CN106011253A - Primer combination for detecting gene PDGFRA mutation in microtissue and application thereof - Google Patents
Primer combination for detecting gene PDGFRA mutation in microtissue and application thereof Download PDFInfo
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Abstract
The invention discloses a primer combination for detecting gene PDGFRA mutation in microtissue. The primer combination comprises a pre-amplification primer group, a primer group for detecting mutation of a twelfth exon, a primer group for detecting mutation of a fourteenth exon and a primer group for detecting mutation of an eighteenth exon. According to the method, it aims at obtaining a high-concentration DNA template through a trace of DNA by means of pre-amplification, and the mutation situation of a gene PDGFRA in a sample is detected through a direct sequencing method. The primer combination is applied to detection reagents for detecting mutation of the gene PDGFRA, can detect samples with the DNA concentration lower to 100 pg and can simultaneously detect all mutation of the twelfth exon, the fourteenth exon and the eighteenth exon of the gene PDGFRA. The detection method is high in sensitivity and specificity, low in cost and suitable for detection of mutation of the gene PDGFRA of a clinical cancer patient and has very high clinical application value.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of in microcomponentPDGFRAGene mutation is examined
The primer surveyed combines and in tumor medication selection and the application of medical diagnosis on disease related fields.
Background technology
PDGFR be molecular weight be the strand membrane glycoprotein of 180kD, extracellular ligand land contain 5 immunoglobulin-likes
Domain, the cysteine residues that tool is conservative, single transmembrane segment；The tyrosine kinase district breaking part of intracellular, for hydrophile amino
Acid insertion sequence.Acceptor molecule is by two kinds of subunit compositions of α, β, and the PDGFR after maturation is with dimer form stable (α α, α β, β β)
Isomer (PDGF-AA, AB, BB) knot corresponding with part PDGF (platelet-derived growth factor, PDGF)
Close.In Colorectal Carcinoma, PDGFR α and PDGFR β all has expression and distribution, PDGFR α to be distributed in Colon and rectum normal structure, breath
On meat tissue and tumor tissues；PDGFR β is expressed in tumor cell, mesenchyma stroma of tumors cell and microvessel cell and (includes blood capillary
Pericyte) upper (Appiah-Kubi K, et al, 2016;Abdel-Rahman O, 2015).
PDGFRAGene mutation is common in the tumors such as GIST, glioblastoma multiforme, pernicious peripheral nervous sheath, wherein in GISTPDGFRAGene mutation rate is about 5 ~ 10%, and sudden change takes place mostly inPDGFRAThe nearly film end regions (exons 1 2) of gene and
Kinases district (exons 14 and exons 1 8), mutation frequency is respectively 0.8% and 3.9%, wherein sports master with exon 18
(Vega F, et al, 2015).PDGFRAAfter gene mutation then by activation AKT, MAPK and stat protein in STAT1 and
STAT3 plays a role.Also studies have found that the A-Raf kinases of activation can regulate PLCG1 and turn through the signal of PDCFR Dependent
Lead, also can regulate the PI3K signal transduction (Farooqi AA, et al, 2015) through PDGFR non-dependent.
Imatinib is a kind of tyrosine protein enzyme inhibitor, can block tyrosine protein kinase KIT function of receptors, thus
The formation of suppression tumor.Research shows,PDGFRAUse after gene extron 12 and exons 18 most gene site mutation
During the treatment with tyrosine kinase inhibitors such as imatinib, Sutent, GIST patient can therefrom benefit.But such as exons 18 gene
Site occurs D842V, RD841-842KI or D1842-843IM sudden change to use the tyrosine kinase such as imatinib, Sutent to press down
During preparation for treating, GIST patient can not therefrom benefit.Therefore, tumour patient was carried out before medicationPDGFRAThe sudden change inspection of gene
Survey, be beneficial to these patients and select best therapeutic modality.
