CN106636396A - Detection method for polymorphism of hyperlipidemia risk associated genes - Google Patents
Detection method for polymorphism of hyperlipidemia risk associated genes Download PDFInfo
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Abstract
The invention relates to a detection method for polymorphism of hyperlipidemia risk associated genes. The detection method comprises the following steps: 1) extracting a DNA (Deoxyribonucleic Acid) template; 2) carrying out a PCR (Polymerase Chain Reaction) amplification reaction: utilizing a PCR reaction system in a kit, wherein the PCR reaction system comprises Taq DNA polymerase, a dNTP (deoxyribonucleoside triphosphate) mixed solution, PCR buffer and ddH2O and the like; 3) purifying a PCR amplified product: utilizing a PCR product purification system in the kit, wherein the PCR product purification system comprises an SAP enzyme, an Exo I enzyme and ddH2O and the like; carrying out a reaction on a PCR amplification device; 4) carrying out DAN sequencing: carrying out a reaction on the PCR amplification device; after reacting, adding an EDTA (Ethylene Diamine Tetraacetic Acid) solution and a 10% ethanol solution; centrifuging and adding an HIDI solution, and putting into a sequencing machine; and 5) carrying out gene type analysis. By adopting the detection method provided by the invention, the effects of taking prevention measures as soon as possible and reducing the morbidity of hyperlipidemia are realized.
Description
[technical field]
The invention belongs to biology field, gene genetic risk assessment is applied to, specifically one kind can be from
Gene level assesses the hyperlipidemia risk association gene pleiomorphism detecting method of hyperlipidemia risk.
[background technology]
With the continuous improvement of people's living standard, ordinary meal structure has great changes, related to nutrient imbalance
Chronic disease is increasingly becoming the principal disease of China's death, particularly cardiovascular and cerebrovascular disease, cardiovascular and cerebrovascular disease mainly by
Atherosclerotic causes, and hyperlipidemia (hyperlipidemia；HLP it is) then to cause atherosclerotic primary danger
Factor, the morbidity of hyperlipidemia is by multifactor impacts such as h and Es.Now studies have found that multiple genes with blood lipid metabolism phase
Close, C112R loci polymorphisms (rs429358) and R158C loci polymorphisms (rs7412) on APOE (apo E) gene,
LPL (lipoprotein lipase) gene Hind Ⅲ loci polymorphism (rs320) and MTHFR (MTHFR) gene
C677T loci polymorphisms (rs1801133) are closely related with HLP, and its polymorphic detection can suffer from hyperlipidemia wind as person under inspection
The basis for forecasting of danger.
APOE (apo E) participates in chylomicron and intermediated-density lipoprotein metabolism.APOE genes mainly have three etc.
Position Gene A POE2, APOE3 and APOE4, the difference of these three allele is the 112nd and the 158th amino acids residue is
Cysteine or arginine.APOE4 genes have more preferable metabolic capability to cholesterol compared to APOE3 and APOE2, and
The efficiency of the protein metabolism cholesterol of APOE2 is minimum in these three allele, so the homozygous people of APOE2 suffer from blood vessel
The probability of disease and III type hyperlipidemia will be far above other people.Therefore detection APOE genotype can be predicted and suffer from hyperlipidemia
Risk.
LPL (lipoprotein lipase) is the rate-limiting enzyme of plasma triglyceride level metabolism, plays extremely important in lipid-metabolism
Effect, there are some researches show the restriction enzyme site polymorphisms of Hind III and the close phase of the generation of hyperlipidemia in the area of lpl gene introne 8
Close, be to sport the restriction enzyme sites of Hind III disappearance that G causes by the T of 495 to cause, will be defined as without the restriction enzyme sites of Hind III
H-, have the restriction enzyme sites of Hind III to be defined as H+, current research shows, with H-Allele is compared, H+Allele and low LPL
Activity is relevant, i.e., the restriction enzyme site T allele of LPL Hind III is the risks and assumptions of hyperlipidemia.
