CN105969876B - Primer for detecting C-KIT gene mutation in microcomponent combines and its application - Google Patents
Primer for detecting C-KIT gene mutation in microcomponent combines and its application Download PDFInfo
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Abstract
The invention discloses one kind for detecting in microcomponentC‑ KITThe primer of gene mutation combines comprising expands primer sets, the primer sets for detecting the mutation of 9 exons, the primer sets for detecting the mutation of 11 exons, the primer sets for detecting the mutation of 13 exons, the primer sets for detecting the mutation of 14 exons, the primer sets for detecting the mutation of 17 exons, the primer sets for detecting the mutation of 18 exons in advance；This method is intended to micro DNA obtaining the DNA profiling of higher concentration by amplification in advance, in conjunction in direct sequencing detection sampleC‑KITThe catastrophe of gene；Primer combination is applied to preparation detectionC‑KITIn the detection reagent of gene mutation, the DNA concentration of sample can detect down to 100pg, and can detect simultaneouslyC‑KITAll mutation of gene the 9th, 11,13,14,17 and 18 exons, detection method high sensitivity provided by the invention, high specificity is at low cost, is suitable for clinical tumor patientC‑KITThe detection of gene mutation has good clinical value.
Description
Technical field
The invention belongs to technical field of biological, and in particular to one kind is in microcomponentC-KITGene mutation inspection
The primer combination of survey and its application in tumour medication selection and medical diagnosis on disease related fields.
Background technique
C-KITGene is located at human chromosome 4q11-21, belongs to proto-oncogene, and the total 5230bp of cDNA overall length contains 21
Exon encodes transmembrane tyrosine kinase receptor (tyrosine kinase receptor, RTK) albumen of a 145KD, life
Name CD117.1st exon coding start codon and signal peptide, 2-9 codon encode the outer ligand domain of film, the
10 exons encode hydrophobic transmembrane domain, and 11-20 exon encodes film intracellular domain.Wherein 11 exons encode
Membrane-proximal region section.C-KIT receptor belongs to type III RTK family, is distributed in cell surface, can be detected with CD117 monoclonal antibody,
Have very with platelet derived growth factor receptor (platelet-derived growth factor receptors, PDGFR)
Strong homology (Patil DT, et al, 2016).
Most gastrointestinal stromal tumors (GIST) are derived fromC-KITGene mutation,C-KITMutation occurs mainly in membrane-proximal region
Exons 11, followed by the exon 9 in outer membrane area, the exons 13,14,17 of tyrosine section can also mutate.Most
Nearly data are shown, can be observed typically to be mutated in about 8 ~ 50% big tumour GIST, the frequency of mutation about 35%(Chen JJ, et
Al, 2015).In different GIST,C-KITGene mutation pattern not fully, most commonly o.11 exons mutation,
The space structure for the nearly spanning domain for causing it to encode changes, and slackens or lose the inhibition function to kinase domain
(Sanlorenzo M, et al, 2015).
Imatinib mesylate (Imatinib mesylate) is a kind of oral tyrosine kinase inhibitor class drug,
It being capable of effectively selective depression all types of abl tyrosine kinase activities, including v-abl, PDGFR and C-KIT albumen etc.
(Capelli L, et al, 2014).Imatinib is listed in May, 2001 in the U.S., and November in the same year lists in Europe, and in
In April, 2002 is in Discussion on Chinese Listed.It is initially applied to (Ph+) chronic myelocytic leukemia (CML) of Philadelphia Chromosome Positive
Treatment, is approved for the treatment of gastrointestinal stromal tumor later, so that GIST treatment enters the molecular targeted epoch.
It is generally acknowledged thatC-KIT/PDGFRAMutation type can predict the curative effect of Imatinib, wherein C-KIT exons 11
The curative effect of mutation person is best；PDGFRAD842V mutation may be to Imatinib and Sutent initial drug-resistant.Sutent is controlled
It is primary to treat GISTC-KIT9 mutation person of exon andC-KITWild type person is better thanC-KITExons 11 is mutated patient；It treats secondary
PropertyC-KITExons 13,14 is mutated patient's curative effect better than secondaryC-KITExons 17,18 mutation persons.Therefore, tumour patient exists
It is carried out before medicationC-KITThe abrupt climatic change of gene is beneficial to these patients and selects best therapeutic modality.