Currently forPDGFRAThe detection method of sudden change has a lot, and such as direct sequencing, sample is required higher by this method, inspection
Survey is limited in scope.Polymorphism analysis method (RFLP), is a kind of method being combined with Restriction Enzyme cut by PCR, this experimental implementation
Loaded down with trivial details, the detection cycle is long, with high costs, there is the false positive that first round enzyme action not exclusively causes.Taqman hydrolysis probes method, makes
Be combined with fluorescent probe with amplification refractory mutation system (ARMS) and detect sudden change, for the primer of the method design so that be prominent
Modification is expanded, and wild type cannot expand, thus enhances jump signal, it is simple to detection.But the application of ARMS technology is last
One base does not mate the amplification that can not block wild type DNA completely, there is false-positive risk, and can only be for specificity
Site is detected.Additionally, such as high performance liquid chromatography, blood capillary electrophoresis etc., need special instrument and equipment, and operation complexity, no
It is beneficial to promote the most on a large scale.Amplification of nucleic acid sequences method (NASBA), author's preface sequence replicating method (3SR) and chain are put
Change duplicating method (SDA) though being isothermal duplication method, but they are the strongest to the specific amplification of aim sequence.
While it is true,PDGFRASudden change detection method still with tumor tissues Samples detection as goldstandard, but clinically
Usually obtain in shortage due to tissue samples so that it is detection has certain limitation.Need badly a kind of the most accurate the most clinically
The most all sidedly in detection microcomponentPDGFRAThe product of gene mutation and method, in order to service for tumor personalized treatment.
To this end, the present invention utilizes the method that constant-temperature amplification combines with direct Sequencing, establish a set of for microcomponent
In samplePDGFRAThe techniqueflow of gene all mutation types detection, this detection method is highly sensitive, high specificity, cost
Low, beneficially Clinical practice and popularization.
Summary of the invention
It is an object of the invention to provide in a kind of detection microcomponent sample with high specific and sensitivityPDGFRAThe primer combination of gene mutation, with tissue DNA for detection object, in conjunction with the pre-amplification technique of constant temperature and regular-PCR technology,
By direct sequencing determine in tumor sample with or withoutPDGFRAGene mutation, can be rightPDGFRAThe sudden change of gene is carried out accurately
Detection.
The primer combination that the present invention provides can detectPDGFRAThere is owning at the 12nd, 14 and 18 exons in gene
Sudden change.
What the present invention provided is used for detectingPDGFRAThe primer combination of gene mutation includes such as SEQ ID NO:1-SEQ ID
Pre-amplification primer sets shown in NO:18, being used for as shown in SEQ ID NO:19-SEQ ID NO:20 detect 12 exons
The primer sets of sudden change, the primer for detection 14 exon sudden change as shown in SEQ ID NO:21-SEQ ID NO:22
Group, the primer sets for detection 18 exon sudden change as shown in SEQ ID NO:23-SEQ ID NO:24.
It is another object of the present invention to above-mentioned primer combination application in preparation detectionPDGFRAThe detection examination of gene mutation
In agent.
It is another object of the present invention to that the combination of above-mentioned primer is applied to preparation be used for detectingPDGFRAThe inspection of gene mutation
In test agent box, described reagent constituents also comprises one or more in following conventional constituents: polymerase, primer combine, PCR
Reaction buffer, dNTP, BSA, deionized water.
The present invention is used for detectingPDGFRAThe detection using method of the primer combination of gene mutation, specifically comprises the following steps that
(1) primer dilution
After being diluted respectively by primer1-prime18, mixing prepares Primer Mix(table 1), wherein primer1-primer16 draws
Final concentration of 0.05-0.1 μM of thing, final concentration of 1-3 μM of primer17-primer18 primer；primer19-primer24
Primer (table 1) is diluted to 5-10 μM.