Key enzyme in MTHFR (MTHFR) encoding folate metabolic pathway, homocysteine
(HCY) methyl turns to methionine, and C of the mthfr gene on 677 sites sports T causes the reduction of its coding enzymatic activity, suppression
HCY processed is converted into methionine, causes high HCY mass formed by blood stasis and low methioninemia, and the rising of HCY levels is to cause hyperlipemia
Important, the independent hazards of disease.
In view of the important function of APOE, LPL and mthfr gene during blood lipid metabolism, said gene polymorphism is made
Carry out the crowd that examination is susceptible to suffer from hyperlipidemia for predisposing factor, take preventive measures as early as possible, for the incidence of disease for reducing hyperlipidemia
It is significant.
[content of the invention]
Present invention aim to solve above-mentioned deficiency and provide a kind of hyperlipidemia risk association gene pleiomorphism
Detection method, can carry out the crowd that examination is susceptible to suffer from hyperlipidemia using forementioned gene polymorphism as predisposing factor, and then can be as early as possible
Take preventive measures, reduce the incidence of disease of hyperlipidemia.
A kind of hyperlipidemia risk association gene pleiomorphism detecting method, including following step are designed for achieving the above object
Suddenly：
1) DNA profiling is extracted.DNA is extracted using DNA extraction kit, the kit includes：On detection APOE genes
C677T on Hind III (rs320) and mthfr gene on C112R (rs429358) and R158C (rs7412), lpl gene
(rs1801133) specific primer and sequencing primer of 4 mononucleotide polymorphism site genotype；
2) pcr amplification reaction.Using the PCR reaction systems in kit, the PCR reaction systems are polymerized including Taq DNA
Enzyme, dNTP mixed liquors, PCR buffer and ddH2O；Reaction condition is：94 DEG C of denaturations 3min, 94 DEG C of denaturation 30 seconds, annealing 30
Second, 72 DEG C of extension 1min expand 35 circulations, and 72 DEG C finally extend 10min, are stored in 4 DEG C；
3) pcr amplification product purifying.Using the PCR primer purification system in kit, the PCR primer purification system includes
SAP enzymes, Exo I enzymes and ddH2O；Reacted in PCR amplification instrument, reaction condition is 37 DEG C of 15min, 72 DEG C of 20min；
4) DNA sequencing.Sequencing reaction system is drawn comprising PCR purified products, 25%BigDye mix, 3.2uM DNA sequencings
Thing, deionized water；Reacted in PCR amplification instrument：98 DEG C of 2min, carry out 25 circulation 96 DEG C 30 seconds, 55 DEG C 30 seconds, 60
℃4min；Reaction adds 125mM EDTA solution and 100% ethanol solution to precipitate 15min at room temperature after terminating；At 4 DEG C,
3600rpm/min is centrifuged 30min, gently removes supernatant, adds 70% ethanol solution, 3600rpm/min that 15min is centrifuged,
Go supernatant, room temperature to place after 20min, HIDI solution is added, in being put into sequenator；
5) genotyping：SNP site genotype can directly be read by Sequencing chromatogram.
Further, step 2) in, in terms of 25 μ L, its component and content are the volume of PCR reaction systems：5×PCR
Buffer5 μ L, 5U/ μ L Taq archaeal dna polymerases 0.25 μ L, 2.5mM dNTP mixed liquors 2 μ L, ddH2The μ L of O 10.75, on 10 μM
The each 2.5 μ L of downstream primer, the μ L of DNA profiling 2.
Further, step 2) in, the annealing temperature of C112R and R158C is 63 DEG C on APOE genes, on lpl gene
The annealing temperature of Hind III is 57 DEG C, and the annealing temperature of C677T is 57 DEG C on mthfr gene.
