CN101140232A - Reagent kit for detecting pulmonary cancer susceptibility with CYP1A1 gene and ERCC2 gene - Google Patents
Reagent kit for detecting pulmonary cancer susceptibility with CYP1A1 gene and ERCC2 gene Download PDFInfo
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- CN101140232A CN101140232A CNA2006100309538A CN200610030953A CN101140232A CN 101140232 A CN101140232 A CN 101140232A CN A2006100309538 A CNA2006100309538 A CN A2006100309538A CN 200610030953 A CN200610030953 A CN 200610030953A CN 101140232 A CN101140232 A CN 101140232A
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Abstract
The present invention discloses a reagent kit to detect lung cancer susceptibility, which comprises a fluorescence probe to synchronously detect specificity tracers and specificity of No. rs1048943 SNP locus on gene CYP1A1 and No. rs13181 SNP locus on gene ERCC2, and a normal assembly to detect fluorescence quantitative PCR and so on. The reagent kit of the present invention synchronously detects and analyzes individual SNPs locus gene carrying type on gene CYP1A1 and ERCC2 to predict susceptibility of the individual to lung cancer.
Description
Technical field
The present invention relates to the kit that a kind of disease susceptibility detects usefulness, especially a kind of kit of detection of lung cancer neurological susceptibility, by detecting Cytochrome P450 1A1 enzyme gene (cytochrome P450 simultaneously, family 1, subfamily A, polypeptide 1, CYP1A1) single nucleotide polymorphism (SNP) site and excision repairing composite 2 (Excision Repair Cross-complementing Rodent Repair Deficiency, Complementation Group 2, mononucleotide polymorphism site ERCC2) predict individual neurological susceptibility to lung cancer.
Background technology
Primary bronchogenic carcinoma of lung is called for short lung cancer, is one of modal malignant tumour in our times various places, is the disease of a kind of serious threat human health and life.Tumour cell comes from tunica mucosa bronchiorum or body of gland, and regional lymph node and hematogenous metastasis are often arranged, and respiratory symptoms such as irritable cough, blood-stained sputum are often arranged in early days, and disease progression speed is relevant with the biological nature of cell.
Since half a century, the incidence of disease of countries in the world lung cancer and case fatality rate all have and obviously increase trend.The World Health Organization's year 2000 report: totally 706.5 ten thousand people of malignant tumour are died from the whole world in 1997, account for 12.6% of death toll, and wherein lung cancer accounts for 19% of malignant tumour death, occupies first of the malignant tumour cause of the death.China sample survey in 1990～1992 years shows, in the tumor mortality of urban population, lung cancer rises to the 1st by original the 4th, and rising the fastest on the rural area also is lung cancer.The lung cancer morbidity rate of Yunnan tin ore is to be to rise to 219.10/10 ten thousand in the period of 197.87/10 ten thousand, 1970～1979 in the period of 28.02/10 ten thousand, 1960～1969 between 1954～nineteen fifty-nine, and this also is rare in the whole world.The famous oncologist R.Peto prophesy of Britain: if untimely control smoking of China and air pollution will become the first in the world lung cancer big country above 1,000,000 to the annual lung cancer of China in 2025.
The cause of disease and pathogenesis are not clear and definite as yet so far.Consistently think that the morbidity of lung cancer is relevant with following factors: 1. smoking (comprising initiatively smoking and passive smoking); 2. professional carcinogen, the occupational factor that causes Human Lung Cancer that has been identified comprise the palycyclic aromatic in asbestos, inorganic arsenic chemicals, dichlormethyl ether, chromium and compound thereof, nickel, radon, yperite, vinyl chloride, coal smoke, tar and the oil, the heating product of tobacco etc.; 3. air pollution (comprising the pollution of indoor subenvironment and outdoor overall situation); 4. ionising radiation; 5. the presentation of results that the perspective crowd that diet and nutrition, USA New York and Chicago are carried out observes, in the food intake of natural vitamin A class, bata-carotene and after the more than ten years cancer be negative correlation, wherein the most outstanding is lung cancer; 6. other, ACS classifies tuberculosis as one of pathogenic factors of lung cancer.It is 10 times of normal population that tuberculosis patient suffers from risk of lung cancer.Its main histological type is a gland cancer.In addition, factors such as virus infections, mycotoxin (aspergillus flavus), body's immunity are low, endocrinopathy and familial inheritance may also play certain combined action to the generation of lung cancer.
