CN109385479A - Detect the kit and method for dredging purine medicaments related gene SNP - Google Patents
Detect the kit and method for dredging purine medicaments related gene SNP Download PDFInfo
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- CN109385479A CN109385479A CN201811654874.3A CN201811654874A CN109385479A CN 109385479 A CN109385479 A CN 109385479A CN 201811654874 A CN201811654874 A CN 201811654874A CN 109385479 A CN109385479 A CN 109385479A
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- C—CHEMISTRY; METALLURGY
- 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
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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 discloses a kind of kits and method for detecting thin purine medicaments related gene SNP, are related to genetic polymorphism detection technical field.The kit includes one of primer combination 1- primer combination 4 or a variety of combinations.The polymorphism of 4 SNP sites of 2 thin purine medicaments related genes can be detected simultaneously using the kit, there is the features such as at low cost, result interpretation is simple, noiseless between SNP site.
Description
Technical field
The present invention relates to genetic polymorphism detection technical fields, dredge purine medicaments phase in particular to a kind of detect
The kit and method of correlation gene SNP.
Background technique
Thin purine belongs to the cell cycle specific drugs for inhibiting purine route of synthesis, chemical structure and hypoxanthine phase
Seemingly, thus hypoxanthic transition process can competitively be inhibited.Thin purine medicaments mainly contain 3 kinds: imuran dredges base
Purine and 6- thioguanine are clinically suitable for chorioepithelioma, chorioadenoma, acute lymphoblastic leukemia and urgency
The rapid change period of property non-lymphocytic leukemia, chronic myelocytic leukemia.
TPMT (thiopurine methyltransferase) also known as dredges base adenine methyl transferase or thin adenine methyl transferase, is thin
One of most important enzyme in the metabolism of purine medicaments can increase a methyl, in turn on the sulphur atom of this kind of compound
Make inaxtivation of drug.The defect of TPMT gene can allow human body that can not inactivate thin purine medicaments, so that the drug not being metabolized is in body
Interior a large amount of accumulations show anaemia, thrombopenia and oligoleukocythemia so as to cause serious or even fatal bone marrow suppression
Disease.
NUDT15 is a kind of hydrolase, can make to dredge purine active metabolite of drug TGTP and TGDP dephosphorylation, Jin Erfang
Only it mixes DNA and negatively affects the cytotoxic effect for dredging purine drug.
The study found that the gross mutation rate of NUDT15 is 2.6%, and the frequency of mutation reaches 10.5% in the crowd of East Asia.
TPMT has 8 groups of allele, and data shows, and 90% people's TPMT enzymatic activity is normal activity, 10% artificial medium activity,
The about 0.3% people enzyme shows as low or inactive, and wherein TPMT*2, TPMT*3C variation account for medium and following active crowd
95%, be the gene loci that must be examined before clinical thin purine drug use.Therefore to obtain optimum therapeuticing effect, doctor should foundation
The genotype data of patient chooses suitable antipsychotics, i.e., according to individual inheritance feature (genotype), selection is effectively controlled
Treatment scheme.Realize " gene targeting type " Individual drug treatment and " amount body medication ".
Traditional detection method is mainly sanger sequencing, fragment analysis or Single base extension technology, and each reaction is only
Detect 1 or several gene locis.Its there are detection efficiencies low, low problem of flux.Traditional detection method is completed primary
Detection cycle is longer, needs experimenter more；It detects in matched reagent simultaneously, needs fluorescent dye primer or ddNTP or length
Fragment primer, higher cost.Since there are more repetitive sequence, traditional detection methods in genome for drug metabolism gene
To the amplification of gene difficulty, difficult detection.Traditional detection method experiment flow is long, and sample well is uncapped repeatedly, is loaded often；It is same simultaneously
Multiple gene loci competitiveness amplifications, relevant interference are big in one reaction.The baseline results that traditional detection method obtains can not be
Visual display detecting result on instrument, needs laboratory technician's self-setting Parameter analysis, and as a result interpretation is complicated.Traditional detection method
The primer or ddNTP or long segment primer for needing to use fluorescent marker, the exposure long period under room temperature or illumination condition,
Or multigelation number excessively easily causes detection to fail, and failure rate is high.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of kits for detecting thin purine medicaments related gene SNP, using the examination
Agent box can simultaneously the polymorphism of 4 SNP sites of 2 thin purine medicaments related genes is detected, have it is at low cost,
As a result the features such as interpretation is simple, noiseless between SNP site.