(2) expand in advance
Primer Mix, 100-1000pg trace DNA templet of the 10 × reaction buffer of 1 μ L, 2.5 μ L is added in amplification pipe
(DNA profiling that the present invention uses derives from colon cancer patient tissue, and according to routine side described in " Molecular Cloning: A Laboratory guide "
Method extract tissue DNA), complement to 8.8 μ L with deionized water；Mixing, 98 DEG C of denaturations 5-10min, it is subsequently placed in 10-on ice
20min；Add 0.5 μ L dNTP (10mM each), 0.2 μ L 100 × BSA and 0.5 μ L phi29 archaeal dna polymerase,
Overnight, 65 DEG C of heating 10-20min make enzyme inactivate in 30-35 DEG C of amplification.
(3) the above-mentioned pre-amplified production of PCR Purification Kit is used.
(4)PDGFRADetection in Gene Mutation
ForPDGFRANo. 12 of gene, No. 14 and the sudden change of 18 exons, be respectively adopted such as SEQ ID NO:19-SEQ ID
Primer sets shown in NO:20 detects 12 exon sudden changes；Primer sets as shown in SEQ ID NO:21-SEQ ID NO:22
Detecting 14 exon sudden changes, the primer sets as shown in SEQ ID NO:23-SEQ ID NO:24 detects 18 exons and dashes forward
Become.
It is 25 μ L that configuration PCR reacts total system: wherein Pfu Mix mixed liquor 12.5 μ L, described forward primer (5-10 μM)
Volume 0.5-1 μ L, the volume 0.5-1 μ L of reverse primer (5-10 μM), pre-amplified production 1-2 μ L, complement to 25 μ L with water.
PCR reaction condition is: 1. 94 DEG C, 5min denaturation；2. 94 DEG C, 30s degeneration；3. 55 DEG C, 30s anneals；4. 72 DEG C, 35s extends；
Circulation is 2. to 4. 35 times；5. 72 DEG C, 5min；Order-checking detection.
The detection advantage of the present invention is:The present invention uses the combination of many primers, utilizes constant-temperature amplification and regular-PCR phase
In conjunction with method, solve initial DNA content in tissue samples low, it is impossible to carry outPDGFRAThe limitation of detection in Gene Mutation
Property.The reagent used in the present invention is cheap, can large batch of process sample.The inventive method amplification efficiency is high, as
The DNA of 100pg is available 1000ng/ μ L after amplification；The catastrophe in many sites can be detected simultaneously.The inventive method DNA
After amplification, DNA fidelity is good.In a word, the inventive method is used can to realize in microcomponent's samplePDGFRAThe sudden change of gene
Situation carries out efficiently, detects accurately, and it is for clinical tumor early screening, instructs clinical application, and monitors the pre-of tumor
After there is good actual application value.
Table 1: the nucleotide sequence of primer 1-24
。
Accompanying drawing explanation
Fig. 1 is the DNA profiling amplification that the present invention is directed to 1000pg；Wherein A figure is pre-amplification (repeating for 3 times)；B
Figure isPDGFRAGene 12,14 and 18 exon amplification.
Fig. 2 is the DNA profiling amplification that the present invention is directed to 500pg；Wherein A figure is pre-amplification (repeating for 3 times)；B
Figure isPDGFRAGene 12,14 and 18 exon amplification.
Fig. 3 is the DNA profiling amplification that the present invention is directed to 100pg.A figure is pre-amplification (repeating for 3 times)；B figure isPDGFRAGene 12,14 and 18 exon amplification.
Fig. 4 is in the DNA profiling that the present invention is directed to 1000pgPDGFRAGene 12,14 and 18 exon sequencing result
Figure.
Fig. 5 is in the DNA profiling that the present invention is directed to 500pgPDGFRAGene 12,14 and 18 exon sequencing result figure.
Fig. 6 is in the DNA profiling that the present invention is directed to 100pgPDGFRAGene 12,14 and 18 exon sequencing result figure.