It is directed at presentC-KITThe detection method of mutation has very much, and such as direct sequencing, the method is more demanding to sample, inspection
Survey is limited in scope.Polymorphism analysis method (RFLP) is a kind of method by PCR in conjunction with restriction enzyme cut phase, this experimental implementation
Cumbersome, detection cycle is long, with high costs, and there are false positives caused by first round digestion not exclusively.Taqman hydrolysis probes method, makes
Mutation is detected in conjunction with fluorescence probe with amplification refractory mutation system (ARMS), for the primer of this method design, so that prominent
Modification is expanded, and wild type can not expand, so that jump signal is enhanced, convenient for detection.But ARMS technical application is last
One base mismatches the amplification that can not block wild type DNA completely, and there are the risks of false positive, and can only be for specificity
Site primer.In addition, such as high performance liquid chromatography, capillary electrophoresis, need special instrument and equipment, and complicated for operation, no
Conducive to clinically being promoted on a large scale.Amplification of nucleic acid sequences method (NASBA), author's preface sequence replicating method (3SR) and chain are set
Replica method (SDA) is changed though being isothermal duplication method, they are not strong to the specific amplification of aim sequence.
Nevertheless,C-KITThe detection method of mutation still using tumor tissues Samples detection as goldstandard, but clinically often
Often since tissue samples acquisition is in shortage, make its detection that there is certain limitation.Therefore a kind of efficiently and accurately is clinically needed
Comprehensively detect in microcomponentC-KITThe product and method of gene mutation, in order to be tumour personalized treatment service.
For this purpose, the method that the present invention is combined using constant-temperature amplification with direct Sequencing, establishes a set of for microcomponent
In sampleC-KITThe techniqueflow of all mutation type detections of gene, the detection method high sensitivity, high specificity is at low cost,
Be conducive to clinical use and popularization.
Summary of the invention
The purpose of the present invention is to provide in a kind of detection microcomponent sample with high specific and sensitivityC-KIT
The primer of gene mutation combines, using tissue DNA as test object, in conjunction with the pre- amplification technique of constant temperature and regular-PCR technology, by straight
Connect PCR sequencing PCR determine in tumor sample whether there is or notC-KITGene mutation, can be rightC-KITThe mutation of gene carries out accurate detection.
Primer combination provided by the invention can detectC-KITGene occurs in No. 9, No. 11, No. 13, No. 14, No. 17
With all mutation of 18 exons.
Provided by the present invention for detectionC-KITThe primer combination of gene mutation includes such as SEQ ID NO:1- SEQ ID
Primer sets, prominent for detecting 9 exons as shown in SEQ ID NO:19- SEQ ID NO:20 are expanded shown in NO:18 in advance
The primer sets of change, as shown in SEQ ID NO:21- SEQ ID NO:22 for detect 11 exons be mutated primer sets,
Primer sets, such as SEQ ID NO as shown in SEQ ID NO:23- SEQ ID NO:24 for detecting the mutation of 13 exons:
Primer sets, such as SEQ ID NO:27- SEQ ID shown in 25- SEQ ID NO:26 for detecting the mutation of 14 exons
For detecting the primer sets of 17 exons mutation, as shown in SEQ ID NO:29- SEQ ID NO:30 shown in NO:28
For detecting the primer sets of 18 exons mutation.
It is another object of the present invention to detect above-mentioned primer combined application in preparationC-KITThe detection reagent of gene mutation
In.
It is another object of the present invention to the combination of above-mentioned primer is applied to preparation to be used to detectC-KITThe detection of gene mutation
In kit, the reagent constituents also include one or more of following conventional constituents: polymerase, primer combination, PCR are anti-
Answer buffer, dNTP, BSA, deionized water.
The present invention is for detectingC-KITThe detection application method of the primer combination of gene mutation, the specific steps are as follows:
(1) primer dilutes
Mixed after primer1-prime18 is diluted respectively and Primer Mix(table 1 be made), wherein primer1-
Final concentration of 0.05-0.1 μM of primer16 primer, final concentration of 1-3 μM of primer17-primer18 primer；
Primer19-primer30 primer (table 1) is diluted to 5-10 μM.
(2) pre- amplification
10 × reaction buffer of 1 μ L, Primer Mix of 2.5 μ L, 100-1000pg minim DNA is added in amplification pipe
(DNA profiling that the present invention uses derives from colon cancer patient tissue to template, and according to normal described in " Molecular Cloning:A Laboratory guide "
Rule method extract tissue DNA), 8.8 μ L are complemented to deionized water；It mixes, 98 DEG C of initial denaturation 5-10min are subsequently placed on ice
10-20min；Add 0.5 μ L dNTP (10mM each), 0.2 100 × BSA of μ L and 0.5 μ L phi29 DNA polymerization
Enzyme, overnight, 65 DEG C of heating 10-20min inactivate enzyme for 30-35 DEG C of amplification.