Another object of the present invention is to provide a kind of method for detecting and dredging purine medicaments related gene SNP, this method
The polymorphism of 4 SNP sites of 2 thin purine medicaments related genes can be detected simultaneously, there is at low cost, result
The features such as interpretation is simple, noiseless between SNP site.
The present invention is implemented as follows:
On the one hand, the present invention provides a kind of kits for detecting thin purine medicaments related gene SNP comprising primer
Combine one of 1- primer combination 4 or a variety of combinations；
Wherein, primer combination 1 includes: detection shown in amplimer shown in SEQ ID NO.1-2 and SEQ ID NO.9
Primer；
Primer combination 2 includes: to detect to draw shown in amplimer shown in SEQ ID NO.3-4 and SEQ ID NO.10
Object；
Primer combination 3 includes: to detect to draw shown in amplimer shown in SEQ ID NO.5-6 and SEQ ID NO.11
Object；
Primer combination 4 includes: to detect to draw shown in amplimer shown in SEQ ID NO.7-8 and SEQ ID NO.12
Object.
It, can be simultaneously to 2 thin purine medicaments in conjunction with ionization time of flight using kit provided by the invention
4 polymorphic sites such as rs1800462, rs1800460, rs1142345 of related gene such as TPMT and NUDT15 and
Rs116855232 polymorphism is detected, and has that at low cost, result interpretation is simple, noiseless between each SNP site testing result
The features such as.
Wherein, the positional relationship of above-mentioned 2 thin purine medicaments related genes and 4 SNP sites adds the following table 1.
Table 1
The sequence of the amplimer of above-mentioned primer combination 1- primer combination 4 see the table below 2.
Table 2
Note: F: upstream primer is used in amplification；R: downstream primer is used in amplification
The sequence of detection primer and the SNP of detection of above-mentioned primer combination 1- primer combination 4 see the table below 3.
Table 3
The detection primer of above-mentioned primer combination 1- primer combination 4 with the peak position of the molecular size range of its amplified production and
Its corresponding SNP site genotype see the table below 4.
Table 4
Further, in some embodiments of the present invention, kit further includes having following component: Mg2+, dNTPs and
Taq archaeal dna polymerase.
On the other hand, the present invention provides it is a kind of detect dredge purine medicaments related gene polymorphism site method,
Include the following steps:
(1) PCR amplification is carried out to sample using the amplimer in mentioned reagent box, obtains the first amplified production；
(2) the first amplified production after alkaline phosphatase treatment is carried out using the detection primer in mentioned reagent box single
Base extension obtains the second amplified production；
(3) polymorphism of second amplified production is analyzed using ionization time of flight.
When judging result, according to the molecular size range of detection primer amplification and peak position, the gene of each each SNP site of determination
Type.
Further, in some embodiments of the present invention, in step (1), the system for carrying out PCR amplification contains:
Primer combines the amplimer of 1- primer combination 4, Mg2+, dNTPs and Taq archaeal dna polymerase.
Further, in some embodiments of the present invention, in step (1), the amplimer is in system
Concentration is 0.8-1.2 μM.
Further, in some embodiments of the present invention, in step (1), the annealing of the program of PCR amplification is carried out
Temperature are as follows: 55-57 DEG C.
Further, in some embodiments of the present invention, in step (2), the body of single base extension is carried out
System is contained: primer combines the detection primer and the first amplified production of 1- primer combination 4.
Further, in some embodiments of the present invention, system of the detection primer in single base extension
In total concentration be 3-5 μM.
Further, in some embodiments of the present invention, system of the detection primer in single base extension
In total concentration be 3.7-4 μM, primer combine 1- primer combination 4 each detection primer in the system of single base extension
Concentration is respectively: 1.052 μM, 1.063 μM, 1.036 μM and 0.681 μM.
