CN108342472A - A kind of coronary heart disease tumor susceptibility gene detection and genotyping kit - Google Patents
A kind of coronary heart disease tumor susceptibility gene detection and genotyping kit Download PDFInfo
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- CN108342472A CN108342472A CN201810331354.2A CN201810331354A CN108342472A CN 108342472 A CN108342472 A CN 108342472A CN 201810331354 A CN201810331354 A CN 201810331354A CN 108342472 A CN108342472 A CN 108342472A
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- heart disease
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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
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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 present invention relates to a kind of coronary heart disease tumor susceptibility gene detection and genotyping kits, including ADTRP, C6orf10 and CDKN2B AS1 gene specific upstream and downstream amplimer sequences, as shown in SEQ ID NO.1 6；And ADTRP, C6orf10 and CDKN2B AS1 alleles A, G specific probe sequences, as shown in SEQ ID NO.7 12.Kit of the present invention can conveniently and efficiently realize the detection to coronary heart disease on a molecular scale, detection efficiency is high, it is with strong points, in coronary disease disease early diagnosis, treatment, intervene and instruct the rational use of medicines, aid forecasting offspring's risk etc. that there is positive meaning.
Description
Technical field
The invention belongs to molecular biology and technical field of medical detection, more particularly to a kind of coronary heart disease tumor susceptibility gene detection
Parting kit.
Background technology
Coronary heart disease is to endanger one of human health or even the common disease of life, it has also become the primary disease of lethality, at present
China's cardiovascular patient about 2.9 hundred million, every year about 3,500,000 people die of cardiovascular disease.Although Chinese cardiovascular death in recent years
The ascendant trend of rate is eased up, but still causes white elephant to society and family.Coronary heart disease is by environmental factor and heredity
Caused by factor interaction, it is up to more than 200 kinds with the relevant risk factor of coronary heart disease, including principal element：Blood fat is different
Often, hypertension, smoking, hyperlipidemia, diabetes B, age-sex is fat, inflammation, eating habit, inherent cause etc., Yi Jiqian
In factor：Chronic inflammation, blood clotting factor level increase, insulin resistance etc..In numerous risk factors, inherent cause is being preced with
It plays an important role in worry pathogenic process.
Single nucleotide polymorphism (single nucleotide polymorphism, SNP) is most normal in human genome
One of hereditary variation seen refers to DNA base polymorphism caused by the change due to single nucleotide acid in the genome.SNP with
The research of coronary disease susceptibility relationship is a hot spot in current coronary heart disease genetic research field, in each race and group all
Have a large amount of research, have discovered that so far largely with the relevant tumor susceptibility gene of incidence of coronary heart disease, including lipid metaboli,
Key gene in immune inflammation, renin-angiotensin system is detected as assessment coronary heart disease genetic risk to tumor susceptibility gene
Important clue is provided with prophylactic treatment.
Whole-genome association research and development discovery, ADTRP, C6orf10, the polymorphism and hat of CDKN2B-AS1 gene locis
The morbidity of worry is significantly correlated, therefore, is detected to these three coronary heart disease tumor susceptibility genes, will appreciate that individual in terms of coronary heart disease
Inherent cause, assess the relative risk of incidence of coronary heart disease, the early diagnosis pair with coronary heart disease, early intervention has actively meaning
Justice.
Invention content
Technical problem to be solved by the invention is to provide a kind of coronary heart disease tumor susceptibility gene detection and genotyping kit, the present invention
Disclose the variant sites for coronary heart disease tumor susceptibility gene ADTRP, C6orf10, CDKN2B-AS1, ADTRP genetic mutation sites
Number is rs6903956, and C6orf10 genetic mutation sites number is rs9268402, CDKN2B-AS1 genetic mutation sites number
For rs10757274, the catastrophe in site and the illness rate of coronary heart disease are significantly correlated.