(3) the above-mentioned pre- amplified production of PCR Purification Kit is used.
(4)C-KITDetection in Gene Mutation
ForC-KITNo. 9, No. 11, No. 13, No. 14, No. 17 of gene and the mutation of 18 exons, are respectively adopted such as SEQ
Primer sets shown in ID NO:19- SEQ ID NO:20 detect the mutation of 9 exons；Such as SEQ ID NO:21- SEQ ID
Primer sets shown in NO:22 detect the mutation of 11 exons, the primer sets as shown in SEQ ID NO:23- SEQ ID NO:24
The mutation of 13 exons is detected, the primer sets as shown in SEQ ID NO:25- SEQ ID NO:26 examine the mutation of 14 exons,
The primer sets as shown in SEQ ID NO:27- SEQ ID NO:28 detect the mutation of 17 exons, such as SEQ ID NO:29-
Primer sets shown in SEQ ID NO:30 detect the mutation of 18 exons.
Configuring PCR reaction total system is 25 μ L: wherein 12.5 μ L of Pfu Mix mixed liquor, the forward primer (5-10 μM)
Volume 0.5-1 μ L, reverse primer (5-10 μM) volume 0.5-1 μ L, pre- amplified production 1-2 μ L, 25 μ L are complemented to water.
PCR reaction condition are as follows: 1. 94 DEG C, 5min initial denaturation；2. 94 DEG C, 30s denaturation；3. 55 DEG C, 30s annealing；4. 72 DEG C, 35s extends；
Circulation is 2. to 4. 35 times；5. 72 DEG C, 5min；Sequencing detection.
Detection advantage of the invention is:The present invention is combined using more primers, utilizes constant-temperature amplification and regular-PCR phase
In conjunction with method, solve in tissue samples that initial DNA content is low, can not carry outC-KITThe limitation of detection in Gene Mutation.Reagent price used in the present invention is cheap, can large batch of processing sample.The method of the present invention amplification efficiency is high, such as
1000ng/ μ L can be obtained in the DNA of 100pg after expanding；The catastrophe of multidigit point can be detected simultaneously.The method of the present invention DNA
DNA fidelity is good after amplification.In short, can be realized using the method for the present invention in microcomponent's sampleC-KITThe mutation of gene
Situation carries out efficient, accurate detection, for clinical tumor early screening, instructs clinical application, and monitor the pre- of tumour
There is good practical application value afterwards.
Table 1: the nucleotide sequence of primer 1-30
。
Detailed description of the invention
Fig. 1 is the DNA profiling amplification that the present invention is directed to 1000pg；Wherein A figure is pre- amplification (3 repetition)；B
Figure isC-KITGene 9, No. 11, No. 13, No. 14, No. 17 and 18 exon amplifications；
Fig. 2 is the DNA profiling amplification that the present invention is directed to 500pg；Wherein A figure is pre- amplification (3 repetition)；B
Figure isC-KITGene 9, No. 11, No. 13, No. 14, No. 17 and 18 exon amplifications；
Fig. 3 is the DNA profiling amplification that the present invention is directed to 100pg.A figure is pre- amplification (3 repetition)；B figure isC-KITGene 9, No. 11, No. 13, No. 14, No. 17 and 18 exon amplifications；
Fig. 4 is the present invention in the DNA profiling of 1000pgC-KITGene No. 9, No. 11, No. 13, No. 14, No. 17 and 18
Exon sequencing result figure；
Fig. 5 is the present invention in the DNA profiling of 500pgC-KITGene No. 9, No. 11, No. 13, No. 14, No. 17 and No. 18
Exon sequencing result figure；
Fig. 6 is the present invention in the DNA profiling of 100pgC-KITGene No. 9, No. 11, No. 13, No. 14, No. 17 and No. 18
Exon sequencing result figure.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention, person skilled in art can be the present invention according to aforementioned present invention content
Some nonessential modifications and adaptations out.In following embodiments, if non-specific show that agents useful for same is to analyze pure, Suo Youshi
Agent can be obtained from commercial channel, and percentage is mass percent.Test method without specific conditions in text, is usually pressed
Implement according to condition proposed by normal condition described in " Molecular Cloning:A Laboratory guide " or reagent manufacturer.Unless separately fixed
Justice, all professions as used herein and scientific words have the same meanings as commonly understood by one of ordinary skill in the art.
