CN109234371A - A kind of c-kit detection method of gene mutation - Google Patents
A kind of c-kit detection method of gene mutation Download PDFInfo
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- CN109234371A CN109234371A CN201811413923.4A CN201811413923A CN109234371A CN 109234371 A CN109234371 A CN 109234371A CN 201811413923 A CN201811413923 A CN 201811413923A CN 109234371 A CN109234371 A CN 109234371A
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Abstract
The invention discloses a kind of c-kit detection method of gene mutation, include the following steps: S1, the DNA sequence dna design specificity amplification primer according to 9,11,13 and No. 17 c-Kit exons；S2, the gene that 9,11,13 and No. 17 c-Kit exons of PCR amplification are carried out using the specificity amplification primer designed in step S1, and pcr amplification product is digested；S3, according to sequencing primer, cyclic amplification is carried out to enzymolysis product and obtains Sanger segment；S4, it is compared with wild-type genotype according to the DNA sequence dna information of 9,11,13 and No. 17 c-Kit exons using analysis on gene instrument is automated using Sanger segment obtained in step S3, finds out mutational site.The present invention devises specificity amplification primer, the length of primer is shortened, specific amplification sensitivity is improved, can intuitively see whether the target gene of PCR amplification is qualified by the specificity amplification primer, to decide whether to carry out to avoid waste reagent in next step.
Description
Technical field
The present invention relates to technical field of biological, and in particular to a kind of c-kit detection method of gene mutation.
Background technique
Gastrointestinal stromal tumor (GIST) is most common leaf source property tumour of alimentary canal.The GIST of the overwhelming majority expresses c-
The Kit albumen (CD117) of kit gene coding.On molecular level, there is c-kit gene mutation in most GIST, thus
The activation of Kit albumen is caused not need the mistake that ligand SCF participates in stimulate the continuous proliferation and anti-apoptotic signal of tumour cell
Control.
C-kit gene mutation rate is about 90% in GIST, and mutant form multiplicity.Wherein it is located at 11 exons
The variation of Lys550-Val560 section is most commonly seen (accounting for about 70-80%), positioned at 9 exon Ala502-Tyr503 sections
6 bases repeat mutation and account for about 5-10%.Clinical research shows that the catastrophe of c-Kit gene and Gleevec are molecular targeted in GIST
The curative effect for the treatment of is related: the curative effect for being mutated patient there are 11 exons is best, and there are patient's curative effects time of 9 exons mutation
It, the curative effect of the patient of 13 exons mutation is also better than wild type, and the curative effect of wild type GIST is worst.In addition, for 9
The patient of exon mutation improves dosage and is remarkably improved curative effect.In addition, the patient of 17 exon D816V mutation
To imatinib-resistant.Therefore c-Kit gene mutation is detected for instructing the rational use of medicines of GIST patient, there is important reference price
Value.
There are Sanger PCR sequencing PCR, NGS for the detection method of ckit at present.
Summary of the invention
In view of the deficiencies of the prior art, the present invention is intended to provide a kind of c-kit detection method of gene mutation, special by design
Specific amplification primers shorten the length of primer, improve specific amplification sensitivity, can intuitively see the purpose of PCR amplification
Whether gene is qualified, to decide whether to carry out in next step, to avoid waste reagent.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of c-kit detection method of gene mutation, includes the following steps:
S1, the DNA sequence dna design specificity amplification primer according to 9,11,13 and No. 17 c-Kit exons；Wherein,
The corresponding specificity amplification primer of No. 9 c-Kit exons are as follows:
ckit9F:TTCTGTACTGCCAGTGGATGTG；
ckit9R:TGGAATGAACTTAAAATCA；
The corresponding specificity amplification primer of No. 11 c-Kit exons are as follows:
kit11F:CTGAGACAATAATTATTAAA；
kit11R:GTGACATGGAAAGCCCCTGT；
The corresponding specificity amplification primer of No. 13 c-Kit exons are as follows:
kit13F:GAAGCCCTCATGTCTGAACTC；
KIT13R:CAATAAAAGGCAGCTTGGACACGGCT；
The corresponding specificity amplification primer of No. 17 c-Kit exons are as follows:
kit17F:AGACTTGGCAGCCAGAAATATCCT；
kit17R:ATCACAGGAAACAATTTTTAT；
S2, using the specificity amplification primer designed in step S1 respectively to 9,11,13 and No. 17 c-Kit exons
Gene DNA carry out PCR amplification, and pcr amplification product is digested respectively；
S3, using sequencing primer, respectively to 9 obtained in step S2, the gene DNA of 11,13 and No. 17 c-Kit exons
Enzymolysis product by cyclic amplification obtain Sanger segment；The sequencing primer are as follows:
ckit9R:TGGAATGAACTTAAAATCA；
kit11R:GTGACATGGAAAGCCCCTGT；
KIT13R:CAATAAAAGGCAGCTTGGACACGGCT；
kit17R:ATCACAGGAAACAATTTTTAT；
S4, it is utilized using Sanger segment obtained in step S3 and is analyzed on automation gene instrument according to 9,11,13 and No. 17
The DNA sequence dna information of c-Kit exon, compares with wild-type genotype, finds out mutational site.
