CN106868110A - A kind of primer and its PCR method of detection PACSIN2rs2413739 gene pleiomorphisms - Google Patents
A kind of primer and its PCR method of detection PACSIN2rs2413739 gene pleiomorphisms Download PDFInfo
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Abstract
The invention discloses a kind of primer pair PrF/PrR that PACSIN2 rs2413739 gene polymorphism sites are detected with PCR method, its sequence is respectively as shown in SEQ ID NO.1 and 2.Also disclose applications of the primer pair PrF/PrR in terms of biological sample PACSIN2 rs2413739 gene polymorphism sites are detected, and PACSIN2 rs2413739 gene polymorphism sites detection kits prepared therefrom.Using primer pair of the invention and its PCR method, the genotype of PACSIN2 rs2413739 gene polymorphism sites can be determined according to amplification electrophoresis result, curative effect and side effect to purinethol class medicine are predicted, can predict that patient uses the validity and security of purinethol class medicine on the basis of genotyping, clinical application is instructed, enables the patient to carry out Personalized medicine.
Description
Technical field
The invention belongs to medical biotechnology field.More particularly, to one kind detection PACSIN2 rs2413739 bases
Because of the primer and its PCR method of polymorphism.
Background technology
Inflammatory bowel disease（inflammatory bowel disease, IBD）, it is a kind of special chronic gut inflammation
Disease, mainly including Crohn disease (CD) and ulcerative colitis (UC).At present clinically, give patient purinethol class medicine more
Thing, because such curative effect of medication is definite, and it is cheap.But, the adverse reaction rate of such medicine is higher, including marrow
Suppression, liver damage, gastrointestinal disorders, pancreatitis and influenza sample symptom etc..
Genetic background may not only influence the generation of disease, and can influence the individual difference of curative effect of medication, and gene is more
State property has important influence for the adverse reaction of medicine.Therefore, using science of heredity screening method, the individuality that realization varies with each individual
Change clinical application target, be prevented effectively from Irrational Use of Drugs, wrong medicine or even drug abuse tendency, reduce for medicine not
The expense of good reaction, it is to avoid the waste that invalid medication is caused, has great importance.
The content of the invention
It is an object of the invention to provide a kind of PACSIN2 rs2413739 gene polymorphism sites are detected with PCR method
Primer pair PrF/PrR.The genotype of PACSIN2 rs2413739 gene polymorphism sites can be determined according to electrophoresis result, is tied
Close individual other biological index to be predicted for the curative effect of purinethol class medicine and side effect, overcome selection of clinical
The blindness of purinethol class medicine, can predict patient's having using purinethol class medicine on the basis of genotyping
Effect property and security, instruct clinical application, enable the patient to carry out Personalized medicine.
Biology sample detection is expanded by PCR in preparation it is a further object of the present invention to provide above-mentioned primer pair PrF/PrR
Application in the reagent of PACSIN2 rs2413739 gene polymorphism sites.
Another object of the present invention is to provide a kind of PACSIN2 rs2413739 bases comprising above-mentioned primer pair PrF/PrR
Because of pleomorphism site detection kit.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of primer pair PrF/PrR that PACSIN2 rs2413739 gene polymorphism sites are detected with PCR method, its sequence point
Not as shown in SEQ ID NO.1 and SEQ ID NO.2.
Above-mentioned primer pair PrF/PrR is in terms of biological sample PACSIN2 rs2413739 gene polymorphism sites are detected
Using also within protection scope of the present invention.
It is a kind of by PCR expand biology sample detection PACSIN2 rs2413739 gene polymorphism sites method, be with
The DNA of biological sample to be measured is template, and entering performing PCR with above-mentioned primer pair PrF/PrR expands, and is determined according to amplification
The genotype of PACSIN2 rs2413739 gene polymorphism sites.
Wherein, the biological sample to be measured is peripheral blood in patients.
Preferably, the reaction system of the PCR amplifications is 25 μ l, including：μ l, 10 × Taq buffer 2.5 of DNA profiling 3
μ l, 0.25mmol/L dNTPs 2 μ l, 10 μm of each μ l of 1 μ l, 5U/ μ l ExTaq 0.15 of ol/L upstream and downstream primers, surplus
ddH2O。
Preferably, the reaction condition of the PCR amplifications is：95℃ 5min；93 DEG C of 30s, 60 DEG C of 15s, 72 DEG C of 30s,
40 circulations；72℃ 7min；4 DEG C of preservations.
