CN107653321A - A kind of kit and method of TaqMan MGB sonde methods detection mankind's TPMT gene pleiomorphisms - Google Patents
A kind of kit and method of TaqMan MGB sonde methods detection mankind's TPMT gene pleiomorphisms Download PDFInfo
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- CN107653321A CN107653321A CN201711138742.0A CN201711138742A CN107653321A CN 107653321 A CN107653321 A CN 107653321A CN 201711138742 A CN201711138742 A CN 201711138742A CN 107653321 A CN107653321 A CN 107653321A
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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
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- 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
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention provides a kind of primer of TaqMan MGB sonde methods detection mankind's TPMT gene pleiomorphisms, including following nucleotide sequence：Amplification TPMT genes (rs1142345, c.719A>G) primer pair of pleomorphism site and the TaqMan MGB probes of detection.The invention also discloses the kit including above-mentioned primer and probe and its application method.As a result accurately the present invention, can be easy to interpretation with quick detection TPMT gene rs1142345 loci polymorphisms.Compared with PCR sequencing PCR, this method need not carry out the subsequent treatment of a series of complex, PCR amplifications and the synchronous progress of detection to PCR primer, whole detection process need not uncap operation, the risk that PCR primer pollutes is reduced, detection time is substantially reduced, reduces testing cost.
Description
Technical field
The invention belongs to technical field of gene detection, and in particular to a kind of TaqMan-MGB sonde methods detect mankind's TPMT bases
Because of the kit and method of polymorphism.
Background technology
Thiopurine medicine such as Ismipur (mercaptopurine, 6-MP), 6- thioguanines (thioguanine,
6-TG) and imuran (azathioprine, AZP) etc. is a kind of antimetabolite with immunosuppressive action.6-TG and 6-
MP is usually used in the chemotherapy of malignant tumour, and AZP is then mainly used in autoimmune disease and Organ Transplantation Patients.AZP is as precursor
Medicine is converted into 6-MP in liver through glutathione transferase.6-MP is metabolized through hypoxanthine-guanine phosphoribosyl transferase
For sulfydryl hypoxanthine monophosphate (thioinosine monophosphate, TIMP), the latter passes through a series of process again
It is metabolized as active metabolite 6- Thioguanosines (6-thioguanine nucleotide, 6-TGN) and plays anti-swell afterwards
Knurl acts on.6-MP can also be metabolized as inactive 6-MMP (6-methyl MP, 6-MMP) through TPMT.TPMT activity
With in red blood cell and hematopoietic tissue 6-MP active metabolites 6-TNG it is horizontal negatively correlated, TPMT activity reduce mercapto can be made fast
Haematological toxicity (serious bone marrow suppression) increase of purine class medicine.
There is pleiomorphism in the distribution of TPMT enzymatic activitys, TPMT hereditary variations are the main originals for causing its enzymatic activity to reduce
Cause.The TPMT of normal activity is encoded by TPMT*1 allele, TPMT*2 (rs1800462,238G>C, Ala80Pro), TPMT*
3A(rs1800460 460G>A, Ala154Thr；Rs1142345,719A>G, Tyr240Cys), TPMT*3B (rs1800460
460G>A, Ala154Thr), TPMT*3C (rs1142345,719A>G, Tyr240Cys) it is cause TPMT activity decreases main
SNP or haplotype.TPMT genotype can be divided into 3 kinds：Wild-type homozygote (TPMT*1/*1), heterozygote and no mutant homozygote.It is wild
Raw type homozygotic individual has normal TPMT activity, and heterozygote individual TPMT activity reduces, and no mutant homozygote TPMT enzyme activity
Property it is extremely low in addition lack.In addition, 2 kinds of mutation allele homozygotes (TPMT*2/TPMT*3A and TPMT*3A/TPMT*3C) are individual
Body also lacks enzymatic activity.In white race crowd and non-descendants' American population, the frequency about 90% of wild-type homozygote genotype, mutation
The frequency of heterozygote genotype about 10%, the frequency about 0.3% of no mutant homozygote genotype.TPMT*3 heterozygotes in Chinese population
Genotype frequency about 2.2%, is not detected by TPMT*2 allele.The therapeutic effect of TPMT gene pleiomorphisms and purine medicaments
Closely related with toxic side effect, doctor should cautiously use purine medicaments with reference to TPMT genotype.
