CN106868109A - One kind detection ITPA94C>The primer and its PCR method of T gene pleiomorphisms - Google Patents
One kind detection ITPA94C>The primer and its PCR method of T gene pleiomorphisms Download PDFInfo
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- CN106868109A CN106868109A CN201710024946.5A CN201710024946A CN106868109A CN 106868109 A CN106868109 A CN 106868109A CN 201710024946 A CN201710024946 A CN 201710024946A CN 106868109 A CN106868109 A CN 106868109A
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- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention discloses one kind ITPA 94C are detected with PCR method>The primer pair ITPAwtF/ITPAwtR and ITPAmtF/ITPAmtR of T gene polymorphism sites, its sequence is respectively as shown in SEQ ID NO.1~4.Primer pair ITPAwtF/ITPAwtR and ITPAmtF/ITPAmtR is also disclosed in detection biological sample ITPA 94C>Application in terms of T gene polymorphism sites, and ITPA 94C prepared therefrom>T gene polymorphism sites detection kits.Using primer pair of the invention and its PCR method, ITPA 94C can be determined according to amplification electrophoresis result>The genotype of T gene polymorphism sites, 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, instruct clinical application, enable the patient to carry out Personalized medicine.
Description
Technical field
The invention belongs to medical biotechnology field.More particularly, to one kind detection ITPA 94C>T gene pleiomorphisms
Primer and its PCR method.
Background technology
Clinically, thiopurine drugs（Purinethol class medicine）It is widely used.But, the adverse reaction hair of such medicine
Raw rate is higher, including bone marrow suppression, liver damage, gastrointestinal disorders, pancreatitis and influenza sample symptom etc..
Current research thinks that the adverse reaction of purinethol class medicine may be with the metabolic pathway of medicine and gene polymorphic
Property is relevant.Thiopurine methyltransferase（Thiopurine methyltransferase, TPMT）With inosine triphosphate Jiao's phosphorus
Sour enzyme（Inosine triphosphate pyrophosphatase, ITPA）It is the metabolic enzyme of purinethol class medicine, wherein
TPMT is the key enzyme of purinethol class drug metabolism, and TPMT and ITPA has obvious genetic polymorphism.But still there is portion
Divide the generation of the adverse reaction of patient, it is impossible to explain.
The content of the invention
It is an object of the invention to provide one kind ITPA 94C are detected with PCR method>The primer pair of T gene polymorphism sites
ITPAwtF/ITPAwtR and ITPAmtF/ITPAmtR.ITPA 94C can be determined according to electrophoresis result>T gene polymorphism sites
Genotype, be predicted for the curative effect of purinethol class medicine and side effect with reference to individual other biological index, gram
The blindness of selection of clinical purinethol class medicine is taken, can predict that patient uses sulfydryl fast on the basis of genotyping
The validity and security of purine class medicine, instruct clinical application, enable the patient to carry out Personalized medicine.
It is a further object of the present invention to provide above-mentioned primer pair ITPAwtF/ITPAwtR and ITPAmtF/ITPAmtR in system
It is standby that biology sample detection ITPA 94C are expanded by PCR>Application in the reagent of T gene polymorphism sites.
Another object of the present invention is to provide one kind comprising above-mentioned primer pair ITPAwtF/ITPAwtR and ITPAmtF/
The ITPA 94C of ITPAmtR>T gene polymorphism sites detection kits.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of PCR method detects ITPA 94C>The primer pair ITPAwtF/ITPAwtR of T gene polymorphism sites and
ITPAmtF/ITPAmtR, its sequence is respectively as shown in SEQ ID NO.1~4.
Above-mentioned primer pair ITPAwtF/ITPAwtR and ITPAmtF/ITPAmtR is in detection biological sample ITPA 94C>T bases
Application in terms of because of pleomorphism site, also within protection scope of the present invention.