In recent years studies show that the inactivation of the generation of lung cancer and activation of some oncogene and tumor suppressor gene is closely related.Verified several oncogene family comprises bcl-2, Kit that the ras family of mutagenesis, the myc family of amplifying gene, C-erB2, mechanism are not clear and by antioncogene p53, the p16 of wild type variation and RB etc.Studies show that in a large number C-myc, L-myc, N-myc and RAF all have overexpression in small-cell carcinoma of the lung.The then overexpression of Chang Kejian ras family gene in the non-small cell lung cancer.These deep researchs will and be treated lung cancer and provide fundamental basis for the gene prevention.
SNP (single nucleotide polymorphism), promptly the single nucleotide polymorphism mark is the dna polymorphism that a class causes based on single nucleotide variation, is called third generation genetic marker by hereditary educational circles.Mainly be meant the dna sequence polymorphism that causes by the variation on the genome nucleotide level, comprise single base conversion, transversion, and the insertion/disappearance of single base etc.It is a class polymorphism mark that the most extensively exists in the genome, accounts for about 90%.The variation of these genome sequences can cause the difference of phenotype between individuality and Different Individual to disease, particularly the neurological susceptibility of complex disease and to the difference of environmental factor, drug response.
The CYP1A1 gene is positioned at chromosome 15q22-q24 position No. 15, total length 6.0kb, and mRNA total length 2601bp has 7 extrons, and 513 amino acid whose Cytochrome P450 1A1 encode.P4501A1 is the important carcinogenic main enzyme of activation multiring aromatic hydrocarbon, the CYP1A1 that is activated can be converted into cytotoxin or other carcinogens as the fragrant hydrocarbon of the many rings of: PAH (polycyclic aromatic hydrocarbons) with the organic substance in the multiple environment, thereby increases the danger that cancer takes place.Polymorphic and the wild type of numerous CYP1A1 of studies show that both domestic and external is compared higher enzyme contact reaction activity and inducibility, thereby increases the risk factor of diseases such as liver cancer, lung cancer, breast cancer, cancer of the esophagus, prostate cancer.
Cytochrome P 450 enzymes (CYP450) is an important I phase metabolic enzyme in the cell, plays an important role in carcinogenic activation and detoxification processes.P4501A1 (CYP1A1) is the carcinogenic main enzyme of activation multiring aromatic hydrocarbon.CYP1A1 is relevant with the bioactivation of some carcinogens as a kind of little neuron body cell enzyme.Simultaneously, CYP1A1 is easy to be comprised laboratory carcinogens such as 3-MECA by the aromatics institute mutagenesis of TCDD and many rings.Present zoopery shows, the activator of the expression of CYP1A1 and aryl hydrocarbon receptor (AhR) and Arnt (AhRNuclear Translocator), and the antagonist of USF1 (Upstream Stimulatory Factor) is relevant.Simultaneously, CYP1A1 participates in estrogenic metabolic activity by the C2 hydroxylation.
Rs1048943 is that an A/G who is positioned on the CYP1A1 gene is polymorphic, is positioned at No. 462 amino acid of the 7th extron and is converted into Val by Ile.The world population frequency of NCBI announcement at present: A: G is 0.896: 0.104, heterozygosity 0.186.World crowd's association analysis shows that CYP1A1Ile462Val site ValVal and the ValIle genotype frequency in lung cancer case group is apparently higher than control group, and risk is especially outstanding in the women.Association analysis research based on a plurality of Chinese population lung cancer morbidities and CYP1A1 Ile462Val site, the analysis of thousands of routine lung cancer individualities and normal healthy controls individuality, show this variation that is positioned at the haem bonding pad of zymoprotein of CYP1A1 Ile462Val, be one of lung cancer inheritance susceptible factor important in the Chinese population, particularly outstanding in the women.Enzyme dynamics shows that the enzymatic activity of 3 kinds of genotypic expressions there are differences.The ability of Val/Val activation poisonous substance is the strongest, and Ile/Val takes second place, and Ile/Ile is the most weak.Studies show that the CYP1A1ValVal genotype is one of lung cancer inheritance susceptible factor important in the Chinese population.