Further, the detailed process of step S2 is
S2.1, PCR amplification: anti-in carrying out expanding on quantitative fluorescent PCR using 10ulPCR reaction solution and 2.5ul gene DNA
It answers；Amplification condition are as follows:
Initial denaturation: it 95 DEG C, 10 minutes, recycles 1 time；
Denaturation: it 94 DEG C, 15 seconds, recycles 45 times；
Annealing extends, fluorescence signal acquisition: 60 DEG C, 45 seconds, recycling 45 times；
Instrument is cooling: 25 DEG C, 1 minute, recycling 1 time；
S2.2, pcr amplification product obtained in step S2.1 is digested:
Take 5 μ L that 2 μ L SAP enzyme mixations are added the pcr amplification product of CT≤30, concussion mixes, after of short duration centrifugation,
37 DEG C of 60 minutes and 80 DEG C of 15 minutes enzymatic hydrolysis are successively carried out in PCR instrument.
Further, the detailed process of step S3 are as follows:
S3.1, sequencing PCR reaction:
It takes 1.5 μ L of enzymolysis product, 1 μ L of sequencing reagent and 18 μ L of sequencing primer to be mixed with pipette tips, PCR expansion is carried out after centrifugation
Increase, obtains Sanger segment；Amplification condition are as follows:
Initial denaturation: it 96 DEG C, 1 minute, recycles 1 time；
Denaturation: it 96 DEG C, 10 seconds, recycles 30 times；
Annealing: it 50 DEG C, 5 seconds, recycles 30 times；
Extend: 50 DEG C, 2 minutes, recycling 30 times；
Instrument is cooling: 25 DEG C, 1 minute, recycling 1 time；
S3.2, Sanger fragment purification:
S3.2.1,50 μ L sodium acetates-alcohol mixture (3M is added into Sanger segment obtained in step S3.1
NaAc: dehydrated alcohol=1:14), it acutely vibrates, is protected from light standing 15 minutes, 4 DEG C of centrifugation half an hour of 12000g or more inhale and abandon upper layer
Liquid；
S3.2.2,70 μ L, 70% pre-cooled ethanol is added, acutely vibrates, 4 DEG C of 12000g or more are centrifuged 15 minutes, inhale on abandoning
Layer liquid；
S3.2.3, it allows ethyl alcohol to volatilize at room temperature completely, 12 μ L Hi-Di Formamide dissolving DNAs is added, obtain pure
Sanger segment after change.
The beneficial effects of the present invention are: the present invention devises specificity amplification primer, shortens the length of primer, improves
Specific amplification sensitivity can intuitively see whether the target gene of PCR amplification closes by the specificity amplification primer
Lattice, to decide whether to carry out in next step, to avoid waste reagent.
Specific embodiment
The invention will be further described below, it should be noted that the present embodiment premised on the technical program,
The detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to the present embodiment.
A kind of c-kit detection method of gene mutation is present embodiments provided, is included the following steps:
S1, the DNA sequence dna design specificity amplification primer according to 9,11,13 and No. 17 c-Kit exons；Wherein,
The corresponding specificity amplification primer of No. 9 c-Kit exons are as follows:
ckit9F:TTCTGTACTGCCAGTGGATGTG
ckit9R:TGGAATGAACTTAAAATCA
The corresponding specificity amplification primer of No. 11 c-Kit exons are as follows:
kit11F:CTGAGACAATAATTATTAAA
kit11R:GTGACATGGAAAGCCCCTGT
The corresponding specificity amplification primer of No. 13 c-Kit exons are as follows:
kit13F:GAAGCCCTCATGTCTGAACTC
KIT13R:CAATAAAAGGCAGCTTGGACACGGCT
The corresponding specificity amplification primer of No. 17 c-Kit exons are as follows:
kit17F:AGACTTGGCAGCCAGAAATATCCT
kit17R:ATCACAGGAAACAATTTTTAT
S2, using the specificity amplification primer designed in step S1 respectively to 9,11,13 and No. 17 c-Kit exons
Gene DNA carry out PCR amplification, and pcr amplification product is digested respectively；Detailed process are as follows:
S2.1, PCR amplification: anti-in carrying out expanding on quantitative fluorescent PCR using 10ulPCR reaction solution and 2.5ul gene DNA
It answers；
Amplification condition is as shown in table 1:
Table 1
S2.2, pcr amplification product obtained in step S2.1 is digested:
Take 5 μ L that 2 μ L SAP enzyme mixations are added the pcr amplification product of CT≤30, concussion mixes, after of short duration centrifugation,