It is a kind of that biology sample detection ITPA 94C are expanded by PCR>The method of T gene polymorphism sites, is with life to be measured
The DNA of thing sample is template, and entering performing PCR with above-mentioned primer pair ITPAwtF/ITPAwtR and ITPAmtF/ITPAmtR expands, root
Determine ITPA 94C according to amplification>The genotype of T 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；94 DEG C of 30s, 66 DEG C/80 DEG C 30s, 75 DEG C
15s, 35 circulations；75℃ 5min；4 DEG C of preservations.
In addition, above-mentioned primer pair ITPAwtF/ITPAwtR and ITPAmtF/ITPAmtR is being prepared by PCR amplification biologies
Sample detection ITPA 94C>Application in the kit of T gene polymorphism sites, and comprising above-mentioned primer pair ITPAwtF/
The ITPA 94C of ITPAwtR and ITPAmtF/ITPAmtR>T gene polymorphism sites detection kits, also in guarantor of the invention
Within the scope of shield.
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 ITPAwtF/ITPAwtR and ITPAmtF/ITPAmtR of the present invention and detect ITPA with PCR method
94C>The method and kit of T gene polymorphism sites, can be used for detection ITPA 94C>The gene of T gene polymorphism sites
Type.
Primer pair ITPAwtF/ITPAwtR and ITPAmtF/ITPAmtR of the invention and can be detected with PCR method
ITPA 94C>T gene pleiomorphisms, ITPA 94C>T gene pleiomorphisms are relevant with thiopurine medicine adverse reaction, can be according to expansion
Increase electrophoresis result and determine ITPA 94C>The genotype of T gene polymorphism sites, curative effect and side effect to purinethol class medicine
Be predicted, prediction patient using purinethol class medicine validity and security, instruct clinical application, enable the patient into
Row Personalized medicine.
The invention has the advantages that：
The invention discloses one kind ITPA 94C are detected with PCR method>The primer pair ITPAwtF/ of T gene polymorphism sites
ITPAwtR and ITPAmtF/ITPAmtR, its sequence is respectively as shown in SEQ ID NO.1~4.Using primer pair of the invention and
Its PCR method, can determine ITPA 94C according to amplification electrophoresis result>The genotype of T gene polymorphism sites, to purinethol
The curative effect of class medicine and side effect are predicted, and can predict that patient uses purinethol class medicine on the basis of genotyping
The validity and security of thing, instruct clinical application, enable the patient to carry out Personalized medicine, overcome selection of clinical purinethol
The blindness of class medicine, 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.
The detection ITPA of embodiment 1 94C>The primer and its PCR method of T 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 limited public affairs
Department, Shanghai）, 5 × TBE electrophoresis liquid storing liquids（0.45 mol/L Tris alkali, 0.45 mol/L boric acid, 0.01 mol/L EDTA,
Adjust 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
Weigh 186.1 g EDTA2H2O is in 1 L beakers, plus 800 ml deionized waters, 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
54 g Tris are weighed, 27.5 g boric acid are placed in beaker, plus appropriate distilled water stirring and dissolving；Add 0.5 M EDTA solution
20 ml, plus distilled water is settled to 1 L, is made 5 × TBE storing solutions.Storing solution will dilute 10 times for 0.5 × TBE solution is used.
（7）6 M NaI solution
45 g 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
Design ITPA 94C>The pcr amplification primer thing of T, as shown in table 1.All primer sequences verify that it is closed through NCBI Blast
Rationality and 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 ITPAwtF/ITPAwtR and ITPAmtF/ITPAmtR and PCR method, can be with Testing and appraisal ITPA 94C>T bases
Because of the genotype of pleomorphism site.
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 ITPAwtF/ITPAwtR and ITPAmtF/ITPAmtR of the invention and PCR method
Detection ITPA 94C>The genotype of T gene polymorphism sites, so as to judge detection object to the bad anti-of purinethol class medicine
Situation is answered, and then instructs personalized medicine.The invention provides a kind of simple and easy to apply and with the PCR of very strong clinical value
Method and primer.