ERCC2 is positioned at chromosome 19q13.3 position No. 19, has 23 extrons, full length gene 19.0kb, and mRNA total length 2355bp, coding contains 761 amino acid whose albumen.Participate in transcribing the nucleotide excision reparation of coupling by the ERCC2 encoded protein, it can be discerned and the irrelevant large-scale damage of repair structure, as big adduct and thymine dimer, and be the constituent of basic transcription factor compound BTF2/TFIIH.This albumen has the dependent DNA of the ATP activity of untwisting, and belongs to RAD3/XPD unwindase subfamily.The afunction of ERCC2 causes the basic transcription factor compound can not correctly discern the dna damage site, thereby makes the base excision repair and can't correctly carry out, and causes the carcinogenic damage accumulation on the DNA.The disease that the sudden change of ERCC2 gene or inactivation may cause comprises: cancer, xeroderma pitmentosum, trichothiodystrophy, Ke Kaiyin syndrome.
Rs13181 is that to be positioned at the T/G of No. 23 extron section Lys751Gln of No. 19 chromosome ERCC2 gene polymorphic.Frequency among the world crowd is about T: G=0.798: 0.202, and heterozygosity 0.322, the frequency in the Chinese population is about T: G=0.91: 0.09.Present multinomial studies show that, the amino acid replacement of Lys751Gln is positioned at ERCC2 PROTEIN C-terminal part, and the polymorphism in this site is relevant with diseases such as lung cancer, carcinoma of urinary bladder, breast cancer, cutaneum carcinomas.The polymorphism in ERCC2 Lys751Gln site and the lung cancer particularly generation of lung squamous cancer exist significantly related.
A large amount of association analysis both domestic and external shows that the lung cancer incidence increased when the rs1048943 SNP loci gene type on the CYP1A1 gene was G; The lung cancer morbidity probability increased when rs13181 SNP loci gene type was G on the ERCC2 gene.The polymorphism in these two SNPs sites can be used for assessing individual neurological susceptibility to lung cancer.
Summary of the invention
Polymorphism based on rs13181 SNP site on rs1048943 SNP site on the CYP1A1 gene and the ERCC2 gene can be used for assessing on the basis of individuality to the neurological susceptibility of lung cancer, the invention provides a kind of kit of detection of lung cancer neurological susceptibility.
This kit comprises: the Auele Specific Primer that detects the rs1048943 SNP site on the CYP1A1 gene is to reaching the specificity fluorescent probe, detecting the Auele Specific Primer in rs13181 SNP site on the ERCC2 gene to reaching specificity fluorescent probe, Taq enzyme, dNTP mixed liquor, MgCl
2Solution, quantitative fluorescent PCR reaction buffer, deionized water.
Auele Specific Primer described in this kit designs being meant at rs13181 SNP site on rs1048943 SNP site on the CYP1A1 gene and the ERCC2 gene, and the primer of dna fragmentation that can specific amplification goes out to harrow the site is right.Design this class primer to being that those skilled in the art can be unlabored.Preferably, comprise in the kit primer with sequence shown in SEQ ID NO:1 and 2 to and to have a primer of sequence shown in SEQ ID NO:3 and 4 right.Auele Specific Primer synthesizes the synthetic technology of available routine.Those skilled in the art will appreciate that primer of the present invention is not limited to this two pairs of primers.
Specificity fluorescent probe described in this kit is meant at rs13181 SNP site on rs1048943 SNP site on the CYP1A1 gene and the ERCC2 gene and designs, can go out the probe of these two SNPs site lung cancer tumor susceptibility gene types by the fluorescent quantitative PCR technique specific detection.Designing this class probe is that those skilled in the art can be unlabored.Preferably, comprise Taqman probe in the kit with sequence shown in SEQ ID NO:5 and 6.The specificity fluorescent probe can synthesize with the synthetic technology of routine.Those skilled in the art can understand, specificity fluorescent probe of the present invention is not limited to this two probes, and all probes that can be used for rs13181 SNP site on rs1048943 SNP site on the fluorescence quantitative PCR detection CYP1A1 gene and the ERCC2 gene all within the scope of the present invention.