Further, in some embodiments of the present invention, in step (2), the journey of single base extension is carried out
The annealing temperature of sequence are as follows: 51-53 DEG C, annealing time are as follows: 4-6s.
Further, in some embodiments of the present invention, the elongating temperature of the program of single base extension is carried out
It is 79-81 DEG C, time 4-6s.
It, can be simultaneously to 2 thin purines medicines in conjunction with ionization time of flight using detection method provided by the invention
4 polymorphic sites such as rs1800462, rs1800460, rs1142345 of object related gene such as TPMT and NUDT15 and
Rs116855232 polymorphism is detected, and has that at low cost, result interpretation is simple, noiseless between each SNP site testing result
The features such as.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the testing result of the control primer pair 1 in experimental example 1.
Fig. 2 is the testing result of the primer combination 1 in experimental example 1.
Fig. 3 is the testing result of the control primer pair 2 in experimental example 2.
Fig. 4 is the testing result of the primer combination 2 in experimental example 2.
Fig. 5 is the testing result of the control primer pair 3 in experimental example 3.
Fig. 6 is the testing result of the primer combination 3 in experimental example 3.
Fig. 7 is the testing result of the control primer pair 4 in experimental example 4.
Fig. 8 is the testing result of the primer combination 4 in experimental example 4.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The kit of thin purine medicaments related gene polymorphism site primer provided in this embodiment comprising for dredging
The primer sets of purine medicaments related gene polymorphism site primer, the primer sets include following primer combination 1- primer combination 4.
Wherein, primer combination 1 includes: detection shown in amplimer shown in SEQ ID NO.1-2 and SEQ ID NO.9
Primer；
Primer combination 2 includes: to detect to draw shown in amplimer shown in SEQ ID NO.3-4 and SEQ ID NO.10
Object；
Primer combination 3 includes: to detect to draw shown in amplimer shown in SEQ ID NO.5-6 and SEQ ID NO.11
Object；
Primer combination 4 includes: to detect to draw shown in amplimer shown in SEQ ID NO.7-8 and SEQ ID NO.12
Object.
The amplimer of each primer combination and the sequence of detection primer and SNP site detected see above table 2- table 4.
Certainly, it should be noted that in other examples, primer sets include in primer combination 1- primer combination 4
Any a kind, 2 kinds or 3 kinds of combination.On the basis of the invention discloses the combination of above-mentioned primer, those skilled in the art can be with
According to the classification and quantity of testing goal gene, any combination is carried out from primer combination 1- primer combination 4.Either which kind of
Combination, as long as it is belonged to the scope of protection of the present invention selected from primer combination 1- primer combination 4.
The kit of the present embodiment can be simultaneously to the 4 of 2 thin purine medicaments related gene TPMT and NUDT15
A polymorphic site, that is, rs1800462, rs1800460, rs1142345 and rs116855232 polymorphism is detected, have at
The features such as this is low, result interpretation is simple, noiseless between each SNP site testing result.
The method detected using the kit is as follows:
(1) PCR amplification is carried out to sample using the amplimer in above-mentioned primer sets, obtains the first amplified production；
PCR amplification system is as follows:
| Reagent | Volume (μ L) | Final concentration |
| ddH2O | 0.8 | n/a |
| 10x PCR buffer | 0.5 | 1x |
| MgCl2 | 0.4 | 2mM |
| dNTP Mix | 0.1 | 500μM |
| Primer Mix | 1.0 | 1μM |
| PCR Enzyme | 0.2 | 0.2U/μL |
| DNA sample | 2.0 | 10-20ng |
| total | 5.0 | n/a |
Note: all detections with reagent are purchased from Agena Bioscience (base receive biotechnology (Shanghai) in 1. present invention
Co., Ltd), similarly hereinafter；
2.Primer Mix is the mixture that is configured to the concentration ratio of 1:1 of all amplimers, and when reaction is all to draw
Final concentration of 1 μM (final concentration of 1 μM of i.e. every amplimer) of object；
3.n/a indicates that this is meaningless.