Detailed description of the invention
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.Following example are merely to illustrate this
Invention rather than restriction the scope of the present invention, the present invention can be done by person skilled in art according to the invention described above content
Go out some nonessential improvement and adjustment.In following embodiment, showing if non-specific, agents useful for same is analytical pure, all examinations
Agent all can obtain from commercial channel, and percentage ratio is mass percent.In literary composition, the experimental technique of unreceipted actual conditions, generally presses
According to normal condition described in " Molecular Cloning: A Laboratory guide ", or the condition proposed by reagent manufacturer is implemented.Unless it is fixed separately
The same meaning that justice, all specialties used in literary composition and scientific words and one skilled in the art are familiar with.
In the DNA profiling of embodiment 1:1000pgPDGFRADetection in Gene Mutation
1, experiment material
Phi29 archaeal dna polymerase, 10 × reaction buffer, dNTP (10nM), the DNA mould of 100 × BSA, 1000pg
Plate, deionized water, primer1-prime18 (i.e. Primer Mix), 2 × Pfu Mix, primer19-primer24, PCR
Instrument, ultra-thin Product Purification Kit (TIANGEN Biotech (Beijing) Co., Ltd. DP203), the agarose gel of 1%, electrophresis apparatus.
2, experimental procedure and result
2.1 expand in advance
(1) primer dilution
After being diluted respectively by primer1-prime18, mixing forms Primer Mix(table 1), wherein primer1-primer16 draws
Final concentration of 0.1 μM of thing, final concentration of 2 μMs of primer17-primer18 primer.Configure following system:
；
(2) after mentioned reagent being mixed, 98 DEG C of denaturations 10min, then it is immediately placed in 20min on ice；
(3) following system is added:
；
After being mixed by said mixture, overnight, 65 DEG C of heating 10-20min make enzyme inactivate in 30-35 DEG C of amplification.The glue detection of paving 1%
(Figure 1A).Result display uses phi29DNA polymerase good to the DNA profiling expanding effect of 1000pg.
2.2 PCR primer purification
(1) column equilibration step: add the balance liquid BL of 500 μ L, 12,000 rpm (～13,400 × g) in adsorption column CB1
Centrifugal 1 min, outwells the waste liquid in collecting pipe, is placed back in collecting pipe by adsorption column；
(2) estimating the volume of PCR reactant liquor, be added thereto to the combination liquid PB of 5 times of volumes, fully mixing is (without removing paraffin
Oil or mineral oil)；
(3) adding in an adsorption column CB1 (adsorption column is put in collecting pipe) by previous step gained solution, room temperature places 2 min,
12,000 rpm (～13,400 × g) centrifugal 30-60s, outwells the waste liquid in collecting pipe, is put in collecting pipe by adsorption column CB1；
(4) the most first check whether before adding 600 μ L rinsing liquid PW(uses in adsorption column CB1 and added dehydrated alcohol), 12,
000 rpm (～13,400 × g) centrifugal 30-60s, outwells the waste liquid in collecting pipe, is put in collecting pipe by adsorption column CB1；
(5) repetitive operation step (4)；
(6) 12,000 rpm (～13,400 × g) centrifugal 2 min, remove rinsing liquid as far as possible, adsorption column is placed in room temperature and places number
Minute, dry up hill and dale, to prevent the rinsing liquid of residual from affecting next step experiment；
(7) taking out adsorption column CB1, put in a clean centrifuge tube, to adsorbed film centre position, unsettled dropping 20-50 μ L washes
De-buffer EB, room temperature places 2 min.12,000rpm (～13,400 × g) centrifugal 2 min, collect DNA solution.
2.3 PDGFRADetection in Gene Mutation
(1) configure following PCR reaction system, expand respectivelyPDGFRAThe 12nd of gene, 14 and 18 exons；
Wherein for 12 exons sudden change primer as shown in SEQ ID NO:19-SEQ ID NO:20；Show for 14 extras
The primer of son sudden change is as shown in SEQ ID NO:21-SEQ ID NO:22；The primer such as SEQ ID NO of 18 exon sudden changes:
Shown in 23-SEQ ID NO:24.
