CN108588207A - Detect the other primer of APOE genotype and method - Google Patents
Detect the other primer of APOE genotype and method Download PDFInfo
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Abstract
Gene related with the cardiovascular and cerebrovascular diseases such as atherosclerosis, coronary heart disease and senile dementia disease is detected the invention discloses a kind of：The other primer of ApoE genotype and method, the primer is used for the 4th exon sequence of amplification gene, and uses Sanger sequencing technologies.The present invention can rapidly detected the base situation of the coding of ApoE genes the 112nd and the 158th amino acids sequence so that it is determined that its gene type.The testing result completed using the present invention is accurate, the early diagnosis to atherosclerosis and senile dementia disease, and carries out individualized treatment with important reference significance.
Description
Technical field
The invention belongs to life sciences and biotechnology, more particularly to detection and atherosclerosis and senile dementia
The other primer of the related APOE genotype of disease and method.
Background technology
Apo E (Apolipoprotein E, ApoE) is the protein of 299 amino acid, molecular weight 34kDa.It is
A kind of main cholesterol carrier has the function of supporting lipid transfer and repair cerebral injury.ApoE genes are located at No. 19 chromosomes
On long-armed, overall length about 3.7kb has 4 exons, the 4th exon to show gene pleiomorphism, ApoE genes is made to have E2, E3, E4
3 kinds of allele can combine and generate 6 kinds of genotype：Three kinds of homozygotes (E2/2, E3/3, E4/4) and three kinds of heterozygotes (E3/2,
E4/2、E4/3).There is ApoE genes isomers specificity, the i.e. protein function of different genes coding to have otherness, to
Lead to that different proteins are different to the metabolic capability of lipid, anti-oxidant and anti-inflammatory ability is different.The amino acid sequence of ApoE
The variation of the 112nd and 158 two kinds of amino acid residue, that is, arginine (Arg) and cysteine (Cys) determines its genotype
Not.ApoE4 is Arg on the two positions；ApoE2 is Cys；It is Arg person is ApoE3 that 112, which are Cys and 158,.From
In right crowd, gene frequency E 3 is distributed highest, ApoE3/3 Phenotype Distributions accounting about 70%.
Alzheimer disease (Alzheimer ' s disease, AD) is a kind of the lethal of the progress sexual development of onset concealment
Nerve system degenerative disease.The cause of disease is unknown so far.Patient was sent out before 65 years old, claimed alzheimer's disease；Patient is sent out after 65 years old
Claim senile dementia.It ranked sixth in the crowd of all death in the U.S., estimate the National People's Congress about 13% of over-65s, 85 years old or more
About 45% has AD.Therefore, the risk factor for inquiring into AD morbidities has important meaning to morning discovery, prevention and the control of Susceptible population
Justice.
In past many decades, the study found that ApoE genes type is related with the morbidity of AD, ApoE gene types are AD
The central genetic determinant of onset risk.It is generally acknowledged that ApoE E2 be protection sexual factor and ApoEE4 is risk factors.
Coronary heart disease (Coronary Atherosclerotic Heart Disease, CHD) is most common in global range
The cause of death, LDL-C levels increase be CHD the most important cause of disease.Effectively reducing LDL-C levels can prevent artery athero-
Hardening progress, to reduce the incidence and the death rate of CHD.Statins are current most effective reduction blood plasma LDL-C water
It puts down to reduce the drug of risk of cardiovascular diseases.Studies have shown that the pharmacokinetics of statins is there are significant individual difference,
And this species diversity may be to statins the effect of and adverse reaction have an impact.This species diversity and ApoE genotype
Also there is not substantial connection.The effect of ApoE2 is to reduce cholesterol concentration, has certain protection to make the development of coronary sclerosis
With, and ApoE 4 can then be such that blood plasma LDL-C, TC concentration increases, and easily induce CHD.
ApoE genes type also occurs with the early stage of atherosclerosis (Atherosclerosis, AS) and develops close
It is related.A large amount of census of population have found that ApoEE4 can significantly increase the total cholesterol concentration of Healthy People, are allowed to be susceptible to suffer from artery congee
Sample hardens, on the contrary, ApoEE2 can reduce cholesterol concentration.
The diagnosis of patient disease can be helped to the other detection of ApoE genotype and instruct personalized medicine, improve patient's
Prognostic level.