PCR amplification program is as follows:
(2) the first amplified production after alkaline phosphatase treatment is carried out using the detection primer in above-mentioned primer sets single
Base extension obtains the second amplified production；
Single base extension system is as follows:
Wherein, IPLEX Primer Mix is the mixture of all detection primers, and detection primer allocation list is (to configure 100 μ
For L, surplus ddH2O complements to 100 μ L) as follows:
Single base extension program is as follows:
(3) polymorphism of second amplified production is analyzed using ionization time of flight.
It can determine the gene of SNP site according to the peak position of detection primer and its molecular size range of the second amplified production
Type is shown in Table 4.
Embodiment 2
Using the kit and method of embodiment 1,6 parts of samples (sample type is whole blood) of known results are examined
It surveys, as a result as shown in table 5 below.
Table 5
Known results in table 5 are using conventional method (fragment analysis, Single base extension and sanger sequencing) detection
Sample results statistics, right side testing result are the result detected using the primer sets of embodiment 1.Embodiment 1 is used as the result is shown
Primer sets and detection method result it is consistent with conventional method result.
Experimental example 1
There are numerous SNP sites before and after the site rs1800462, within the scope of 3 bases of distance, there are
Then there are 10 SNP sites within the scope of 10 bases, at 20 in rs771921981 and rs190484211 totally 2 SNP sites
There are SNP site 16 within the scope of base.Furthermore there are a part section deletion mutations at rs1800462 site upstream 27bp
(ra750040431), easily lead to sample amplification failure, while being also likely to cause detection failure.In addition, being deposited in genome
There is pseudogene, extremely easy amplification obtains non-target fragment, therefore general strategy is amplification piece as long as possible in design of primers
Section, then detect, easily cause single detection site few, flux is few (such as sequencing analysis, fragment analysis)；If reducing amplification length, easily
Detection failure.The design needs of the various primers of these cause descriptions primer sets of the present invention make the creative labor.
Based on this, for the site rs1800462, design compares primer pair 1:
F (represents upstream primer, similarly hereinafter): CCCTCTATTTAGTCATTTGAAA；
R (represents downstream primer, similarly hereinafter): AAAACTTTTGTGGGGATATGG.
Amplified production 241bp can be obtained using control primer pair 1.It is as shown in Figure 1 using the result of mass spectral analysis to product.
Rs1800462 represents detection primer in figure, and right side marks black C/G and represents detection product, can determine that into 3 kinds not according to the presence or absence of peak
Homogenic type can not judgement sample genotype due to no product peak in result as shown in the figure.As a result it is difficult to judge, parting failure.
And the amplimer pair of the primer combination 1 for the site rs1800462 provided using the embodiment of the present invention 1:
F:ACTCTAATATAACCCTCTA；
R:ACCAACTACACTGTGTCCCC。
It is expanded, it is as shown in Figure 2 to be analyzed by mass spectrometry result to product.Left side rs1800462 represents detection and draws in figure
Object, right side mark black C/G and represent detection product, can determine that into 3 kinds of different genotypes, result sample as shown in the figure according to the presence or absence of peak
This genotype is CC homozygous.
Testing result is preferable as the result is shown, and Genotyping is clear, and non-false positive product generates.It indicates to use this primer detection
The site, can succeed parting.
Experimental example 2
There are 1 insert type SNP site rs1419856318, the positions within the scope of 3 bases before and after the site rs1800460
Point is the main bugbear for designing rs1800460 detection primer, can largely cause the detection false positive and vacation of target site
Feminine gender, and have SNP site 5 within the scope of 10 bases of target site, there are 12 SNP sites before the site rs4986893
Afterwards between 20 bases, including a high frequency mutational site rs2842934.
For the site rs1800460, design compares primer pair 2:
F:GGGACGCTGCTCATCTTCT；
R:TTCAAACTCATAGAAGTCTAAGCTGAT。
Amplified production 242bp can be obtained using the control amplification of primer pair 2.Result such as Fig. 4 of mass spectral analysis is used product
It is shown.Left side rs1800460 represents detection primer in figure, and right side C/T represents detection product, can determine that into 3 according to the presence or absence of peak
Kind different genotype, may be simultaneously present 3 kinds of genotype as the result is shown as shown in the figure.