(2) reaction condition: 1. 94 DEG C, 5min denaturation；2. 94 DEG C, 30s degeneration；3. 55 DEG C, 30s anneals；4. 72 DEG C,
35s extends；Circulation is 2. to 4. 35 times；5. 72 DEG C, 5min；
(3), after PCR reaction terminates, glue detection (Figure 1B) of paving 1%, result shows that second takes turns PCR pairPDGFRAGene the 12nd,
All there is specific amplification in 14 and 18 exons；It is then sent to Sangon Biotech (Shanghai) Co., Ltd. check order
Detection.Sequencing result as shown in Figure 4,PDGFRAGene the 12nd, 14 and 18 exon is all not detected by sudden change.
In the DNA profiling of embodiment 2:500pgPDGFRADetection in Gene Mutation
1, experiment material
Phi29 archaeal dna polymerase, 10 × reaction buffer, dNTP (10nM), the DNA mould of 100 × BSA, 1000pg
Plate, deionized water, primer1-prime18 (i.e. Primer Mix), 2 × Pfu Mix, primer19-primer24, PCR
Instrument, ultra-thin Product Purification Kit (TIANGEN Biotech (Beijing) Co., Ltd. DP203), the agarose gel of 1%, electrophresis apparatus.
2, experimental procedure
2.1 expand in advance
(1) primer dilution.After being diluted respectively by primer1-prime18, mixing forms Primer Mix(table 1), wherein
Final concentration of 0.1 μM of primer1-primer16 primer, final concentration of 2 μMs of primer17-primer18 primer.Configuration is such as
Lower system:
(2) after mentioned reagent being mixed, 98 DEG C of denaturations 10min, then it is immediately placed in 20min on ice.
(3) following system is added:
After being mixed by said mixture, overnight, 65 DEG C of heating 10-20min make enzyme inactivate in 30-35 DEG C of amplification.The glue detection of paving 1%
(Fig. 2 A).Result display uses phi29DNA polymerase good to the DNA profiling expanding effect of 500pg.
2.2 PCR primer purification
(1) column equilibration step: add the balance liquid BL of 500 μ L, 12,000 rpm (~ 13,400 × g) in adsorption column CB1
Centrifugal 1 min, outwells the waste liquid in collecting pipe, is placed back in collecting pipe by adsorption column.
(2) estimating the volume of PCR reactant liquor, be added thereto to the combination liquid PB of 5 times of volumes, fully mixing is (without removing
Paraffin oil or mineral oil).
(3) adding in an adsorption column CB1 (adsorption column is put in collecting pipe) by previous step gained solution, room temperature places 2
Min, 12,000 rpm (~ 13,400 × g) are centrifuged 30-60 sec, outwell the waste liquid in collecting pipe, adsorption column CB1 is put into receipts
In collector.
(4) the most first check whether before adding 600 μ L rinsing liquid PW(uses in adsorption column CB1 and added dehydrated alcohol),
12,000 rpm (~ 13,400 × g) are centrifuged 30-60 sec, outwell the waste liquid in collecting pipe, adsorption column CB1 is put into collecting pipe
In.
(5) repetitive operation step 4.
(6) 12,000 rpm (~ 13,400 × g) are centrifuged 2 min, remove rinsing liquid as far as possible.Adsorption column is placed in room temperature put
Put several minutes, dry up hill and dale, to prevent the rinsing liquid of residual from affecting next step experiment.
(7) take out adsorption column CB1, put in a clean centrifuge tube, the unsettled dropping 20-50 to adsorbed film centre position
μ L elution buffer EB, room temperature places 2 min.12,000rpm (~ 13,400 × g) are centrifuged 2 min, collect DNA solution.
2.3 PDGFRADetection in Gene Mutation
(1) configure following PCR reaction system, expand respectivelyPDGFRAThe 12nd of gene, 14 and 18 exons.