Invention content
The object of the present invention is to provide a kind of other primers of detection APOE genotype, using round pcr, can be used for quickly examining
Survey cardiovascular and cerebrovascular diseases and the senile dementia disease patient's body APOE gene types such as atherosclerosis, coronary heart disease.The inspection
The other primer of APOE genotype is surveyed, including：
ApoE-Exon4-F：AACAACTGACCCCGGTGGCG
ApoE-Exon4-R：ACCTGCTCCTTCACCTCGTC
Further include sequencing primer, base sequence is：
Sequencing primer 1：AACAACTGACCCCGGTGGCG
Sequencing primer 2：ACCTGCTCCTTCACCTCGTC
The present invention provides detection APOE genotype method for distinguishing, include the following steps：
1. extracting the genomic DNA in peripheral blood；
2. with the DNA extracted in PCR amplification step 1；
3. the amplified production in pair step 2 is sequenced；
4. a pair sequencing result judges, APOE gene types are determined：
The present invention also provides a kind of other kit of detection APOE genotype, including Whole Blood Genomic DNA extraction agent,
Detection architecture PCR reaction solution and sequencing system reagent；Amplimer sequence is：
ApoE-Exon4-F：AACAACTGACCCCGGTGGCG
ApoE-Exon4-R：ACCTGCTCCTTCACCTCGTC.
Wherein it is for the reaction system of PCR：Total volume 20ul, 2*PCR Buffer10ul, d NTP (2mM) 4ul,
APOE-Exon4-F (10uM) 0.5uL, APOE-Exon4-R (10uM) 0.5uL, KOD FX (1U/ul) 0.4ul, Template
DNA 1ul, ddH2O 3.6uL。
The kit is 10ng/ μ l for Template DNA minimum concentrations in the reaction system of PCR.
The kit is for the reaction condition of PCR：
Advantageous effect：Since ApoE polymorphisms have biologic importance, the research to ApoE polymorphisms is current medicine
Hot issue.There are many other detection method of ApoE genotype, successively use isoelectric focusing electrophoresis and genetic analysis art for many years
It is detected.And this method is easy, at low cost, effect is good.Pair of primers is only needed just to can determine that ApoE gene types.And phase
It is fairly simple to the judgement of result compared with for other methods.It is easy to spread.
Description of the drawings
Fig. 1 is the gel electrophoresis figure for examining an example normal population peripheral blood APOE gene primer effects, totally 20 repetitions, M
For Marker DL 2000, as shown in Figure 1, primer amplification is effective, and band is single.
Fig. 2 is the electrophoretogram of the optimization of annealing temperature：APOE gene grads PCR Gel electrophoresis results.M is Marker DL
1000, as shown in Fig. 2, optimum annealing temperature is 69 DEG C.
Fig. 3 is the electrophoretogram of the optimization of the template concentrations of three parallel samples, and M is Marker DL 2000, by upper figure
Electrophoresis result is it is found that primer is that occur band at 1ng/ μ l very dark or without band in template concentrations, thus minimum template concentrations
For 10ng/ μ l.(1 representative sample 1,2 representative samples 2,3 representative samples 3 in figure)
The 112nd amino acids site generation sequencing result of ApoE genes of Fig. 4 clinical samples, as shown in figure 4,388 alkali
Base site is T.
The 158th amino acids site generation sequencing result of ApoE genes of Fig. 5 clinical samples, as shown in figure 5,526 alkali
Base site is C.
APOE gene types known to complex chart 4 and Fig. 5 results are E3.
Specific implementation mode
With reference to specific embodiments and the drawings, the present invention is further explained.It should be noted that not specified in embodiment
Normal condition and method, usually according to fields experimenter routinely use method：For example, Ao Sibai and James Kingston chief editor
's《Fine works molecular biology experiment guide》Fourth edition, or according to the step and condition proposed by manufacturer.
Embodiment 1 detects the other primer of APOE genotype and is to combine
A kind of other primer of detection APOE genotype, the design of the primer are the amplifications for the design of the 4th exons of APOE
Primer：
ApoE-Exon4-F：AACAACTGACCCCGGTGGCG
ApoE-Exon4-R：ACCTGCTCCTTCACCTCGTC
Sequencing primer is：
Sequencing primer 1：AACAACTGACCCCGGTGGCG
Sequencing primer 2：ACCTGCTCCTTCACCTCGTC
A kind of kit of detection APOE genes-type, including
(i) Whole Blood Genomic DNA extraction agent；
(ii) detection architecture PCR reaction solution；
(iii) system reagent is sequenced；
Wherein, peripheral blood extracting genome DNA can be used the kit of Tiangeng bio tech ltd, and blood/cell/
The DNA extractings of tissue gene group can refer to the operating process of kit (Tiangeng biology).