Mainly there is PCR- RFLPs (PCR- currently for the method for polymorphic position point analysis
Restriction Length Polymorphism, PCR-RFLP), amplification refractory mutation system-PCR (Amplification
Refractory mutation system PCR, ARMS-PCR), flight mass spectrum etc., but due to these method operating procedures
It is more, waste time and energy, or need PCR to post-process, pollution is also easy to produce, therefore be not suitable for the large-scale quick detection of crowd.Survey
Sequence method is the goldstandard of nucleic acid sequencing, although accurately, complex for operation step and cost is higher.The present invention, using TaqMan-
MGB probe techniques, a kind of fast and accurately methods of genotyping and kit are invented, can be the individualized treatment of medicine
Play positive impetus.The technology not only realizes the real-time monitoring to nucleic acid specificity amplification, and has sensitivity
With the features such as specificity is high, automaticity is high, pollution-free, cost is cheap.
The content of the invention
It is more that the technical problems to be solved by the invention are to provide a kind of TaqMan-MGB sonde methods detection mankind's TPMT genes
The kit and its method of state property.The present invention plays positive impetus for the individualized treatment of medicine.The technology is not only real
Showed the real-time monitoring to nucleic acid specificity amplification, and with sensitivity and specificity are high, automaticity is high, it is pollution-free,
The features such as cost is cheap.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
A kind of primer of TaqMan-MGB sonde methods detection mankind's TPMT gene pleiomorphisms, including following nucleotide sequence：
(1) primer pair of TPMT gene rs1142345 pleomorphism sites, its nucleotide sequence SEQ ID NO. are expanded：1 He
SEQ ID NO.：Shown in 2；
(2) it is used for the TaqMan-MGB probes for detecting TPMT gene rs1142345 pleomorphism sites, its nucleotide sequence
SEQ ID NO.：3 and SEQ ID NO.：Shown in 4, wherein SEQ ID NO.：3 in 5 end mark Fam signals, SEQ ID NO.：
4 in 5 end mark VIC signals；
Above-mentioned primer pair and fluorescence probe in one-time detection to being used in conjunction with.
The primer of above-mentioned TaqMan-MGB sonde methods detection mankind's TPMT gene pleiomorphisms is in detection TPMT gene pleiomorphisms
In apply within protection scope of the present invention.
The present invention also provides the kit that a kind of TaqMan-MGB sonde methods are used to detect mankind's TPMT gene pleiomorphisms, should
Kit includes the primer and probe and 2x PCR reaction mixtures described in claim 1, positive criteria product.
The present invention also provides another technical solution：A kind of TaqMan-MGB sonde methods are more for detecting mankind's TPMT genes
The application method of the kit of state property, comprises the following steps：
(1) human peripheral genomic DNA is extracted；
(2) reacted respectively using the human peripheral genomic DNA of extraction and positive criteria product as template, primer, probe, PCR
Mixed liquor, carry out real-time fluorescence quantitative PCR reaction；
(3) genotype judgement is carried out to rs1142345 sites according to the fluorescence signal value detected.
Further, in the step (2), the amplification system of real-time fluorescence quantitative PCR reaction is：Cumulative volume 20 μ L, 2x
PCR reaction mixtures 10uL, SEQ ID NO.：1 0.2uM, SEQ ID NO.：2,0.2uM, SEQ ID NO.：10.2uM SEQ
ID NO.：1 0.2uM, template DNA 50ng.