In addition, above-mentioned primer pair PrF/PrR is being prepared by PCR amplification biology sample detection PACSIN2 rs2413739
Application in the kit of gene polymorphism sites, and the PACSIN2 rs2413739 comprising above-mentioned primer pair PrF/PrR
Gene polymorphism sites detection kit, also within protection scope of the present invention.
Preferably, the kit also contains dNTPs, ExTaq polymerase.
Preferably, the ExTaq polymerases of dNTPs, 5U/ μ l of the kit also containing 0.25mmol/L.
The above-mentioned primer pair PrF/PrR of the present invention and detect PACSIN2 rs2413739 gene pleiomorphisms position with PCR method
The method and kit of point, can be used for detecting the genotype of PACSIN2 rs2413739 gene polymorphism sites.
Gene PACSIN2 can adjust thiopurine methyltransferase（Thiopurine methyltransferase,
TPMT）Activity, and TPMT is the key enzyme of purinethol class drug metabolism, and its gene pleiomorphism has with adverse drug reaction
Close, therefore, it can determine according to amplification electrophoresis result the genotype of PACSIN2 rs2413739 gene polymorphism sites, to mercapto
The curative effect of base purine medicaments and side effect are predicted, and prediction patient uses the validity and safety of purinethol class medicine
Property, clinical application is instructed, enable the patient to carry out Personalized medicine.
The invention has the advantages that：
The invention discloses a kind of primer pair PrF/ that PACSIN2 rs2413739 gene polymorphism sites are detected with PCR method
PrR, its sequence is respectively as shown in SEQ ID NO.1 and 2.Using primer pair of the invention and its PCR method, can be according to amplification
Electrophoresis result determines the genotype of PACSIN2 rs2413739 gene polymorphism sites, to the curative effect of purinethol class medicine and
Side effect is predicted, and can predict that patient uses the validity and peace of purinethol class medicine on the basis of genotyping
Quan Xing, instructs clinical application, enables the patient to carry out Personalized medicine, overcomes the blindness of selection of clinical purinethol class medicine
Property, it is to avoid bad kickback of using medicine.
And, the method for the present invention is simple and easy to apply, and application prospect is extensive.
Brief description of the drawings
Fig. 1 is the genotype electrophoresis pattern of genotypic results（PCR-RFLP）；HW is wild-type homozygote, and HM is mutation
Type homozygote, HT is heterozygous mutation.
Specific embodiment
The present invention, but embodiment are further illustrated below in conjunction with Figure of description and specific embodiment not to the present invention
Limit in any form.Unless stated otherwise, reagent, the method and apparatus that the present invention is used are for the art is routinely tried
Agent, method and apparatus.
Unless stated otherwise, agents useful for same of the present invention and material are purchased in market.
Embodiment 1 detects the primer and its PCR method of PACSIN2 rs2413739 gene pleiomorphisms
1st, reagent and its configuration needed for testing
（1）DNA extracts reagents
6mol/L NaI（Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, Shanghai）, chloroform, isopropanol, isoamyl alcohol, ethanol
It is commercially available analysis net product.
（2）PCR amplifing reagents
Ex Taq enzymes（Takara companies, Japan）, synthetic primer（Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, on
Sea）.
（3）Agarose gel electrophoresis reagent
Plain agar sugar（Biowest companies, Spain）, low melting-point agarose（Shanghai biotechnology Services Co., Ltd,
Shanghai）, 5 × TBE electrophoresis liquid storing liquids（0.45 mol/L Tris alkali, 0.45 mol/L boric acid, 0.01 mol/L EDTA are adjusted
PH to 8.0；Tris, boric acid, EDTA are purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd）10 times of dilution is used,
GoldviewTM Nucleic Acid stain（Beijing SBS Genetech bio-engineering corporation, Beijing）.
（4）DL1000、DL2000 DNA Ladder Marker（Takara companies, Japan）.
（5）0.5 M EDTA solution
186.1 g EDTA2H2O are weighed in 1 L beakers, plus 800 ml deionized waters, it is sufficiently stirred for, plus NaOH regulations pH
To 8.0, stirring is to being completely dissolved, plus deionized water is settled to 1 L, autoclave sterilization.
（6）5 × TBE solution
54g Tris are weighed, 27.5 g boric acid are placed in beaker, plus appropriate distilled water stirring and dissolving；Add 0.5M EDTA solution
20ml, plus distilled water is settled to 1L, is made 5 × TBE storing solutions.Storing solution will dilute 10 times for 0.5 × TBE solution is used.