As a result there is false positive, product peak is unobvious, but is higher than background value, it is not easy to determine the exact genotype of sample,
Therefore using this primer detection site result inaccuracy.
And the amplimer pair of the primer combination 2 for the site rs1800460 provided using the embodiment of the present invention 1:
F:CTGGTAGGACAAATATTGGC；
R:ACTTACCATTTGCGATCACC。
It is expanded, it is as shown in Figure 4 to be analyzed by mass spectrometry result to product.Left side rs1800460 represents detection and draws in figure
Object, right side C/T represent detection product, can determine that into 3 kinds of different genotypes according to the presence or absence of peak, are as shown in the figure as the result is shown C
Homozygous genotype.
Testing result is preferable as the result is shown, and Genotyping is clear, and non-false positive product generates.It indicates to use this primer detection
The site, can succeed parting.
Experimental example 3
The connected base in the site rs1142345 is known SNP site rs1333327023 and rs1455177009, by
It is unknown in the base type of the polymorphic site of sample herein, therefore in detection primer detection easily due to 3 end ends of most critical
Base can not form pairing and detection is caused to fail, and furthermore also contain 10 SNP within the scope of 20 bases of target site
Site.
For the site rs1142345, design compares primer pair 3:
F:GAATCCCTGATGTCATTCTTCA；
R:CATTACATTTTCAGGCTTTAGCA。
Amplified production 248bp can be obtained using the control amplification of primer pair 3.Result such as Fig. 5 of mass spectral analysis is used product
It is shown.Left side rs1142345 represents detection primer in figure, and right side C/T represents detection product, can determine that into 3 according to the presence or absence of peak
Kind different genotype, as shown in the figure detection failure as the result is shown.As a result detection failure, therefore tied using this primer detection site
Fruit inaccuracy.
And the amplimer pair of the primer combination 3 for the site rs1142345 provided using the embodiment of the present invention 1:
F:CAAAAACATGTCAGTGTGAT；
R:TAAAAGTTGGGGAATTGACTG。
It is expanded, it is as shown in Figure 6 to be analyzed by mass spectrometry result to product.Left side rs1142345 represents detection and draws in figure
Object, right side C/T represent detection product, can determine that into 3 kinds of different genotypes according to the presence or absence of peak, are as shown in the figure as the result is shown T
Homozygous genotype.Testing result is preferable as the result is shown, and Genotyping is clear, and non-false positive product generates.It indicates to use this primer
The site is detected, can succeed parting.
Experimental example 4
Have before and after the site rs116855232 within the scope of 3 bases rs1199238822, rs1026010382,
Totally 4 point mutation SNP sites, rs749170586 and rs770524351 are inserted into for 2 totally rs147390019 and rs1436122860
Type SNP site easily causes detection to be lost in detection primer detection since 3 end end bases of most critical can not form pairing
It loses.And there are SNP site 12 within the scope of the base of 10, the site distance rs1057910, while including 4 insert type SNP sites.
There are 17 SNP sites within 20 bases.
For the site rs116855232, design compares primer pair 4:
F:TGGGTTCCTTGGGAAGAACTA；
R:ATCCCACCAGA TGGTTCAGAT。
Amplified production 112bp can be obtained using the control amplification of primer pair 4.Result such as Fig. 7 of mass spectral analysis is used product
It is shown.Left side rs116855232 represents detection primer in figure, and right side C/T represents detection product, be can determine that into according to the presence or absence of peak
3 kinds of different genotypes, as shown in the figure detection failure as the result is shown.As a result detection failure, therefore use this primer detection site
As a result inaccurate.
And the amplimer pair of the primer combination 4 for the site rs116855232 provided using the embodiment of the present invention 1:
F:AAGAACTACCTCCCCTGGAC；
R:TATCCCACCAGATGGTTCAG。
It is expanded, it is as shown in Figure 8 to be analyzed by mass spectrometry result to product.Left side rs116855232 represents detection in figure
Primer, right side C/T represent detection product, can determine that into 3 kinds of different genotypes according to the presence or absence of peak, be as the result is shown as shown in the figure
C homozygous genotype.Testing result is preferable as the result is shown, and Genotyping is clear, and non-false positive product generates.It indicates to use this primer
The site is detected, can succeed parting.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Guangzhou Kingmed Center for Clinical Laboratory Co., Ltd.