Embodiment 2 detects APOE genotype method for distinguishing
APOE genotype method for distinguishing is detected, is included the following steps：
(1) Whole Blood Genomic DNA is extracted
1) 20 μ l QIAGEN Protease (or proteinase K) are added in 1.5ml centrifugation bottom of the tube..
2) 200 μ l blood plasma are added in centrifuge tube.
3) 200 μ l Buffer AL are added, vibrate 15s.(pay attention to：It can not be by QIAGEN Protease or proteinase
K is added directly into Buffer AL.If sample size is larger, QIAGEN Protease and Buffer AL are scaling up.)
4) 56 DEG C of water-bath 10min, of short duration centrifugation later, to remove the liquid on edge in centrifuge tube lid.
5) 200 μ l ethyl alcohol (96%-100%) are added, vibrate 15s, of short duration centrifugation, to remove in centrifuge tube lid along liquid.
6) carefully mixed liquor obtained as above (including sediment) is carefully added into QIAamp Mini spin column
Centrifugal column is put into 2ml collecting pipes by (without wetting the rim), centrifuge tube lid is covered tightly, with 6000 × g
(8000rpm) centrifuges 1min.Centrifuge tube is taken out, is put into a clean 2ml collecting pipe (kit offer).It abandons and collects
Pipe and wherein liquid.
7) (without wetting in 500 μ l Buffer AW1 to QIAamp Mini spin column are carefully added into
the rim).Centrifuge tube lid is covered tightly, 1min is centrifuged with 6000 × g (8000rpm).Centrifuge tube is taken out, be put into one it is clean
In 2ml collecting pipes (kit offer).Abandon collecting pipe and wherein liquid.
8) (without wetting in 500 μ l Buffer AW2 to QIAamp Mini spin column are carefully added into
the rim).Centrifuge tube lid is covered tightly, 3min is centrifuged with 20000 × g (14000rpm).
9) QIAamp Mini spin column are placed in a new 2ml collecting pipe and (be provided for oneself), abandon collecting pipe and
Wherein liquid.Centrifugation 1min at full speed.
10) QIAamp Mini spin column are placed in a cleaning 1.5ml collecting pipe and (are provided for oneself), abandoned and collect
Pipe and wherein liquid.100 μ l Buffer AE or distilled water are carefully added in QIAamp Mini spin column.Room temperature
Under (15-25 DEG C) standing 1min, 6000 × g (8000rpm) centrifuge 1min.Collecting pipe is covered, is stored in -20 DEG C.
(2) reagent configures：Each X μ l of detection architecture PCR reaction solution are configured by detection person-portion number, per 19 μ l packing of person-portion：
X=19 μ l reaction solutions × (+1 part of blank control of n parts of samples)
N is detection number of samples.
(3) it is loaded：DNA is added in the PCR reaction tubes dispensed, blank control adds 1 μ l physiological saline or is not added with any
Substance.Sample-adding requires as follows：
(4) it expands：Detection carries out on Standard PCR instrument, can include ABI veriti (U.S. Applied with instrument
Biosystems companies) etc..Reaction condition is as follows：
KOD FOX used in PCR are provided by TOYOBO companies.Primer is synthesized by Shanghai Invitrogen Corp..
PCR amplification system preparation of reagents method is as follows：
(5) electrophoresis：1.5% agarose gel electrophoresis, 110V, 25min, gel imaging system observation.
As shown in Figure 1, as after primer amplification products therefrom electrophoresis pattern.By electrophoretogram analysis shows the present invention
The amplification is effective, and band is single.
(6) Sanger is sequenced：
Take 9 μ l PCR products and 2 μ l purification systems.It is purified according to following procedure：
2 μ l purified products are mixed with upper and lower sequencing primer according to following system respectively：
Sequencing reaction program：
Precipitate link：
The EDTA of 2 μ l 125mmol is added into the product for completing sequencing reaction, stands 5min；The 15 anhydrous second of μ l are added
Alcohol, whirlpool mixing；3700rpm centrifuges 40min；It is inverted centrifugation 15sec, 50 μ l are added and freeze 75% ethyl alcohol, whirlpool mixing；
3700rpm centrifuges 20min；It is inverted centrifugation 15sec, is placed on 95 DEG C of metal baths；Denatured test is carried out after 10 μ l Hi-Di are added.
Denaturation program：95 DEG C of 5min, after 4 DEG C of 5min denaturation programs, upper sequenator (ABI3730) sequencing.