In the DNA profiling of embodiment 1:1000pgC-KITDetection in Gene Mutation
1, experimental material
Phi29 archaeal dna polymerase, 10 × reaction buffer, dNTP (10nM), 100 × BSA, 1000pg's
DNA profiling, deionized water, primer1-prime18 (i.e. Primer Mix), 2 × Pfu Mix, primer19-primer30,
PCR instrument, ultra-thin Product Purification Kit (TIANGEN Biotech (Beijing) Co., Ltd. DP203), 1% Ago-Gel, electrophoresis
Instrument.
2, experimental procedure and result
2.1 pre- amplifications
(1) primer dilutes
Mixed after primer1-prime18 is diluted respectively and form Primer Mix(table 1), wherein primer1-
Final concentration of 0.1 μM of primer16 primer, final concentration of 2 μM of primer17-primer18 primer.Configure following system:
；
(2) after mixing mentioned reagent, then 98 DEG C of initial denaturation 10min are immediately placed in 20min on ice；
(3) following system is added:
；
After said mixture is mixed, overnight, 65 DEG C of heating 10min inactivate enzyme for 30-35 DEG C of amplification.The glue detection of paving 1%
(Figure 1A).It is good to the DNA profiling expanding effect of 1000pg using phi29DNA polymerase as the result is shown.
The purifying of 2.2 PCR products
(1) column equilibration step: be added into adsorption column CB1 500 μ L equilibrium liquid BL, 12,000 rpm (~13,400 ×
G) it is centrifuged 1 min, the waste liquid in collecting pipe is outwelled, adsorption column is placed back in collecting pipe；
(2) volume for estimating PCR reaction solution, is added the combination liquid PB of 5 times of volumes thereto, mixes well (without removal
Paraffin oil or mineral oil)；
(3) previous step acquired solution is added in an adsorption column CB1 (adsorption column is put into collecting pipe), is placed at room temperature for 2
Min, 12,000 rpm (~13,400 × g) are centrifuged 30s, outwell the waste liquid in collecting pipe, adsorption column CB1 is put into collecting pipe
In；
(4) 600 μ L rinsing liquid PW(be added into adsorption column CB1 please first checked whether before and dehydrated alcohol has been added),
12,000 rpm (~13,400 × g) are centrifuged 30s, outwell the waste liquid in collecting pipe, adsorption column CB1 is put into collecting pipe；
(5) repetitive operation step (4)；
(6) 12,000 rpm (~13,400 × g) are centrifuged 2 min, as far as possible removing rinsing liquid, and adsorption column is placed in room temperature and is put
It sets several minutes, thoroughly dries, to prevent remaining rinsing liquid from influencing the experiment of next step；
(7) adsorption column CB1 is taken out, is put into a clean centrifuge tube, 20-50 is vacantly added dropwise to adsorbed film middle position
μ L elution buffer EB, is placed at room temperature for 2 min.12,000rpm (~13,400 × g) are centrifuged 2 min, collect DNA solution.
2.3 C-KITDetection in Gene Mutation
(1) following PCR reaction system is configured, is expanded respectivelyC-KITNo. 9, No. 11, No. 13, No. 14, No. 17 of gene and
18 exons；
；
Wherein for the primer of 9 exons mutation as shown in SEQ ID NO:19- SEQ ID NO:20；For No. 11
The primer of exons mutation is as shown in SEQ ID NO:21- SEQ ID NO:22；The primer such as SEQ ID of 13 exons mutation
Shown in NO:23- SEQ ID NO:24；The primer of 14 exons mutation is as shown in SEQ ID NO:25- SEQ ID NO:26；
The primer of 17 exons mutation is as shown in SEQ ID NO:27- SEQ ID NO:28；The primer of 18 exons mutation is such as
Shown in SEQ ID NO:29- SEQ ID NO:30.