Further, step 3) in, in terms of 25 μ L, its component and content are the volume of PCR primer purification system：20μ
LPCR products, the μ L of 1U/ μ L SAP enzymes 0.75,10U/ μ L Exo I enzyme 0.375U/ μ L, ddH2O 3.875μL。
Further, step 4) in, in terms of 5 μ L, its component and content are the cumulative volume of sequencing reaction system：PCR is purified
The μ L of product 1,25%BigDye mix1 μ L, the μ L of 3.2uM DNA sequencings primer 1, the μ L of deionized water 2, the μ L of 125mM EDTA solution 1,
The μ L of 100% ethanol solution 15, the μ L of 70% ethanol solution 30, the μ L of HIDI solution 8.
Further, step 4) in, each gene sequencing primer is：
APOE(C112R/R158C)：5′-CACTGTGCGACACCCTCCCC-3′；
LPL(HindⅢ)：5′-TCATTTGGCACTGTTTCTTGC-3′；
MTHFR(C677T)：5′-TCAAGGCAGGACAGTGTGGGAGTTC-3′.
The present invention is compared with the existing technology, there is provided a kind of hyperlipidemia risk association gene pleiomorphism detecting method, should
Method initially with DNA extraction kit extract DNA, kit include detection APOE genes on C112R (rs429358) and
4 mononucleotides of C677T (rs1801133) are more on Hind III (rs320) and MTHFR on R158C (rs7412), lpl gene
The specific primer and sequencing primer of state property (SNP) loci gene type, while being produced using the PCR reaction systems in kit, PCR
Thing purification system, DNA sequencing system, and with the use of the rational component of each system and content, it is straight eventually through Sequencing chromatogram
Reading SNP site genotype is connect, according to testing result, with reference to the work of APOE, LPL and mthfr gene during blood lipid metabolism
With, carry out the crowd that examination is susceptible to suffer from hyperlipidemia using said gene polymorphism as predisposing factor, and then can as early as possible take prevention to arrange
Apply, reduce the incidence of disease of hyperlipidemia.
[description of the drawings]
Fig. 1 is the testing result schematic diagram of (C112R) on embodiment of the present invention APOE gene；
Fig. 2 is the testing result schematic diagram of (R158C) on embodiment of the present invention APOE gene.
[specific embodiment]
The invention provides a kind of hyperlipidemia risk association gene pleiomorphism detecting method, arrives used in method
DNA extraction kit includes：Hind on C112R (rs429358) and R158C (rs7412), lpl gene on detection APOE genes
The specificity of 4 SNP (SNP) loci gene types of C677T (rs1801133) on III (rs320) and MTHFR
Primer and sequencing primer.PCR reaction systems, PCR primer purification system in kit, DNA sequencing system are respectively：PCR is anti-
System is answered to include Taq archaeal dna polymerases, dNTP mixed liquors, PCR buffer and ddH2O etc.；PCR primer purification system includes SAP
Enzyme, Exo I enzymes and ddH2O etc.；DNA sequencing system includes Big Dye Mix, EDTA solution, 100% ethanol solution, 70% second
Alcoholic solution, HIDI solution and ddH2O etc..
The concrete component and content of the kit be：25 μ LPCR reaction systems：5×PCR buffer 5μL；5U/μL
The μ L of Taq archaeal dna polymerases 0.25；The μ L of 2.5mM dNTP mixed liquors 2；ddH2O 10.75μL；The each 2.5 μ L of 10 μM of upstream and downstream primers；
The μ L of DNA profiling 2.25 μ LPCR purification systems：20 μ LPCR products；The μ L of 1U/ μ L SAP enzymes 0.75；10U/ μ L Exo I enzymes
0.375U/μL；ddH2O 3.875μL.Sequencing reaction system：The μ L of 25%BigDye mix 1；3.2 μM of μ L of DNA sequencing primer 1；
The μ L of 125mM EDTA solution 1；The 100% μ L of ethanol solution 15；The μ L of 70% ethanol solution 30；The μ L of HIDI solution 8；ddH2O 2μL。
Further explained below is made to the present invention with reference to specific embodiment：
1. DNA profiling is extracted
Scraping Buccal mucosa cell, is DNA extraction kit extracting DNA with health.