<120>kit and method for dredging purine medicaments related gene SNP are detected
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>artificial sequence
<400> 1
actctaatat aaccctcta 19
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
accaactaca ctgtgtcccc 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> 3
ctggtaggac aaatattggc 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence
<400> 4
acttaccatt tgcgatcacc 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence
<400> 5
caaaaacatg tcagtgtgat 20
<210> 6
<211> 21
<212> DNA
<213>artificial sequence
<400> 6
taaaagttgg ggaattgact g 21
<210> 7
<211> 20
<212> DNA
<213>artificial sequence
<400> 7
aagaactacc tcccctggac 20
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<211> 20
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tatcccacca gatggttcag 20
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ggtgtatgat tttatgcagg ttt 23
<210> 10
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<212> DNA
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<400> 10
gtgacatgat ttgggataga gga 23
<210> 11
<211> 23
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<213>artificial sequence
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tcatttactt ttctgtaagt aga 23
<210> 12
<211> 16
<212> DNA
<213>artificial sequence
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gcttttctgg ggactg 16
Claims (8)
1. a kind of detect the kit for dredging purine medicaments related gene SNP, which is characterized in that it includes primer combination 1- primer
One of combination 4 or a variety of combinations；
Wherein, primer combination 1 includes: to detect to draw shown in amplimer shown in SEQ ID NO.1-2 and SEQ ID NO.9
Object；
Primer combination 2 includes: detection primer shown in amplimer shown in SEQ ID NO.3-4 and SEQ ID NO.10；
Primer combination 3 includes: detection primer shown in amplimer shown in SEQ ID NO.5-6 and SEQ ID NO.11；
Primer combination 4 includes: detection primer shown in amplimer shown in SEQ ID NO.7-8 and SEQ ID NO.12.
2. a kind of detect the method for dredging purine medicaments related gene SNP, which is characterized in that it includes the following steps:
(1) PCR amplification is carried out to sample using the amplimer in kit described in claim 1, obtains the first amplification production
Object；
(2) the first amplification after alkaline phosphatase treatment is produced using the detection primer in kit described in claim 1
Object carries out single base extension, obtains the second amplified production；
(3) polymorphism of second amplified production is analyzed using ionization time of flight.
3. according to the method described in claim 2, it is characterized in that, the system for carrying out PCR amplification contains: drawing in step (1)
Object combines the amplimer of 1- primer combination 4, Mg2+, dNTPs and Taq archaeal dna polymerase.
4. according to the method described in claim 2, it is characterized in that, the detection primer is in the system of single base extension
Total concentration be 3-5 μM.
5. according to the method described in claim 4, it is characterized in that, the detection primer is in the system of single base extension
Total concentration be 3.7-4 μM, primer combine 1- primer combination 4 each detection primer it is dense in the system of single base extension
Degree is respectively: 1.052 μM, 1.063 μM, 1.036 μM and 0.681 μM.
6. according to the method described in claim 2, it is characterized in that, concentration of the amplimer in system is 0.8-1.2 μ
M。
7. according to the method described in claim 2, it is characterized in that, carrying out the annealing of the program of PCR amplification in step (1)
Temperature are as follows: 55-57 DEG C.
8. according to the method described in claim 2, it is characterized in that, carrying out the system of single base extension in step (2)
Contain: primer combines the detection primer and the first amplified production of 1- primer combination 4.
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| CN111394440A (en) * | 2020-04-30 | 2020-07-10 | 北京和合医学诊断技术股份有限公司 | Primer group for detecting TPMT gene polymorphism and application method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111235264A (en) * | 2020-02-20 | 2020-06-05 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting polymorphism of human TPMT gene and NUDT15 gene |
| CN111235264B (en) * | 2020-02-20 | 2023-05-16 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting polymorphism of human TPMT gene and NUDT15 gene |
| CN111394440A (en) * | 2020-04-30 | 2020-07-10 | 北京和合医学诊断技术股份有限公司 | Primer group for detecting TPMT gene polymorphism and application method thereof |
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