The component and the content of kit of the present invention comprise:
1 μ l 10X quantitative fluorescent PCR reaction buffer,
0.1 μ l 25mM dNTP mixed liquor,
0.5 μ l 25mM MgCl
2Solution,
0.02 μ l (5units/ μ l) Taq archaeal dna polymerase,
20 μ M Auele Specific Primers are to (four) each 0.225 μ l,
Each 0.25 μ l of 10 μ M specificity fluorescent probes (two),
Deionized water 4.98 μ l.
This kit detects for a person-portion and uses, and the storage temperature of 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
But use the fluorescent quantificationally PCR detecting kit of detection of lung cancer neurological susceptibility, wherein, contain following primer to and fluorescence probe:
It is 56 ℃ that adopted primer 1:5 '-GTGTTAAGTGAGAAGGTGAT-3 ' (SEQ ID NO:1) Tm value is arranged
Antisense primer 1:5 '-AGGATAGCCAGGAAGAGAAA-3 ' (SEQ ID NO:2) Tm value is 58 ℃
It is 58 ℃ that adopted primer 2: 5 '-ACTCAGGAGTCACCAGGAA-3 ' (SEQ ID NO:3) Tm value is arranged
Antisense primer 2:5 '-TCTGTTCTCTGCAGGAGGA-3 ' (SEQ ID NO:4) Tm value is 58 ℃
Fluorescence probe 1:5 '-TATCGGTGAGACCGTTGCCC-3 ' (SEQ ID NO:5) Tm value is 64 ℃
Fluorescence probe 2:5 '-AATCTGCTCTATCCTCTGCAGC-3 ' (SEQ ID NO:6) Tm value is 66 ℃
Adopted primer 1 is arranged, and antisense primer 1 and fluorescence probe 1 are specifically at the rs1048943 SNP loci polymorphism that detects on the CYP1A1 gene; Adopted primer 2 is arranged, and antisense primer 2 and fluorescence probe 2 are specifically at detecting rs13181 SNP loci polymorphism on the ERCC2 gene.
The quantitative fluorescent PCR reaction system is cumulative volume 10 μ l, and comprising concentration is dna profiling 2 μ l, 1 μ l 10X quantitative fluorescent PCR reaction buffer, 0.1 μ l 25mM dNTP mixed liquor, the 0.5 μ l 25mM MgCl of 20ng/ μ l
2The antisense primer 1 that adopted primer 1 and 2 each 0.225 μ l, 20 μ M are arranged of solution, 0.02 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M and the fluorescence probe 1 of 2 each 0.225 μ l, 10 μ M and 2 each 0.25 μ l, deionized water 4.98 μ l.
React on ABI9700 type 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 ABI7900 type quantitative real time PCR Instrument.
Step 3:SNP genotyping
The final sample fluorescence volume and the normal control that show on the quantitative real time PCR Instrument are compared, the final fluorescence signal of fluorescence probe 1 is apparently higher than normal control, illustrate that the rs1048943 SNP loci gene type on the CYP1A1 gene of detected DNA carries the G type, is lung cancer susceptible type; The final fluorescence signal of fluorescence probe 2 is apparently higher than normal control, illustrates that rs13181 SNP loci gene type carries the G type on the ERCC2 gene of detected DNA, is lung cancer susceptible type.
Embodiment 2. instructs people initiatively to prevent the service of lung cancer
Carried out this service in Shanghai Zhujian Biological Engineering Co., Ltd at present.
Step 1:DNA extracts
The detected person is served preceding consulting, sign Informed Consent Form, fill in living and diet custom questionnaire by the detected person.Using the oral cavity sampling to wipe away according to the indication in the sampling box carries out the mouth epithelial cells sampling, adopts silica gel adsorption to carry out the DNA extracting of mouth epithelial cells.
Step 2: Genotyping detects
Use kit provided by the invention, fluorescence quantitative PCR detection is carried out in rs13181 SNP site on rs1048943 SNP site on the CYP1A1 gene of detected person DNA and the ERCC2 gene simultaneously, determine the genotype in these two SNPs sites.