Wherein for 12 exons sudden change primer as shown in SEQ ID NO:19-SEQ ID NO:20；Show for 14 extras
The primer of son sudden change is as shown in SEQ ID NO:21-SEQ ID NO:22；The primer such as SEQ ID NO of 18 exon sudden changes:
Shown in 23-SEQ ID NO:24.
(2) reaction condition: 1. 94 DEG C, 5min denaturation；2. 94 DEG C, 30s degeneration；3. 55 DEG C, 30s anneals；4. 72 DEG C,
35s extends；Circulation is 2. to 4. 35 times；5. 72 DEG C, 5min；
(3), after PCR reaction terminates, glue detection (Fig. 2 B) of paving 1%, result shows that second takes turns PCR pairPDGFRAGene the 12nd,
All there is specific amplification in 14 and 18 exons；It is then sent to Sangon Biotech (Shanghai) Co., Ltd. check order
Detection.Sequencing result as it is shown in figure 5,PDGFRAGene the 12nd, 14 and 18 exon is all not detected by sudden change.
In the DNA profiling of embodiment 3:100pgPDGFRADetection in Gene Mutation
1, experiment material
Phi29 archaeal dna polymerase, 10 × reaction buffer, dNTP (10nM), the DNA mould of 100 × BSA, 1000pg
Plate, deionized water, primer1-prime18 (i.e. Primer Mix), 2 × Pfu Mix, primer19-primer24, PCR
Instrument, ultra-thin Product Purification Kit (TIANGEN Biotech (Beijing) Co., Ltd. DP203), the agarose gel of 1%, electrophresis apparatus.
2, experimental procedure
2.1 expand in advance
(1) primer dilution.After being diluted respectively by primer1-prime18, mixing forms Primer Mix(table 1), wherein
Final concentration of 0.1 μM of primer1-primer16 primer, final concentration of 2 μMs of primer17-primer18 primer.
Configure following system:
(2) after mentioned reagent being mixed, 98 DEG C of denaturations 10min, then it is immediately placed in 20min on ice.
(3) following system is added:
After being mixed by said mixture, overnight, 65 DEG C of heating 10-20min make enzyme inactivate in 30-35 DEG C of amplification.The glue detection of paving 1%
(Fig. 3 A).Result display uses phi29DNA polymerase good to the DNA profiling expanding effect of 100pg.
2.2 PCR primer purification
(1) column equilibration step: add the balance liquid BL of 500 μ L, 12,000 rpm (~ 13,400 × g) in adsorption column CB1
Centrifugal 1 min, outwells the waste liquid in collecting pipe, is placed back in collecting pipe by adsorption column.
(2) estimating the volume of PCR reactant liquor, be added thereto to the combination liquid PB of 5 times of volumes, fully mixing is (without removing
Paraffin oil or mineral oil).
(3) adding in an adsorption column CB1 (adsorption column is put in collecting pipe) by previous step gained solution, room temperature places 2
Min, 12,000 rpm (~ 13,400 × g) are centrifuged 30-60 sec, outwell the waste liquid in collecting pipe, adsorption column CB1 is put into receipts
In collector.
(4) the most first check whether before adding 600 μ L rinsing liquid PW(uses in adsorption column CB1 and added anhydrous second
Alcohol), 12,000 rpm (~ 13,400 × g) are centrifuged 30-60 sec, outwell the waste liquid in collecting pipe, are put into by adsorption column CB1
In collecting pipe.
(5) repetitive operation step 4.
(6) 12,000 rpm (~ 13,400 × g) are centrifuged 2 min, remove rinsing liquid as far as possible.Adsorption column is placed in room temperature put
Put several minutes, dry up hill and dale, to prevent the rinsing liquid of residual from affecting next step experiment.
(7) take out adsorption column CB1, put in a clean centrifuge tube, the unsettled dropping 20-50 to adsorbed film centre position
μ L elution buffer EB, room temperature places 2 min.12,000rpm (~ 13,400 × g) are centrifuged 2 min, collect DNA solution.