（7）6 M NaI solution
45g NaI are weighed to be placed in beaker, plus distilled water stirring and dissolving, 50 ml are settled to, keep in dark place.
2nd, key instrument needed for testing
ABI GeneAmp PCR System 2700 (Applied Biosystems companies, the U.S.)
Eppendorf MasterCycler epGradient PCR instruments（Eppendorf companies, Germany）
DYY-11131C type Horizontal electrophoresis tanks（Liuyi Instruments Plant, Beijing, Beijing）
DYY-6C type voltage stabilization and current stabilization electrophoresis apparatuses（Liuyi Instruments Plant, Beijing, Beijing）
EC3 gel imaging systems（UVP companies, the U.S.）
Eppendorf 5417R refrigerated centrifuges（Eppendorf companies, Germany）
Fuhua SHA-C type constant temperature oscillation bain-maries（Changzhou Fei Pu laboratory apparatus factory, Jiangsu）
DZF-6050 vacuum drying chambers（The permanent Science and Technology Ltd. in Shanghai one, Shanghai）
Ultra low temperature freezer（-80℃）（Thermo companies, Germany）
3rd, experimental technique
（1）Peripheral blood DNA is extracted
1）The μ l of EDTA anticoagulated whole bloods 100 are taken, plus the μ l of aseptic double-distilled water 200 are mixed；
2）The μ l of 6 mol/L sodium iodides 200 are added, is mixed；
3）Add chloroform/isoamyl alcohol（24:1, v/v）400 μ l, mix repeatedly；Then 12000 rpm are centrifuged 10 min, careful to inhale
Go out supernatant and put another centrifuge tube；
4）Add the μ l of isopropanol 300 to mix to supernatant, be stored at room temperature 3 min, 10 min then are centrifuged in 12000 rpm, abandon
Supernatant；
5）μ l, 12000 rpm 3 min of centrifugation of 70% ethanol 500 are added, ethanol is outwelled；
6）Repeat step（5）Once, it is inverted a moment；
7）After tube wall dries, the μ l dissolving DNAs of TE buffer solutions 40 are added, being blown and beaten with suction pipe makes DNA fully dissolve.
（2）DNA concentration is determined and purified
The μ l of sample 20, plus ultra-pure water are taken to 600 μ l, is fully mixed, determining wavelength with ultraviolet specrophotometer is respectively 260
OD values when nm, 280 nm, 320 nm, calculate the concentration and purity of DNA.
DNA concentration (μ g/ml)=(OD260-OD320) × 50 μ g/ml × extension rate (30)
DNA purity=(OD260-OD320)/(OD280-OD320)
DNA purity represents that purity is good when being 1.8；<Represent that protein content is too high when 1.6, purified using chloroform/isoamyl alcohol extraction；>
Indicate that RNA pollutes when 1.9, can be processed with RNase.
（3）PCR reacts
1）Design of primers
The pcr amplification primer thing of design PACSIN2 rs2413739, as shown in table 1.All primer sequences are tested through NCBI Blast
Its reasonability is demonstrate,proved with specificity.
The primer sequence of table 1
2）PCR reaction systems
PCR reaction systems are 25 μ l, including 3 μ l, 10 × Taq buffer of DNA profiling 2.5,2 μ l of μ l, dNTPs
(0.25mmol/L), each 1 μ l of upstream and downstream primer (10 μm of ol/L), the μ l of ExTaq 0.15 (5U/ μ l), uses ddH2O supplements are total
Volume is to 25 μ l.
3）PCR reaction conditions are as shown in table 2
The PCR reaction conditions of table 2
4th, the detection of PCR primer：Agarose gel electrophoresis
Glue：Appropriate agarose is weighed, adds 0.5 × TBE solution to be configured to 1% suspension, be heated in micro-wave oven completely
Dissolving, adds the Goldview of final concentration of 0.05 μ l/ml, in the abundant rubber moulding for mixing placing flat of falling back, gel thicknesses
About 5 mm, plug comb, static 20 ~ 30 min.
Loading：The gel that will be made is put into electrophoresis tank, electrophoretic buffer（With 0.5 × TBE of batch）Liquid level is higher by gel table
The mm of face 1 ~ 2, takes after 5 μ l amplified productions mix with 1 μ 6 × Loading of l buffer, in sequentially adding well；190 V
The min of electrophoresis 15.