A kind of coronary heart disease tumor susceptibility gene detection and genotyping kit of the present invention, including：
ADTRP gene specific upstream and downstream amplimer sequences, as shown in SEQ ID NO.1-2：
Upstream amplification primer：5'-ACAGAGAGAGATTCCATCTC-3',
Downstream amplification primer：5'-GGGTTGAATTTATAGTATGG-3'；
C6orf10 gene specific upstream and downstream amplimer sequences, as shown in SEQ ID NO.3-4：
Upstream amplification primer：5'-CTTAATCAATGGAATATTAT-3',
Downstream amplification primer：5'-AATATATCTTGGAGTATGTC-3'；
CDKN2B-AS1 gene specific upstream and downstream amplimer sequences, as shown in SEQ ID NO.5-6：
Upstream amplification primer：5'-ATGGGAGGTACTGGTATTAC-3',
Downstream amplification primer：5'-CCCTCTGATTAGAATTCCC-3'；
ADTRP alleles A, G specific probe sequences, as shown in SEQ ID NO.7-8：
Allele A specific probes：5'-FAM-AGTGCCATAGATTATTACTTA-BHQ-3',
Allele G specific probes：5'-HEX-AGTGCCATAGGTTATTACTTA-BHQ-3'；
C6orf10 alleles A, G specific probe sequences are as shown in SEQ ID NO.9-10：
Allele A specific probes：5'-FAM-TGAACATCCAGATTGAAGAC-BHQ-3',
Allele G specific probes：5'-HEX-TGAACATCCGGATTGAAGAC-BHQ-3'；
The nucleotide sequence of CDKN2B-AS1 alleles A, G specific probes is as shown in SEQ ID NO.11-12：
Allele A specific probes：5'-FAM-TGAGTGTTGAGACATAATTG-BHQ-3',
Allele G specific probes：5'-HEX-TGAGTGTTGGGACATAATTG-BHQ-3'.
The kit further includes negative quality-control product, positive quality control product and blank control.
The detection object of the kit is human genome DNA.
Advantageous effect
Compared with prior art, present invention firstly discloses can be used for detect coronary heart disease correlation tumor susceptibility gene ADTRP,
The kit of C6orf10, CDKN2B-AS1 variant sites.Due to ADTRP, C6orf10, CDKN2B-AS1 gene polynorphisms become
Different significantly correlated with the illness rate of coronary heart disease, the kit based on detecting this three gene pleiomorphisms can conveniently exist
The detection to coronary heart disease risk is realized on molecular level, detection efficiency is high, with strong points, contributes to the early stage of coronary heart disease pre-
Anti- and treatment, at the same can also aided coronary heart disease diagnosis, instruct the rational use of medicines, aid forecasting offspring's risk etc..
Description of the drawings
Fig. 1 is the sites ADTRP gene rs6903956 genotyping result figure in embodiment 1.
Fig. 2 is the sites C6orf10 gene rs9268402 genotyping result figure in embodiment 1.
Fig. 3 is the sites CDKN2B-AS1 gene rs10757274 genotyping result figure in embodiment 1.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, people in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Embodiment 1
Utilize fluorescent quantitation detection of platform coronary heart disease tumor susceptibility gene polymorphism.
1, research object is raised：The ordinary circumstance of volunteer is investigated in the form of application form, while taking mucous membrane of mouth
Cell is put into 1.5ml centrifuge tubes, 4 DEG C of storages.
2, genomic DNA is extracted：Use the buccal swab extracting genome DNA reagent of Tiangeng biochemical technology Co., Ltd
Box, product identification DP322 extract Oral Mucosal Cells genomic DNA according to operating instruction.With 0.8% agarose gel electrophoresis
Determine DNA mass and concentration.