(2) reaction condition: 1. 94 DEG C, 5min initial denaturation；2. 94 DEG C, 30s denaturation；3. 55 DEG C, 30s annealing；4. 72 DEG C,
35s extends；Circulation is 2. to 4. 35 times；5. 72 DEG C, 5min；
(3) PCR after reaction, spreads 1% glue detection (Figure 1B), as the result is shown the second PCR pairs of wheelC-KITThe 9th of gene
Number, No. 11, No. 13, No. 14, No. 17 and 18 exons there is specific amplification；It is then sent to raw work bioengineering (Shanghai)
Limited liability company carries out sequencing detection.Sequencing result as shown in figure 4,C-KITGene the 9,11,13,14th and 17 exons are equal
Be not detected mutation, the 18th exon occur same sense mutation c.2586G > C.
In the DNA profiling of embodiment 2:500pgC-KITDetection in Gene Mutation
1, experimental material
Phi29 archaeal dna polymerase, 10 × reaction buffer, dNTP (10nM), 100 × BSA, 1000pg's
DNA profiling, deionized water, primer1-prime18 (i.e. Primer Mix), 2 × Pfu Mix, primer19-primer30,
PCR instrument, ultra-thin Product Purification Kit (TIANGEN Biotech (Beijing) Co., Ltd. DP203), 1% Ago-Gel, electrophoresis
Instrument.
2, experimental procedure
2.1 pre- amplifications
(1) primer dilutes.Mixed after primer1-prime18 is diluted respectively and form Primer Mix(table 1), wherein
Final concentration of 0.1 μM of primer1-primer16 primer, final concentration of 2 μM of primer17-primer18 primer.Configuration is such as
Lower system:
；
(2) after mixing mentioned reagent, then 98 DEG C of initial denaturation 10min are immediately placed in 20min on ice.
(3) following system is added:
；
After said mixture is mixed, overnight, 65 DEG C of heating 20min inactivate enzyme for 30-35 DEG C of amplification.The glue detection of paving 1%
(Fig. 2A).It is good to the DNA profiling expanding effect of 500pg using phi29DNA polymerase as the result is shown.
The purifying of 2.2 PCR products
(1) column equilibration step: be added into adsorption column CB1 500 μ L equilibrium liquid BL, 12,000 rpm (~ 13,400 ×
G) it is centrifuged 1 min, the waste liquid in collecting pipe is outwelled, adsorption column is placed back in collecting pipe.
(2) volume for estimating PCR reaction solution, is added the combination liquid PB of 5 times of volumes thereto, mixes well (without removal
Paraffin oil or mineral oil).
(3) previous step acquired solution is added in an adsorption column CB1 (adsorption column is put into collecting pipe), is placed at room temperature for 2
Min, 12,000 rpm (~13,400 × g) are centrifuged 60 sec, outwell the waste liquid in collecting pipe, adsorption column CB1 is put into collection
Guan Zhong.
(4) 600 μ L rinsing liquid PW(be added into adsorption column CB1 please first checked whether before and dehydrated alcohol has been added),
12,000 rpm (~13,400 × g) are centrifuged 30 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, as far as possible removing rinsing liquid.Adsorption column is placed in room temperature to put
It sets several minutes, thoroughly dries, to prevent remaining rinsing liquid from influencing the experiment of next step.
(7) adsorption column CB1 is taken out, is put into a clean centrifuge tube, 20-50 is vacantly added dropwise to adsorbed film middle position
μ L elution buffer EB, is placed at room temperature for 2 min.12,000rpm (~13,400 × g) are centrifuged 2 min, collect DNA solution.
2.3 C-KITDetection in Gene Mutation
(1) following PCR reaction system is configured, is expanded respectivelyC-KITNo. 9, No. 11, No. 13, No. 14, No. 17 of gene and
18 exons.
；
Wherein for the primer of 9 exons mutation as shown in SEQ ID NO:19- SEQ ID NO:20；For No. 11
The primer of exons mutation is as shown in SEQ ID NO:21- SEQ ID NO:22；The primer such as SEQ ID of 13 exons mutation
Shown in NO:23- SEQ ID NO:24；The primer of 14 exons mutation is as shown in SEQ ID NO:25- SEQ ID NO:26；
The primer of 17 exons mutation is as shown in SEQ ID NO:27- SEQ ID NO:28；The primer of 18 exons mutation is such as
Shown in SEQ ID NO:29- SEQ ID NO:30.