Further, in the step (2), the amplification program in real-time fluorescence quantitative PCR reaction is：95 DEG C, 2min；
95 DEG C, 5s, 60 DEG C, 45s, collect fluorescence, 40 circulations.
The invention provides a kind of TaqMan-MGB sonde methods detection mankind's TPMT gene pleiomorphisms primer and kit,
Realized using TaqMan-MGB sonde methods and quick, simple detection is carried out to TPMT gene rs1142345 sites.Present invention inspection
It is accurate to survey result, is easy to interpretation.Compared with PCR sequencing PCR, this method need not carry out the follow-up place of a series of complex to PCR primer
Reason, PCR amplifications and detection is synchronous carries out, whole detection process need not uncap operation, reduce the wind that PCR primer pollutes
Danger, substantially reduces detection time, reduces testing cost.
Brief description of the drawings
Fig. 1 is result diagram corresponding to different genotype.
Embodiment
According to following embodiments, the present invention may be better understood.It is however, as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and be merely to illustrate the present invention, without should be also without limitation on sheet described in detail in claims
Invention.
Embodiment 1：
Step 1：DNA is extracted
200 μ L peripheral bloods are taken, it is (new purchased from Hangzhou to carry out DNA extractions according to Whole Blood Genomic DNA extracts kit specification
Prompt bio tech ltd), extraction DNA is used for subsequent experimental or deposits in -20 DEG C.
Step 2：Quantitative pcr amplification and detection
(1) primer pair of TPMT gene rs1142345 pleomorphism sites, its nucleotide sequence SEQ ID NO. are expanded：1 He
SEQ ID NO.：Shown in 2；
(2) it is used for the TaqMan-MGB probes for detecting TPMT gene rs1142345 pleomorphism sites, its nucleotide sequence
SEQ ID NO.：3 and SEQ ID NO.：Shown in 4, wherein SEQ ID NO.：3 in 5 end mark Fam signals, SEQ ID NO.：
4 in 5 end mark VIC signals.
In 96 orifice plates, reaction system is formulated as follows：2x PCR reaction mixtures 10uL, SEQ ID NO.：1 0.2uM,
SEQ ID NO.：2 0.2uM, SEQ ID NO.：3 0.2uM, SEQ ID NO.：4 0.2uM, template DNA 50ng, add water to mend
Foot sets up positive criteria product and negative control to the μ L of cumulative volume 20.Amplification program in real-time fluorescence quantitative PCR reaction
For：95 DEG C, 2min；95 DEG C, 5s, 60 DEG C, 45s, collect fluorescence, 40 circulations.
Step 3：Interpretation of result
After reaction terminates, carry software using instrument and carry out data analysis.According to amplification, sample genotype is carried out
Judge, specific testing result see the table below.
Step 4：As a result count
The genotype results statistics of 10 samples is as follows：
It is described above, it is preferred embodiments of the present invention, and the limitation that non-invention is any formal or substantial, should
Point out, for those skilled in the art, under the premise of the scope of the present invention is not departed from, it can also be modified
And improvement, these are improved and supplement should also be considered as protection scope of the present invention.