2.3 PDGFRADetection in Gene Mutation
(1) configure following PCR reaction system, expand respectivelyPDGFRAThe 12nd of gene, 14 and 18 exons.
；
Wherein for 12 exons sudden change primer as shown in SEQ ID NO:19-SEQ ID NO:20；Show for 14 extras
The primer of son sudden change is as shown in SEQ ID NO:21-SEQ ID NO:22；The primer such as SEQ ID NO of 18 exon sudden changes:
Shown in 23-SEQ ID NO:24.
(2) reaction condition: 1. 94 DEG C, 5min denaturation；2. 94 DEG C, 30s degeneration；3. 55 DEG C, 30s anneals；4. 72 DEG C,
35s extends；Circulation is 2. to 4. 35 times；5. 72 DEG C, 5min；
(3), after PCR reaction terminates, glue detection (Fig. 3 B) of paving 1%, result shows that second takes turns PCR pairPDGFRAGene the 12nd,
All there is specific amplification in 14 and 18 exons；It is then sent to Sangon Biotech (Shanghai) Co., Ltd. check order
Detection；Sequencing result as shown in Figure 6,PDGFRAGene the 12nd, 14 and 18 exon is all not detected by sudden change.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.All within the spirit and principles in the present invention, that is made any repaiies
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Sequence table
<110>Kunming University of Science and Technology
<120>it is used for detecting in microcomponentPDGFRAThe primer combination of gene mutation and application thereof
<160> 24
<170> PatentIn version 3.3
<210> 1
<211> 15
<212> DNA
<213>artificial sequence
<400> 1
cactgaggcc aagta 15
<210> 2
<211> 15
<212> DNA
<213>artificial sequence
<400> 2
gatatccaat cacag 15
<210> 3
<211> 15
<212> DNA
<213>artificial sequence
<400> 3
caggaagttg gtagc 15
<210> 4
<211> 15
<212> DNA
<213>artificial sequence
<400> 4
acggccagat ccagt 15
<210> 5
<211> 15
<212> DNA
<213>artificial sequence
<400> 5
gataatgact cacct 15
<210> 6
<211> 15
<212> DNA
<213>artificial sequence
<400> 6
agcctgcacc aagtc 15
<210> 7
<211> 15
<212> DNA
<213>artificial sequence
<400> 7
ggacacatgt ggttg 15
<210> 8
<211> 15
<212> DNA
<213>artificial sequence
<400> 8
cacacatggc aggga 15
<210> 9
<211> 15
<212> DNA
<213>artificial sequence
<400> 9
ctatttacca gaagt 15
<210> 10
<211> 15
<212> DNA
<213>artificial sequence
<400> 10
aaccaagaat atgac 15
<210> 11
<211> 15
<212> DNA
<213>artificial sequence
<400> 11
atcacatatc agtcc 15
<210> 12
<211> 15
<212> DNA
<213>artificial sequence
<400> 12
tcagttcaga catga 15
<210> 13
<211> 15
<212> DNA
<213>artificial sequence
<400> 13
ctcccagcaa gttta 15
<210> 14
<211> 15
<212> DNA
<213>artificial sequence
<400> 14
gtgtccagtg aaaat 15
<210> 15
<211> 15
<212> DNA
<213>artificial sequence
<400> 15
atggagagtg gagga 15
<210> 16
<211> 15
<212> DNA
<213>artificial sequence
<400> 16
agtcagctcc attca 15
<210> 17
<211> 10
<212> DNA
<213>artificial sequence
<400> 17
gggcaggang 10
<210> 18
<211> 8
<212> DNA
<213>artificial sequence
<400> 18
nnatgtgg 8
<210> 19
<211> 40
<212> DNA
<213>artificial sequence
<400> 19
agggttttcc cagtcacggt ccagtcactg tgctgcttca 40
<210> 20
<211> 22
<212> DNA
<213>artificial sequence
<400> 20
aaagggagtc ttgggaggtt ac 22
<210> 21
<211> 42
<212> DNA
<213>artificial sequence
<400> 21
agggttttcc cagtcacgct gaggccaagt agctatctgc tt 42