3, genetic polymorphism detection：Parting, the original substantially of this technology are carried out to polymorphic site using fluorescent quantitation technology
Reason：The oligonucleotides (probe) that 2 kinds of ends 5' use different fluorochrome labels, two probes point are introduced in PCR reaction systems
It can not be matched with the base of 2 kinds of genotype, the ends 3' of probe connect quenching group.Under normal circumstances, due to probe
With the ends 3' quenching group together with, fluorescence is quenched the ends 5' fluorophor.With the progress that PCR reacts, with genome sample
Originally probe that can be complementary is gradually cut by the 5 prime excision enzyme activity of archaeal dna polymerase 5' → 3', leads to the fluorescent base on the ends probe 5'
Group detaches with the quenching group at the ends 3', and quenching effect failure is detected glimmering to which fluorescent reporter group is activated by corresponding instrument
Light value can be detected；And can not be polymerize cleavage with another probe that template cannot be complementary, therefore can't detect phase
The fluorescence signal answered, according to the fluorescent signals wavelengths detected, it can be determined that the genotype of sample.This research uses
Primer3 online softwares carry out design of primers, then carry out primer synthesis, and specific primer is as shown in table 1.
The PCR primer of 1 ADTRP, C6orf10 and CDKN2B-AS1 gene magnification of table
4, fluorescent PCR detects：50ng DNA are added separately in respective 50ul PCR systems, wherein each reactant
System includes each pair of special primers of 100nmol, each pair of specific probes of 150nmol, 1X sonde method partings Mix (Tiangeng is biochemical, China,
FP211).Negative control is set simultaneously.Fluorescent PCR detector is CFX96 (biorad), and PCR response procedures are：95 DEG C of denaturation 3
Minute；95 DEG C are denaturalized 30 seconds, and 50 DEG C of renaturation extend 30 seconds, 35 cycles.Fluorescence signal is detected after final cycle.
5, Analysis of test results：Sample is detected using above-mentioned reaction system, uses Bio-Rad CFX Manager
(biorad) software automatically analyzes gathered data, ADTRP gene rs6903956 site phenotypic analysis result such as Fig. 1 institutes
Show, orbicular spot indicates that FAM signal values are significantly higher than HEX signal values, is enriched in X-coordinate axle, and interpretation is AA genotype, triangle table
Show that FAM signal values are close with HEX signal values, be enriched in middle part, interpretation is AG genotype, and square indicates that HEX signal values are significantly high
In FAM signal values, it is enriched in Y-coordinate axle, interpretation is GG genotype, and diamond shape is enriched in coordinate origin, is negative control.
The results are shown in Figure 2 for the sites C6orf10 gene rs9268402 phenotypic analysis, and orbicular spot indicates that FAM signal values are significantly higher than
HEX signal values are enriched in X-coordinate axle, and interpretation is AA genotype, and triangle indicates that FAM signal values are close with HEX signal values, enrichment
In middle part, interpretation is AG genotype, and square indicates that HEX signal values are significantly higher than FAM signal values, is enriched in Y-coordinate axle, interpretation is
GG genotype, diamond shape are enriched in coordinate origin, are negative control.The sites CDKN2B-AS1 gene rs10757274 phenotypic analysis knot
Fruit is enriched in X-coordinate axle, interpretation is AA genes as shown in figure 3, its orbicular spot indicates that FAM signal values are significantly higher than HEX signal values
Type, triangle indicate that FAM signal values are close with HEX signal values, are enriched in middle part, and interpretation is AG genotype, and square indicates HEX signals
Value is significantly higher than FAM signal values, is enriched in Y-coordinate axle, and interpretation is GG genotype, and diamond shape is enriched in coordinate origin, is negative right
According to.
Comparative example 1
Using other common detection method (PCR-LDR, SNaPshot, Sanger sequencing, restriction fragment length polymorphisms
Property) sample is detected, and carry out comparing result with the detection scheme of kit of the present invention and be shown in Table 2, it is known that it is comprehensive every right
Than the kit of the present invention known to parameter, time-consuming most short, detection efficiency is apparently higher than other detection methods.