Sequence table
<110>Jinan Dean medical test centered finite company
<120>A kind of kit and method of TaqMan-MGB sonde methods detection mankind's TPMT gene pleiomorphisms
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
cgttgtcttg agaaggttga tg 22
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
tcctcaaaaa catgtcagtg tg 22
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ttgaaaagtt atatctactt tg 22
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ttgaaaagtt atgtctactt tg 22
<210> 5
<211> 141
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
cgttgtcttg agaaggttga tgcttttgaa gaacgacata aaagttgggg aattgactgt 60
ctttttgaaa agttatatct acttacagaa aagtaaatga gacatagata aaataaaatc 120
acactgacat gtttttgagg a 141
<210> 6
<211> 141
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
cgttgtcttg agaaggttga tgcttttgaa gaacgacata aaagttgggg aattgactgt 60
ctttttgaaa agttatgtct acttacagaa aagtaaatga gacatagata aaataaaatc 120
acactgacat gtttttgagg a 141
Claims (6)
1. a kind of primer of TaqMan-MGB sonde methods detection mankind's TPMT gene pleiomorphisms, it is characterised in that including following core
Acid sequence：
(1) primer pair of TPMT gene rs1142345 pleomorphism sites, its nucleotide sequence SEQ ID NO. are expanded：1 and SEQ
ID NO.：Shown in 2；
(2) it is used for the TaqMan-MGB probes for detecting TPMT gene rs1142345 pleomorphism sites, its nucleotide sequence SEQ ID
NO.：3 and SEQ ID NO.：Shown in 4, wherein SEQ ID NO.：3 in 5 end mark Fam signals, SEQ ID NO.：4 at 5 ends
End mark VIC signals；
Above-mentioned primer pair and fluorescence probe in one-time detection to being used in conjunction with.
2. a kind of primer of TaqMan-MGB sonde methods detection mankind's TPMT gene pleiomorphisms described in claim 1 is detecting
Application in TPMT gene pleiomorphisms.
3. a kind of TaqMan-MGB sonde methods are used for the kit for detecting mankind's TPMT gene pleiomorphisms, it is characterised in that the examination
Agent box includes the primer and probe described in claim 1, and 2x PCR reaction mixtures, positive criteria product.
4. the TaqMan-MGB sonde methods described in a kind of claim 3 are used for the kit for detecting mankind's TPMT gene pleiomorphisms
Application method, it is characterised in that comprise the following steps：
(1) human peripheral genomic DNA is extracted；
(2) respectively using the human peripheral genomic DNA of extraction and positive criteria product as the primer in template, claim 1 and 2,
Probe, PCR reaction mixtures, carry out real-time fluorescence quantitative PCR reaction；
(3) genotype judgement is carried out to rs1142345 sites according to the fluorescence signal value detected.
5. TaqMan-MGB sonde methods according to claim 4 are used for the kit for detecting mankind's TPMT gene pleiomorphisms
Application method, it is characterised in that in the step (2), the amplification system of real-time fluorescence quantitative PCR reaction is：The μ L of cumulative volume 20,
2x PCR reaction mixtures 10uL, SEQ ID NO.：1 0.2uM, SEQ ID NO.：2,0.2uM, SEQ ID NO.：3
0.2uM, SEQ ID NO.：4 0.2uM, template DNA 50ng.
6. TaqMan-MGB sonde methods according to claim 4 are used for the kit for detecting mankind's TPMT gene pleiomorphisms
Application method, it is characterised in that in the step (2), the amplification program in real-time fluorescence quantitative PCR reaction is：95 DEG C,
2min；95 DEG C, 5s, 60 DEG C, 45s, collect fluorescence, 40 circulations.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182460A (en) * | 2018-10-22 | 2019-01-11 | 北京华夏时代生物工程有限公司 | Fluorescence in situ hybridization sequencing approach and the application in the detection of TPMT gene SNP |
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| CN103131776A (en) * | 2013-02-05 | 2013-06-05 | 武汉艾迪康医学检验所有限公司 | A719G single nucleotide polymorphism detection kit of thiopurine methyltransferase (TPMT) * 3C |
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| KR101747422B1 (en) * | 2015-12-11 | 2017-06-14 | 대한민국 | Reagent for SNP-genotyping of a gene related with anticancer drug-metabolizing enzyme and signal transduction pathway, the kit comprising the same, and the method for the SNP-genotyping |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182460A (en) * | 2018-10-22 | 2019-01-11 | 北京华夏时代生物工程有限公司 | Fluorescence in situ hybridization sequencing approach and the application in the detection of TPMT gene SNP |
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Application publication date: 20180202 |