SEQUENCE LISTING
<110>Zhongshan University
<120>One kind detection ITPA 94C>The primer and its PCR method of T gene pleiomorphisms
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 26
<212> DNA
<213>Primer I TPAwtF
<400> 1
cgttcagatt ctaggagata agttca 26
<210> 2
<211> 26
<212> DNA
<213>Primer I TPAwtR
<400> 2
cgttcagatt ctaggagata agttcc 26
<210> 3
<211> 25
<212> DNA
<213>Primer I TPAmtF
<400> 3
ttccacgaac atgtgtgaat gcagc 25
<210> 4
<211> 25
<212> DNA
<213>Primer I TPAmtR
<400> 4
gcttagcaca agcagagacc tgacg 25
Claims (9)
1. one kind detects ITPA 94C with PCR method>The primer pair ITPAwtF/ITPAwtR of T gene polymorphism sites and
ITPAmtF/ITPAmtR, it is characterised in that its sequence is respectively as shown in SEQ ID NO.1~4.
2. primer pair ITPAwtF/ITPAwtR and ITPAmtF/ITPAmtR described in claim 1 is in detection biological sample ITPA
94C>Application in terms of T gene polymorphism sites.
3. it is a kind of that biology sample detection ITPA 94C are expanded by PCR>The method of T gene polymorphism sites, it is characterised in that
DNA with biological sample to be measured as template, with primer pair ITPAwtF/ITPAwtR and ITPAmtF/ described in claim 1
ITPAmtR enters performing PCR amplification, and ITPA 94C are determined according to amplification>The genotype of T 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；
94 DEG C of 30s, 66 DEG C/80 DEG C 30s, 75 DEG C of 15s, 35 circulations；75℃ 5min；4 DEG C of preservations.
6. primer pair ITPAwtF/ITPAwtR and ITPAmtF/ITPAmtR described in claim 1 is being prepared by PCR amplification lifes
Thing sample detection ITPA 94C>Application in the reagent of T gene polymorphism sites.
7. a kind of ITPA 94C>T gene polymorphism sites detection kits, it is characterised in that comprising drawing described in claim 1
Thing is to ITPAwtF/ITPAwtR and ITPAmtF/ITPAmtR.
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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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107574239A (en) * | 2017-10-25 | 2018-01-12 | 广州和康医疗技术有限公司 | A kind of detection method and kit for detecting sulphur purine medicine SNP site genotype |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994629A (en) * | 2011-09-15 | 2013-03-27 | 爱科来株式会社 | Method for detecting mutations at genes IL28B (RS8099917) and ITPA (RS1127354) |
| CN105506099A (en) * | 2015-12-30 | 2016-04-20 | 广州金域检测科技股份有限公司 | Primer and method for detecting ITPA gene polymorphism |
| CN105543349A (en) * | 2015-12-30 | 2016-05-04 | 广州金域检测科技股份有限公司 | Primers and method for simultaneously detecting IL28B and ITPA gene polymorphism |
-
2017
- 2017-01-13 CN CN201710024946.5A patent/CN106868109A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994629A (en) * | 2011-09-15 | 2013-03-27 | 爱科来株式会社 | Method for detecting mutations at genes IL28B (RS8099917) and ITPA (RS1127354) |
| CN105506099A (en) * | 2015-12-30 | 2016-04-20 | 广州金域检测科技股份有限公司 | Primer and method for detecting ITPA gene polymorphism |
| CN105543349A (en) * | 2015-12-30 | 2016-05-04 | 广州金域检测科技股份有限公司 | Primers and method for simultaneously detecting IL28B and ITPA gene polymorphism |
Non-Patent Citations (3)
| Title |
|---|
| W.R.WANROSALINA ET AL.: "Polymorphism of ITPA94C>A and risk of adverse effects among patients with acute lymphoblastic leukaemia treated with 6-mercaptopurine", 《JOURNAL OF CLINICAL PHARMACY AND THERAPEUTICS》 * |
| 冯静等: "影响硫嘌呤类药物个体化应用的主要代谢酶遗传多态性的研究进展", 《中国药师》 * |
| 辛华雯等: "肾移植受者TPMT和ITPA基因多态性", 《华南国防医学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107574239A (en) * | 2017-10-25 | 2018-01-12 | 广州和康医疗技术有限公司 | A kind of detection method and kit for detecting sulphur purine medicine SNP site genotype |
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