(2) reaction condition: 1. 94 DEG C, 5min initial denaturation；2. 94 DEG C, 30s denaturation；3. 55 DEG C, 30s annealing；4. 72 DEG C,
35s extends；Circulation is 2. to 4. 35 times；5. 72 DEG C, 5min；
(3) PCR after reaction, spreads 1% glue detection (Fig. 2 B), as the result is shown the second PCR pairs of wheelC-KITThe 9th of gene
Number, No. 11, No. 13, No. 14, No. 17 and 18 exons there is specific amplification；It is then sent to raw work bioengineering (Shanghai)
Limited liability company carries out sequencing detection.Sequencing result as shown in figure 5,C-KITGene the 9,11,13,14th and 17 exons are equal
Be not detected mutation, the 18th exon occur same sense mutation c.2586G > C.
In the DNA profiling of embodiment 3:100pgC-KITDetection in Gene Mutation
1, experimental material
Phi29 archaeal dna polymerase, 10 × reaction buffer, dNTP (10nM), 100 × BSA, 1000pg's
DNA profiling, deionized water, primer1-prime18 (i.e. Primer Mix), 2 × Pfu Mix, primer19-primer30,
PCR instrument, ultra-thin Product Purification Kit (TIANGEN Biotech (Beijing) Co., Ltd. DP203), 1% Ago-Gel, electrophoresis
Instrument.
2, experimental procedure
2.1 pre- amplifications
(1) primer dilutes.Mixed after primer1-prime18 is diluted respectively and form Primer Mix(table 1), wherein
Final concentration of 0.1 μM of primer1-primer16 primer, final concentration of 2 μM of primer17-primer18 primer.
Configure following system:
；
(2) after mixing mentioned reagent, then 98 DEG C of initial denaturation 10min are immediately placed in 20min on ice.
(3) following system is added:
；
After said mixture is mixed, overnight, 65 DEG C of heating 20min inactivate enzyme for 30-35 DEG C of amplification.The glue detection of paving 1%
(Fig. 3 A).It is good to the DNA profiling expanding effect of 100pg using phi29DNA polymerase as the result is shown.
The purifying of 2.2 PCR products
(1) column equilibration step: be added into adsorption column CB1 500 μ L equilibrium liquid BL, 12,000 rpm (~ 13,400 ×
G) it is centrifuged 1 min, the waste liquid in collecting pipe is outwelled, adsorption column is placed back in collecting pipe.
(2) volume for estimating PCR reaction solution, is added the combination liquid PB of 5 times of volumes thereto, mixes well (without removal
Paraffin oil or mineral oil).
(3) previous step acquired solution is added in an adsorption column CB1 (adsorption column is put into collecting pipe), is placed at room temperature for 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) it 600 μ L rinsing liquid PW(is added into adsorption column CB1 is please first checked whether before and anhydrous second has been added
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
In collecting pipe.
(5) repetitive operation step 4.
(6) 12,000 rpm (~13,400 × g) are centrifuged 2 min, as far as possible removing rinsing liquid.Adsorption column is placed in room temperature to put
It sets several minutes, thoroughly dries, to prevent remaining rinsing liquid from influencing the experiment of next step.
(7) adsorption column CB1 is taken out, is put into a clean centrifuge tube, 20-50 is vacantly added dropwise to adsorbed film middle position
μ L elution buffer EB, is placed at room temperature for 2 min.12,000rpm (~13,400 × g) are centrifuged 2 min, collect DNA solution.
2.3 C-KITDetection in Gene Mutation
(1) following PCR reaction system is configured, is expanded respectivelyC-KITNo. 9, No. 11, No. 13, No. 14, No. 17 of gene and
18 exons.
；
Wherein for the primer of 9 exons mutation as shown in SEQ ID NO:19- SEQ ID NO:20；For No. 11
The primer of exons mutation is as shown in SEQ ID NO:21- SEQ ID NO:22；The primer such as SEQ ID of 13 exons mutation
Shown in NO:23- SEQ ID NO:24；The primer of 14 exons mutation is as shown in SEQ ID NO:25- SEQ ID NO:26；
The primer of 17 exons mutation is as shown in SEQ ID NO:27- SEQ ID NO:28；The primer of 18 exons mutation is such as
Shown in SEQ ID NO:29- SEQ ID NO:30.
(2) reaction condition: 1. 94 DEG C, 5min initial denaturation；2. 94 DEG C, 30s denaturation；3. 55 DEG C, 30s annealing；4. 72 DEG C,
35s extends；Circulation is 2. to 4. 35 times；5. 72 DEG C, 5min；
(3) PCR after reaction, spreads 1% glue detection (Fig. 3 B), as the result is shown the second PCR pairs of wheelC-KITThe 9th of gene
Number, No. 11, No. 13, No. 14, No. 17 and 18 exons there is specific amplification；It is then sent to raw work bioengineering (Shanghai)
Limited liability company carries out sequencing detection；Sequencing result as shown in fig. 6,C-KITGene the 9,11,13,14th and 17 exons are equal
Be not detected mutation, the 18th exon occur same sense mutation c.2586G > C.