Step 3: instruct people initiatively to prevent lung cancer
By to the genotypic analysis of detected person SNPs, provide the report of Genotyping examining report and detected person's individuation health guidance.The genetic test report describes the height of detected person's lung cancer susceptible degree and ill probability size in detail.The report of individuation health guidance is based on detected person's Genotyping testing result, in conjunction with its living and diet custom survey result, the relative risk of assessment detected person lung cancer inheritance susceptible.Make the personalized healthy action scheme that is directed to the detected person simultaneously, scheme is included in the recommendation on improvement on eating habit, the life style etc., and popularizes the health knowledge of prevention lung cancer for the detected person.
Sequence table
＜110〉Shanghai Zhujian Biological Engineering Co., Ltd
＜120〉a kind of kit by CYP1A1 gene and ERCC2 genetic test lung cancer susceptibility
<160>6
<210>1
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>1
gtgttaagtg?agaaggtgat 20
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>2
aggatagcca?ggaagagaaa 20
<210>3
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>3
actcaggagt?caccaggaa 19
<210>4
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>4
tctgttctct?gcaggagga 19
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉probe
<400>5
tatcggtgag?accgttgccc 20
<210>6
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉probe
<400>6
aatctgctct?atcctctgca?gc 22
Claims (6)
1. the kit of a detection of lung cancer neurological susceptibility comprises: the Auele Specific Primer that detects rs13181 SNP site on rs1048943 SNP site on the CYP1A1 gene and the ERCC2 gene simultaneously to and specificity fluorescent probe, Taq enzyme, dNTP mixed liquor, MgCl
2Solution, quantitative fluorescent PCR reaction buffer, deionized water.
2. kit according to claim 1, it is characterized in that: described Auele Specific Primer designs being meant at rs13181 SNP site on rs1048943 SNP site on the CYP1A1 gene and the ERCC2 gene, and the primer that can specific amplification goes out to comprise the dna fragmentation in rs13181 SNP site on rs1048943 SNP site on the CYP1A1 gene and the ERCC2 gene is right.
3. kit according to claim 1, it is characterized in that: described specificity fluorescent probe is meant at rs13181 SNP site on rs1048943 SNP site on the CYP1A1 gene and the ERCC2 gene and designs, can go out the probe of these two SNPs site lung cancer tumor susceptibility gene types by the fluorescent quantitative PCR technique specific detection.
4. kit according to claim 1 is characterized in that: contained Auele Specific Primer to be selected from primer with sequence shown in SEQ ID NO:1 and 2 to and to have a primer of sequence shown in SEQ ID NO:3 and 4 right.
5. kit according to claim 1 is characterized in that: contained specificity fluorescent probe is selected from the Taqman probe with sequence shown in SEQ ID NO:5 and 6.
6. kit according to claim 1 is characterized in that: the component of kit and content comprise 1 μ l 10X quantitative fluorescent PCR reaction buffer, 0.1 μ l 25mM dNTP mixed liquor, 0.5 μ l 25mM MgCl
2Solution, 0.02 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M Auele Specific Primers are to (four) each 0.225 μ l, and 10 μ M specificity fluorescent probes (two) are 0.25 μ l respectively, deionized water 4.98 μ l.This kit detects for a person-portion and uses, and the storage temperature of kit is-20 ℃.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100309538A CN101140232A (en) | 2006-09-08 | 2006-09-08 | Reagent kit for detecting pulmonary cancer susceptibility with CYP1A1 gene and ERCC2 gene |
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2006100309538A CN101140232A (en) | 2006-09-08 | 2006-09-08 | Reagent kit for detecting pulmonary cancer susceptibility with CYP1A1 gene and ERCC2 gene |
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| CN101140232A true CN101140232A (en) | 2008-03-12 |
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| CNA2006100309538A Pending CN101140232A (en) | 2006-09-08 | 2006-09-08 | Reagent kit for detecting pulmonary cancer susceptibility with CYP1A1 gene and ERCC2 gene |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662968A (en) * | 2020-06-23 | 2020-09-15 | 南方科技大学 | Detection method of PAM-sequence-free DNA based on CRISPR and application thereof |
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- 2006-09-08 CN CNA2006100309538A patent/CN101140232A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662968A (en) * | 2020-06-23 | 2020-09-15 | 南方科技大学 | Detection method of PAM-sequence-free DNA based on CRISPR and application thereof |
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Application publication date: 20080312 |