5th, result
Electrophoresis result is observed under uviol lamp, and in automated imaging on gel imaging system, as a result as shown in Figure 1, using this hair
Bright primer pair PrF/PrR and PCR method, can be with the gene of Testing and appraisal PACSIN2 rs2413739 gene polymorphism sites
Type.
From the point of view of clinical observation, no mutant homozygote is to the adverse reaction rate of purinethol class medicine apparently higher than mutation
Heterozygote, and no mutant homozygote and the rate of adverse reactions both heterozygous mutation are better than wild type.
Therefore, it can using primer pair PrF/PrR of the invention and PCR method detection PACSIN2 rs2413739 genes
The genotype of pleomorphism site, so as to judge adverse reaction situation of the detection object to purinethol class medicine, and then instructs individual
Property medication.The invention provides a kind of simple and easy to apply and with the PCR method and primer of very strong clinical value.
SEQUENCE LISTING
<110>Zhongshan University
<120>A kind of primer and its PCR method of detection PACSIN2 rs2413739 gene pleiomorphisms
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Primer PrF
<400> 1
gcatgggccc aaaatagcaa 20
<210> 2
<211> 20
<212> DNA
<213>Primer PrR
<400> 2
tccagagggg tactcctgac 20
Claims (9)
1. it is a kind of with PCR method detect PACSIN2 rs2413739 gene polymorphism sites primer pair PrF/PrR, its feature
It is that its sequence is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.
2. primer pair PrF/PrR described in claim 1 is in detection biological sample PACSIN2 rs2413739 gene pleiomorphisms position
Application in terms of point.
3. a kind of method that biology sample detection PACSIN2 rs2413739 gene polymorphism sites are expanded by PCR, it is special
Levy and be, the DNA with biological sample to be measured enters performing PCR and expands as template, with primer pair PrF/PrR described in claim 1, according to
Amplification determines the genotype of PACSIN2 rs2413739 gene polymorphism sites.
4. method according to claim 3, it is characterised in that the reaction system of the PCR amplifications is 25 μ l, including：
3 μ l, 10 × Taq buffer of DNA profiling, 2.5 μ l, 0.25mmol/L dNTPs 2 μ l, 10 μm of each 1 μ l of ol/L upstream and downstream primers,
5U/ μ l ExTaq 0.15 μ l, surplus ddH2O。
5. method according to claim 3, it is characterised in that the reaction condition of the PCR amplifications is：95℃ 5min；
93 DEG C of 30s, 60 DEG C of 15s, 72 DEG C of 30s, 40 circulations；72℃ 7min；4 DEG C of preservations.
6. primer pair PrF/PrR described in claim 1 is being prepared by PCR amplification biology sample detections PACSIN2
Application in the reagent of rs2413739 gene polymorphism sites.
7. a kind of PACSIN2 rs2413739 gene polymorphism sites detection kits, it is characterised in that comprising claim 1
The primer pair PrF/PrR.
8. kit according to claim 8, it is characterised in that also include dNTPs, ExTaq polymerase.
9. kit according to claim 7, it is characterised in that also include dNTPs, 5U/ μ l's of 0.25mmol/L
ExTaq polymerases.
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| CN101210268A (en) * | 2006-12-29 | 2008-07-02 | 北京华安佛医药研究中心有限公司 | Kit for detecting mercaptopurine methyltransferase genetype and method thereof |
| CN101591699A (en) * | 2008-05-27 | 2009-12-02 | 北京华安佛医药研究中心有限公司 | Detect test kit, method and the purposes of purinethol methyltransferase gene G 460 A polymorphism locus |
| CN205347424U (en) * | 2016-01-27 | 2016-06-29 | 上海睿玻生物科技有限公司 | TPMT gene polymorphism detection kit |
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| CN101210268A (en) * | 2006-12-29 | 2008-07-02 | 北京华安佛医药研究中心有限公司 | Kit for detecting mercaptopurine methyltransferase genetype and method thereof |
| CN101591699A (en) * | 2008-05-27 | 2009-12-02 | 北京华安佛医药研究中心有限公司 | Detect test kit, method and the purposes of purinethol methyltransferase gene G 460 A polymorphism locus |
| CN205347424U (en) * | 2016-01-27 | 2016-06-29 | 上海睿玻生物科技有限公司 | TPMT gene polymorphism detection kit |
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Application publication date: 20170620 |