<210> 22
<211> 24
<212> DNA
<213>artificial sequence
<400> 22
tgaaaatcct cactccaggt cagt 24
<210> 23
<211> 39
<212> DNA
<213>artificial sequence
<400> 23
agggttttcc cagtcacgac catggatcag ccagtcttg 39
<210> 24
<211> 21
<212> DNA
<213>artificial sequence
<400> 24
agcctgacca gtgagggaag t 21
Claims (3)
1. it is used for detecting in microcomponentPDGFRAThe primer combination of gene mutation, it is characterised in that include such as SEQ ID NO:
Pre-amplification primer sets shown in 1-SEQ ID NO:18, being used for as shown in SEQ ID NO:19-SEQ ID NO:20 detect
The primer sets of 12 exon sudden changes, dashing forward for detection 14 exons as shown in SEQ ID NO:21-SEQ ID NO:22
The primer sets become, the primer sets for detection 18 exon sudden change as shown in SEQ ID NO:23-SEQ ID NO:24.
2. the primer sets described in claim 1 is combined in preparation detectionPDGFRAApplication in the detectable of gene mutation.
3. the primer sets described in claim 1 is combined in preparation detectionPDGFRAApplication in the detection kit of gene mutation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108374045A (en) * | 2018-01-04 | 2018-08-07 | 广州金域医学检验集团股份有限公司 | Primer group, kit and method for detecting PDGFRA gene mutation |
| CN114058701A (en) * | 2021-10-27 | 2022-02-18 | 上海科亦生物科技有限公司 | C-kit and PDGFR mutation detection kit |
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| CN102634510A (en) * | 2012-04-27 | 2012-08-15 | 昆明理工大学 | Pre-amplification method for trace DNA applied in medicolegal expertise |
| CN104328182A (en) * | 2014-10-30 | 2015-02-04 | 武汉百泰基因工程有限公司 | Primer, probe, locked nucleic acid probe, kit and detection method for detecting PDGFRA gene hotspot mutation |
| CN104561250A (en) * | 2013-10-22 | 2015-04-29 | 联合基因生物医药有限公司 | Method and primer for detecting imatinib targeted medication gene |
| CN105087792A (en) * | 2015-08-13 | 2015-11-25 | 广州和实生物技术有限公司 | Rapid single-tube detection method and rapid single-tube detection kit for multipoint mutation of PDGFRA (platelet-derived growth factor receptor alpha) gene |
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| CN102634510A (en) * | 2012-04-27 | 2012-08-15 | 昆明理工大学 | Pre-amplification method for trace DNA applied in medicolegal expertise |
| CN104561250A (en) * | 2013-10-22 | 2015-04-29 | 联合基因生物医药有限公司 | Method and primer for detecting imatinib targeted medication gene |
| CN104328182A (en) * | 2014-10-30 | 2015-02-04 | 武汉百泰基因工程有限公司 | Primer, probe, locked nucleic acid probe, kit and detection method for detecting PDGFRA gene hotspot mutation |
| CN105087792A (en) * | 2015-08-13 | 2015-11-25 | 广州和实生物技术有限公司 | Rapid single-tube detection method and rapid single-tube detection kit for multipoint mutation of PDGFRA (platelet-derived growth factor receptor alpha) gene |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108374045A (en) * | 2018-01-04 | 2018-08-07 | 广州金域医学检验集团股份有限公司 | Primer group, kit and method for detecting PDGFRA gene mutation |
| CN114058701A (en) * | 2021-10-27 | 2022-02-18 | 上海科亦生物科技有限公司 | C-kit and PDGFR mutation detection kit |
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