The different detection method comparing results of table 2
SEQUENCE LISTING
<110>Donghua University
<120>A kind of coronary heart disease tumor susceptibility gene detection and genotyping kit
<130> 1
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
acagagagag attccatctc 20
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<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
gggttgaatt tatagtatgg 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
cttaatcaat ggaatattat 20
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<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
aatatatctt ggagtatgtc 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<400> 5
atgggaggta ctggtattac 20
<210> 6
<211> 19
<212> DNA
<213>Artificial sequence
<400> 6
ccctctgatt agaattccc 19
<210> 7
<211> 21
<212> DNA
<213>Artificial sequence
<400> 7
agtgccatag attattactt a 21
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<211> 21
<212> DNA
<213>Artificial sequence
<400> 8
agtgccatag gttattactt a 21
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<400> 9
tgaacatcca gattgaagac 20
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<400> 10
tgaacatccg gattgaagac 20
<210> 11
<211> 20
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<213>Artificial sequence
<400> 11
tgagtgttga gacataattg 20
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<400> 12
tgagtgttgg gacataattg 20
Claims (3)
1. a kind of coronary heart disease tumor susceptibility gene detection and genotyping kit, it is characterised in that：Including ADTRP, C6orf10 and CDKN2B-
AS1 gene specific upstream and downstream amplimer sequences, as shown in SEQ ID NO.1-6；And ADTRP, C6orf10 and
CDKN2B-AS1 alleles A, G specific probe sequences, as shown in SEQ ID NO.7-12.
2. a kind of coronary heart disease tumor susceptibility gene detection and genotyping kit according to claim 1, it is characterised in that：The reagent
Box further includes negative quality-control product, positive quality control product and blank control.
3. a kind of coronary heart disease tumor susceptibility gene detection and genotyping kit according to claim 1, it is characterised in that：The reagent
The detection object of box is human genome DNA.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810331354.2A CN108342472A (en) | 2018-04-13 | 2018-04-13 | A kind of coronary heart disease tumor susceptibility gene detection and genotyping kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810331354.2A CN108342472A (en) | 2018-04-13 | 2018-04-13 | A kind of coronary heart disease tumor susceptibility gene detection and genotyping kit |
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| Publication Number | Publication Date |
|---|---|
| CN108342472A true CN108342472A (en) | 2018-07-31 |
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ID=62954946
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810331354.2A Pending CN108342472A (en) | 2018-04-13 | 2018-04-13 | A kind of coronary heart disease tumor susceptibility gene detection and genotyping kit |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101668865A (en) * | 2007-02-21 | 2010-03-10 | 解码遗传学私营有限责任公司 | Genetic susceptibility variants associated with cardiovascular disease |
| CN102757954A (en) * | 2012-06-07 | 2012-10-31 | 中国医学科学院阜外心血管病医院 | Combination of multiple genetic single nucleotide polymorphisms related to coronary heart disease and application of combination |
| US20170145486A1 (en) * | 2015-11-25 | 2017-05-25 | Integrated Dna Technologies, Inc. | Methods for variant detection |
| CN106987618A (en) * | 2016-01-21 | 2017-07-28 | 张培祥 | The multiple gene mononucleotide polymorphism Sites Combinations related to statins personalized medicine and its application |
-
2018
- 2018-04-13 CN CN201810331354.2A patent/CN108342472A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101668865A (en) * | 2007-02-21 | 2010-03-10 | 解码遗传学私营有限责任公司 | Genetic susceptibility variants associated with cardiovascular disease |
| CN102757954A (en) * | 2012-06-07 | 2012-10-31 | 中国医学科学院阜外心血管病医院 | Combination of multiple genetic single nucleotide polymorphisms related to coronary heart disease and application of combination |
| US20170145486A1 (en) * | 2015-11-25 | 2017-05-25 | Integrated Dna Technologies, Inc. | Methods for variant detection |
| CN106987618A (en) * | 2016-01-21 | 2017-07-28 | 张培祥 | The multiple gene mononucleotide polymorphism Sites Combinations related to statins personalized medicine and its application |
Non-Patent Citations (2)
| Title |
|---|
| BIO-RAD: "Real-Time PCR Application Guide", 《BIO-RAD LABORATORIES INC》 * |
| XIANG-FENG LU等: "Genome-wide association study in Han Chinese identifies four new", 《NAT GENET.》 * |
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Application publication date: 20180731 |