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 table
<110>Kunming University of Science and Technology
<120>for detecting in microcomponentC-KITThe primer of gene mutation combines and its application
<160>30
<170> PatentIn version 3.3
<210> 1
<211> 15
<212> DNA
<213>artificial sequence
<400> 1
ggtgtactga atact 15
<210> 2
<211> 15
<212> DNA
<213>artificial sequence
<400> 2
gcaacactat agtat 15
<210> 3
<211> 15
<212> DNA
<213>artificial sequence
<400> 3
ttacaagtta aaatg 15
<210> 4
<211> 15
<212> DNA
<213>artificial sequence
<400> 4
cacagagact tggca 15
<210> 5
<211> 15
<212> DNA
<213>artificial sequence
<400> 5
gatcacaaag atttg 15
<210> 6
<211> 15
<212> DNA
<213>artificial sequence
<400> 6
gtggttaaag gaaac 15
<210> 7
<211> 15
<212> DNA
<213>artificial sequence
<400> 7
agtcctgcaa aggat 15
<210> 8
<211> 15
<212> DNA
<213>artificial sequence
<400> 8
cctgtctagg gatat 15
<210> 9
<211> 15
<212> DNA
<213>artificial sequence
<400> 9
tagtgttgct tatat 15
<210> 10
<211> 15
<212> DNA
<213>artificial sequence
<400> 10
cttgtaaaga gtgaa 15
<210> 11
<211> 15
<212> DNA
<213>artificial sequence
<400> 11
gaatacacta ttagg 15
<210> 12
<211> 15
<212> DNA
<213>artificial sequence
<400> 12
cgaccatgag taagg 15
<210> 13
<211> 15
<212> DNA
<213>artificial sequence
<400> 13
accacataat tagaa 15
<210> 14
<211> 15
<212> DNA
<213>artificial sequence
<400> 14
gcaggactgt caagc 15
<210> 15
<211> 15
<212> DNA
<213>artificial sequence
<400> 15
ggatttacat tatga 15
<210> 16
<211> 15
<212> DNA
<213>artificial sequence
<400> 16
gctgaagtat actta 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 cagtcacgag tatgccacat cccaagtgtt 40
<210> 20
<211> 22
<212> DNA
<213>artificial sequence
<400> 20
cctaaacatc cccttaaatt gg 22
<210> 21
<211> 45
<212> DNA
<213>artificial sequence
<400> 21
agggttttcc cagtcacgct gacctacaaa tatttacagg taacc 45
<210> 22
<211> 20
<212> DNA
<213>artificial sequence
<400> 22
tgacatggaa agcccctgtt 20
<210> 23
<211> 40
<212> DNA
<213>artificial sequence
<400> 23
agggttttcc cagtcacgca agcgtaagtt cctgtatggt 40
<210> 24
<211> 22
<212> DNA
<213>artificial sequence
<400> 24
caataaaagg cagcttggac ac 22
<210> 25
<211> 38
<212> DNA
<213>artificial sequence
<400> 25
agggttttcc cagtcacgca tgaccaccct tgggtatt 38
<210> 26
<211> 25
<212> DNA
<213>artificial sequence
<400> 26
aactgcctgt caacagctaa ctatt 25
<210> 27
<211> 40
<212> DNA
<213>artificial sequence
<400> 27
agggttttcc cagtcacgca tcattcaagg cgtacttttg 40
<210> 28
<211> 22
<212> DNA
<213>artificial sequence
<400> 28
ctttgcagga ctgtcaagca ga 22
<210> 29
<211> 40
<212> DNA
<213>artificial sequence
<400> 29
agggttttcc cagtcacgca gcaacagcag catctataag 40
<210> 30
<211> 25
<212> DNA
<213>artificial sequence
<400> 30
ctgagtctaa tgaagttgtc tttgg 25
Claims (3)
1. for detecting in microcomponentC-KITThe primer of gene mutation combines, it is characterised in that: by such as SEQ ID NO:1-
Primer sets are expanded shown in SEQ ID NO:18 in advance, are used to detect No. 9 as shown in SEQ ID NO:19- SEQ ID NO:20
The primer sets of exons mutation, as shown in SEQ ID NO:21- SEQ ID NO:22 for detect 11 exons be mutated
Primer sets, the primer sets as shown in SEQ ID NO:23- SEQ ID NO:24 for detecting the mutation of 13 exons, such as SEQ
Primer sets, such as SEQ ID NO:27- shown in ID NO:25- SEQ ID NO:26 for detecting the mutation of 14 exons
Primer sets, such as SEQ ID NO:29- SEQ ID NO:30 shown in SEQ ID NO:28 for detecting the mutation of 17 exons
Shown in for detect 18 exons mutation primer sets form；