2.PCR amplified reactions
Using PCR reaction components in kit, wherein each gene primer sequence is respectively such as following table：
PCR reaction systems：5×PCR buffer 5μL；The μ L of 5U/ μ L Taq archaeal dna polymerases 0.25；2.5mM dNTP are mixed
Close the μ L of liquid 2；ddH2O 10.75μL；The each 2.5 μ L of 10 μM of upstream and downstream primers；The μ L of DNA profiling 2.
Reaction condition：94 DEG C of denaturations 3min, 94 DEG C of denaturation 30 seconds, (different genes are by corresponding annealing temperature within 30 seconds for annealing
Degree), 72 DEG C of extension 1min expand 35 circulations, and 72 DEG C finally extend 10min, are stored in 4 DEG C.
3.PCR amplified productions are purified
Using the PCR primer purification componentry in kit：20 μ LPCR products；The μ L of 1U/ μ L SAP enzymes 0.75；10U/μL
Exo I enzyme 0.375U/ μ L；ddH2O 3.875μL。
Reacted in PCR amplification instrument：Reaction condition is 37 DEG C of 15min, 72 DEG C of 20min.
4.DNA is sequenced：
Each gene sequencing primer is as follows：
APOE(C112R/R158C)：5′-CACTGTGCGACACCCTCCCC-3′
LPL(HindⅢ)：5′-TCATTTGGCACTGTTTCTTGC-3′
MTHFR(C677T)：5′-TCAAGGCAGGACAGTGTGGGAGTTC-3′
The system of reaction is the μ L of cumulative volume 5, comprising the μ L of PCR purified products 1,25%BigDye mix1 μ L, 3.2uM DNA
The μ L of sequencing primer 1, the μ L of deionized water 2.
Reacted in PCR amplification instrument：98 DEG C of 2min, carry out 25 circulation 96 DEG C 30 seconds, 55 DEG C 30 seconds, 60 DEG C
4min。
Reaction adds the μ L and μ L of 100% ethanol solution 15 of 125mM EDTA solution 1 to precipitate 15min at room temperature after terminating；
At 4 DEG C, 3600rpm/min centrifugation 30min gently remove supernatant, add 70% ethanol solution 30 μ L, 3600rpm/min
Centrifugation 15min, goes supernatant, room temperature to place after 20min, the μ L of HIDI solution 8 is added, in being put into sequenator.
5. genotyping
SNP site genotype can directly be read by Sequencing chromatogram.For example to the testing result of APOE (C112R/R158C)
As shown in Figures 1 and 2, wherein, accompanying drawing 1 is C112R (466T>C) schematic diagram, accompanying drawing 2 is R158C (604C>T) illustrate
Figure, because 112 and 158 sites are not undergone mutation, therefore the tester carries APOE3 allele, without suffering from senile dementia risk.
The present invention is not limited by above-mentioned embodiment, other any Spirit Essences and principle without departing from the present invention
Lower made change, modification, replacement, combination, simplification, should be equivalent substitute mode, be included in the protection model of the present invention
Within enclosing.