(7) result judges：Respectively by sequencing result and APOE gene reference sequences (Genbankaccn：NG_007084) into
Row compares, and is reported result according to practical catastrophe.
Embodiment 3：The APOE gene types of clinical sample detect
1, clinical tissue sample to be checked is taken, APOE gene types point are carried out according to the method in Examples 1 and 2
Analysis, as a result as shown in table 4 and 5.As shown in figure 4,388 bit base sites are T.As shown in figure 5,526 bit base sites are C.It is comprehensive
It is E3 to close APOE gene types known to Fig. 4 and Fig. 5 results.
Sequence table
<110>Nanchang Ai Dikang Laboratory of medical test Co., Ltd
<120>Detect the other primer of APOE genotype and method
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
aacaactgac cccggtggcg 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
acctgctcct tcacctcgtc 20
Claims (7)
1. detecting the other primer of APOE genotype, it is characterised in that：Expand the amplimer sequence of the 4th exon of ApoE genes
For：
ApoE-Exon4-F：AACAACTGACCCCGGTGGCG
ApoE-Exon4-R：ACCTGCTCCTTCACCTCGTC.
2. primer as described in claim 1, which is characterized in that further include sequencing primer, sequencing primer sequence is：
Sequencing primer 1：AACAACTGACCCCGGTGGCG
Sequencing primer 2：ACCTGCTCCTTCACCTCGTC.
3. detecting ApoE genotype method for distinguishing, include the following steps：
(1) genomic DNA in peripheral blood is extracted；
(2) with the DNA extracted in PCR amplification step 1；
(3) amplified production in step 2 is sequenced；
(4) sequencing result is judged, determines APOE gene types；
Wherein, the PCR amplification primer sequence of step (2) is：
ApoE-Exon4-F：AACAACTGACCCCGGTGGCG
ApoE-Exon4-R：ACCTGCTCCTTCACCTCGTC.
4. method as stated in claim 3, which is characterized in that further include sequencing primer：
Sequencing primer 1：AACAACTGACCCCGGTGGCG
Sequencing primer 2：ACCTGCTCCTTCACCTCGTC.
5. method as claimed in claim 3, which is characterized in that type site of analysis is the 112nd and 158 amino acids of coding
The base situation of sequence.
6. the method as described in claim 1, which is characterized in that type judgment method is：
7. method as claimed in claim 6, which is characterized in that the reaction condition of PCR is：
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2730745A1 (en) * | 1995-02-17 | 1996-08-23 | Eurobio Lab | METHOD FOR DETECTION OF A MUTATION INVOLVING IN ALZHEIMER'S DISEASE AND NUCLEOTIDIC SEQUENCES FOR ITS IMPLEMENTATION |
| CN102676669A (en) * | 2012-05-04 | 2012-09-19 | 周宏灏 | Kit and method for detecting apolipoprotein E (ApoE) gene polymorphisms by means of pyro sequencing method |
| CN104862402A (en) * | 2015-05-29 | 2015-08-26 | 沈阳优吉诺生物科技有限公司 | Primers for detecting ApoE gene polymorphism, kit and PCR (polymerase chain reaction) method for primers or kit |
-
2018
- 2018-04-02 CN CN201810284721.8A patent/CN108588207A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2730745A1 (en) * | 1995-02-17 | 1996-08-23 | Eurobio Lab | METHOD FOR DETECTION OF A MUTATION INVOLVING IN ALZHEIMER'S DISEASE AND NUCLEOTIDIC SEQUENCES FOR ITS IMPLEMENTATION |
| CN102676669A (en) * | 2012-05-04 | 2012-09-19 | 周宏灏 | Kit and method for detecting apolipoprotein E (ApoE) gene polymorphisms by means of pyro sequencing method |
| CN104862402A (en) * | 2015-05-29 | 2015-08-26 | 沈阳优吉诺生物科技有限公司 | Primers for detecting ApoE gene polymorphism, kit and PCR (polymerase chain reaction) method for primers or kit |
Non-Patent Citations (3)
| Title |
|---|
| FEDERICA AGOSTA等: "Apolipoprotein E _4 is associated withdisease-specific effects on brain atrophy in Alzheimer’s disease and frontotemporal dementia", 《PNAS》 * |
| 贺淹才: "《基因工程概论》", 30 April 2008, 清华大学出版社 * |
| 韩永凯等: "卒中后抑郁患者认知功能与ApoE基因多态性的关联研究", 《中华行为医学与脑科学杂志》 * |
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Application publication date: 20180928 |