For detectingC-KITThe detection application method of the primer combination of gene mutation, the specific steps are as follows:
(1) primer dilutes
It is mixed after primer1-prime18 is diluted respectively and Primer Mix is made, wherein primer1-primer16 primer
Final concentration of 0.05-0.1 μM, final concentration of 1-3 μM of primer17-primer18 primer；Primer19-primer30 primer
It is diluted to 5-10 μM；
(2) pre- amplification
10 × reaction buffer of 1 μ L, Primer Mix of 2.5 μ L, 100-1000pg minim DNA mould is added in amplification pipe
Plate complements to 8.8 μ L with deionized water；It mixes, 98 DEG C of initial denaturation 5-10min are subsequently placed in 10-20min on ice；It adds
0.5 μ L dNTP 10mM each, 0.2 100 × BSA of μ L and 0.5 μ L phi29 archaeal dna polymerase, 30-35 DEG C of amplification are stayed overnight,
65 DEG C of heating 10-20min inactivate enzyme；
(3) the above-mentioned pre- amplified production of PCR Purification Kit is used；
(4)C-KITDetection in Gene Mutation
ForC-KITNo. 9, No. 11, No. 13, No. 14, No. 17 of gene and the mutation of 18 exons, are respectively adopted such as SEQ ID
Primer sets shown in NO:19- SEQ ID NO:20 detect the mutation of 9 exons；Such as SEQ ID NO:21- SEQ ID NO:22
Shown in primer sets detect 11 exons mutation, the primer sets as shown in SEQ ID NO:23- SEQ ID NO:24 detection 13
Exon mutation, the primer sets as shown in SEQ ID NO:25- SEQ ID NO:26 examine the mutation of 14 exons, such as SEQ
Primer sets shown in ID NO:27- SEQ ID NO:28 detect the mutation of 17 exons, such as SEQ ID NO:29- SEQ ID
Primer sets shown in NO:30 detect the mutation of 18 exons；
Configuring PCR reaction total system is 25 μ L: wherein 12.5 μ L of Pfu Mix mixed liquor, 5-10 μM of forward primer 0.5-1 μ L, 5-
10 μM of reverse primer 0.5-1 μ L, pre- amplified production 1-2 μ L, 25 μ L are complemented to water；
PCR reaction condition are as follows: 1. 94 DEG C, 5min initial denaturation；2. 94 DEG C, 30s denaturation；3. 55 DEG C, 30s annealing；4. 72 DEG C, 35s
Extend；Circulation is 2. to 4. 35 times；5. 72 DEG C, 5min；Sequencing detection.
2. primer combination described in claim 1 is detected in preparationC-KITApplication in the detection reagent of gene mutation.
3. primer combination described in claim 1 is detected in preparationC-KITApplication in the detection kit of gene mutation.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102061333A (en) * | 2009-11-13 | 2011-05-18 | 上海宝藤生物医药科技有限公司 | Primer used for detecting c-kit gene mutation and application thereof |
| CN102634510A (en) * | 2012-04-27 | 2012-08-15 | 昆明理工大学 | Pre-amplification method for trace DNA applied in medicolegal expertise |
| CN104328184A (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 C-kit gene 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 |
-
2016
- 2016-06-15 CN CN201610418064.2A patent/CN105969876B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102061333A (en) * | 2009-11-13 | 2011-05-18 | 上海宝藤生物医药科技有限公司 | Primer used for detecting c-kit gene mutation and application thereof |
| 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 |
| CN104328184A (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 C-kit gene mutation |
Non-Patent Citations (1)
| Title |
|---|
| Multiple displacement amplification as an adjunct to PCR-based detection of Staphylococcus aureus in synovial fluid;Sandeep Kathju et al.;《BMC Research Notes》;20101231;1-5 * |
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