Claims (6)
1. a kind of hyperlipidemia risk association gene pleiomorphism detecting method, it is characterised in that comprise the following steps：
1) DNA profiling is extracted
DNA is extracted using DNA extraction kit, the kit includes：Detection APOE genes on C112R (rs429358) and
On R158C (rs7412), lpl gene on Hind III (rs320) and mthfr gene C677T (rs1801133) 4 monokaryons
The specific primer and sequencing primer of nucleotide polymorphism loci gene type；
2) pcr amplification reaction
Using the PCR reaction systems in kit, the PCR reaction systems include Taq archaeal dna polymerases, dNTP mixed liquors, PCR
Buffer and ddH2O；
Reaction condition is：94 DEG C of denaturations 3min, 94 DEG C of denaturation 30 seconds are annealed 30 seconds, and 72 DEG C of extension 1min, amplification 35 is followed
Ring, 72 DEG C of last extension 10min, is stored in 4 DEG C；
3) pcr amplification product purifying
Using the PCR primer purification system in kit, the PCR primer purification system includes SAP enzymes, Exo I enzymes and ddH2O；
Reacted in PCR amplification instrument, reaction condition is 37 DEG C of 15min, 72 DEG C of 20min；
4) DNA sequencing：
Sequencing reaction system includes PCR purified products, 25%BigDye mix, 3.2uM DNA sequencing primers, deionized water；
Reacted in PCR amplification instrument：98 DEG C of 2min, carry out 25 circulation 96 DEG C 30 seconds, 55 DEG C 30 seconds, 60 DEG C of 4min；
Reaction adds 125mM EDTA solution and 100% ethanol solution to precipitate 15min at room temperature after terminating；At 4 DEG C,
3600rpm/min is centrifuged 30min, gently removes supernatant, adds 70% ethanol solution, 3600rpm/min that 15min is centrifuged,
Go supernatant, room temperature to place after 20min, HIDI solution is added, in being put into sequenator；
5) genotyping
SNP site genotype can directly be read by Sequencing chromatogram.
2. the method for claim 1, it is characterised in that：Step 2) in, the volume of PCR reaction systems in terms of 25 μ L, its
Component and content are：The μ L of 5 × PCR buffer 5,5U/ μ L Taq archaeal dna polymerases 0.25 μ L, the μ L of 2.5mM dNTP mixed liquors 2,
ddH2The μ L of O 10.75,10 μM of upstream and downstream primers each 2.5 μ L, the μ L of DNA profiling 2.
3. method as claimed in claim 2, it is characterised in that：Step 2) in, the annealing of C112R and R158C on APOE genes
Temperature is 63 DEG C, and the annealing temperature of Hind III is 57 DEG C on lpl gene, and the annealing temperature of C677T is 57 DEG C on mthfr gene.
4. the method for claim 1, it is characterised in that：Step 3) in, the volume of PCR primer purification system is with 25 μ L
Meter, its component and content are：20 μ LPCR products, the μ L of 1U/ μ L SAP enzymes 0.75,10U/ μ L Exo I enzyme 0.375U/ μ L, ddH2O
3.875μL。
5. the method for claim 1, it is characterised in that：Step 4) in, the cumulative volume of sequencing reaction system in terms of 5 μ L,
Its component and content are：The μ L of PCR purified products 1,25%BigDye mix1 μ L, the μ L of 3.2uM DNA sequencings primer 1, deionized water
The μ L of 2 μ L, 125mM EDTA solution 1, the μ L of 100% ethanol solution 15, the μ L of 70% ethanol solution 30, the μ L of HIDI solution 8.
6. method as claimed in claim 5, it is characterised in that step 4) in, each gene sequencing primer is：
APOE(C112R/R158C)：5′-CACTGTGCGACACCCTCCCC-3′；
LPL(HindⅢ)：5′-TCATTTGGCACTGTTTCTTGC-3′；
MTHFR(C677T)：5′-TCAAGGCAGGACAGTGTGGGAGTTC-3′.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852956A (en) * | 2021-03-23 | 2021-05-28 | 上海康黎诊断技术有限公司 | Kit for guiding medication of human hyperlipidemia |
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- 2016-12-21 CN CN201611189889.8A patent/CN106636396A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852956A (en) * | 2021-03-23 | 2021-05-28 | 上海康黎诊断技术有限公司 | Kit for guiding medication of human hyperlipidemia |
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