Cytochrome P450 1A1 gene (CYP1A1) are gone up MspI SNP site, Cytochrome P450 1B1 gene (CYP1B1) is gone up Leu432Val SNP site and carried out fluorescence quantitative PCR detection, determine the genotype in these 4 SNP sites.
Step 3: the analysis of individual uterus carcinoma genetic predisposition
By to the genotypic analysis of detected person SNP, provide the analysis report list of individual uterus carcinoma genetic predisposition.Describe the height of detected person's uterus carcinoma genetic risk in the report in detail, and describe and understand individual uterus carcinoma genetic predisposition analysis report list in detail to the examinee by the doctor.
Claims (2)
1. test kit that detects individual uterus carcinoma genetic predisposition is characterized in that: comprise detect simultaneously estrogen receptor alpha gene (ER-α) go up codon10T/C SNP site, Cytochrome P450 17A1 gene (CYP17A1) go up MspA1I SNP site, Cytochrome P450 1A1 gene (CYP1A1) go up the Auele Specific Primer of Msp I SNP site, Cytochrome P450 1B1 gene (CYP1B1) going up Leu432Val SNP site to and the specificity fluorescent probe to, Taq enzyme, dNTP mixed solution, MgCl
2Solution, quantitative fluorescent PCR reaction buffer, deionized water.
2. test kit according to claim 1, it is characterized in that: the component and the content of test kit comprise: 10X quantitative fluorescent PCR reaction buffer 4 μ l, 25mM dNTP mixed solution 0.4 μ l, 25mM MgCl2 solution 2.4 μ l, 5units/ μ l Taq archaeal dna polymerase 0.01 μ l, 20 μ M Auele Specific Primers are to each 0.225 μ l of every primer, and 10 μ M specificity fluorescent probes are to each 0.25 μ l of every probe, deionized water 21.3 μ l.
This test kit detects for a person-portion and uses, and the storage temperature of test kit is-20 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100392727A CN101608230A (en) | 2008-06-20 | 2008-06-20 | Kit for genetic detection of uterine cancer |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100392727A CN101608230A (en) | 2008-06-20 | 2008-06-20 | Kit for genetic detection of uterine cancer |
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| Publication Number | Publication Date |
|---|---|
| CN101608230A true CN101608230A (en) | 2009-12-23 |
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| CNA2008100392727A Pending CN101608230A (en) | 2008-06-20 | 2008-06-20 | Kit for genetic detection of uterine cancer |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703591A (en) * | 2012-05-30 | 2012-10-03 | 北京艾迪康医学检验所有限公司 | Primer used for performing pyrophosphoric acid detection on PvuII and XbaI polymorphism of intron 1 of estrogen receptor gene alpha |
| CN102732608A (en) * | 2011-03-31 | 2012-10-17 | 中国科学院上海生命科学研究院 | Marker for diagnosing liver cancer and application thereof |
| CN104513828A (en) * | 2014-11-25 | 2015-04-15 | 新疆医科大学第一附属医院 | Coronary heart disease (CHD) related gene namely CYP17A1 gene, and in-vitro detection reagent, preparation or kit and application thereof |
-
2008
- 2008-06-20 CN CNA2008100392727A patent/CN101608230A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732608A (en) * | 2011-03-31 | 2012-10-17 | 中国科学院上海生命科学研究院 | Marker for diagnosing liver cancer and application thereof |
| CN102732608B (en) * | 2011-03-31 | 2014-12-10 | 中国科学院上海生命科学研究院 | Marker for diagnosing liver cancer and application thereof |
| CN102703591A (en) * | 2012-05-30 | 2012-10-03 | 北京艾迪康医学检验所有限公司 | Primer used for performing pyrophosphoric acid detection on PvuII and XbaI polymorphism of intron 1 of estrogen receptor gene alpha |
| CN104513828A (en) * | 2014-11-25 | 2015-04-15 | 新疆医科大学第一附属医院 | Coronary heart disease (CHD) related gene namely CYP17A1 gene, and in-vitro detection reagent, preparation or kit and application thereof |
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Application publication date: 20091223 |