37 DEG C of 60 minutes and 80 DEG C of 15 minutes enzymatic hydrolysis are successively carried out in PCR instrument.
S3, using sequencing primer, respectively to 9 obtained in step S2, the gene DNA of 11,13 and No. 17 c-Kit exons
Enzymolysis product by cyclic amplification obtain Sanger segment；The sequencing primer are as follows:
ckit9R:TGGAATGAACTTAAAATCA
kit11R:GTGACATGGAAAGCCCCTGT
KIT13R:CAATAAAAGGCAGCTTGGACACGGCT
kit17R:ATCACAGGAAACAATTTTTAT
The cyclic amplification obtains the detailed process of Sanger segment are as follows:
S3.1, sequencing PCR reaction:
It takes 1.5 μ L of enzymolysis product, 1 μ L of sequencing reagent and 18 μ L of sequencing primer to be mixed with pipette tips, PCR expansion is carried out after centrifugation
Increase, obtains Sanger segment；The sequencing reagent is the Bigdye of AB company.
Specific amplification condition is as shown in table 2:
Table 2
S3.2, Sanger fragment purification:
S3.2.1,50 μ L sodium acetates-alcohol mixture (3M is added into Sanger segment obtained in step S3.1
NaAc: dehydrated alcohol=1:14), it acutely vibrates, is protected from light standing 15 minutes, 4 DEG C of centrifugation half an hour of 12000g or more inhale and abandon upper layer
Liquid；
S3.2.2,70 μ L, 70% pre-cooled ethanol is added, acutely vibrates, 4 DEG C of 12000g or more are centrifuged 15 minutes, inhale on abandoning
Layer liquid；
S3.2.3, it allows ethyl alcohol to volatilize at room temperature completely, 12 μ L Hi-Di Formamide dissolving DNAs is added, obtain pure
Sanger segment after change.
S4, it is utilized using Sanger segment obtained in step S3 and is analyzed on automation gene instrument according to 9,11,13 and No. 17
The DNA sequence dna information of c-Kit exon, compares with wild-type genotype, finds out mutational site；Gleevec is carried out according to mutation type
Medication guide.
In the method for the present embodiment, it can intuitively see whether the target gene of PCR amplification closes after increasing probe
Lattice, to decide whether to carry out in next step, to avoid waste reagent.
For those skilled in the art, it can be provided various corresponding according to above technical solution and design
Change and modification, and all these change and modification, should be construed as being included within the scope of protection of the claims of the present invention.
Claims (3)
1. a kind of c-kit detection method of gene mutation, which comprises the steps of:
S1, the DNA sequence dna design specificity amplification primer according to 9,11,13 and No. 17 c-Kit exons；Wherein,
The corresponding specificity amplification primer of No. 9 c-Kit exons are as follows:
ckit9F:TTCTGTACTGCCAGTGGATGTG；
ckit9R:TGGAATGAACTTAAAATCA；
The corresponding specificity amplification primer of No. 11 c-Kit exons are as follows:
kit11F:CTGAGACAATAATTATTAAA；
kit11R:GTGACATGGAAAGCCCCTGT；
The corresponding specificity amplification primer of No. 13 c-Kit exons are as follows:
kit13F:GAAGCCCTCATGTCTGAACTC；
KIT13R:CAATAAAAGGCAGCTTGGACACGGCT；
The corresponding specificity amplification primer of No. 17 c-Kit exons are as follows:
kit17F:AGACTTGGCAGCCAGAAATATCCT；
kit17R:ATCACAGGAAACAATTTTTAT；
S2, using the specificity amplification primer designed in step S1 respectively to the base of 9,11,13 and No. 17 c-Kit exons
Because DNA carries out PCR amplification, and pcr amplification product is digested respectively；
S3, using sequencing primer, respectively to 9 obtained in step S2, the enzyme of the gene DNA of 11,13 and No. 17 c-Kit exons
It solves product and obtains Sanger segment by cyclic amplification；The sequencing primer are as follows:
ckit9R:TGGAATGAACTTAAAATCA；
kit11R:GTGACATGGAAAGCCCCTGT；
KIT13R:CAATAAAAGGCAGCTTGGACACGGCT；
kit17R:ATCACAGGAAACAATTTTTAT；
S4, it is utilized using Sanger segment obtained in step S3 and is analyzed on automation gene instrument according to 9,11,13 and No. 17 c-Kit
The DNA sequence dna information of exon, compares with wild-type genotype, finds out mutational site.
2. c-kit detection method of gene mutation according to claim 1, which is characterized in that the detailed process of step S2 is
S2.1, PCR amplification: using 10ulPCR reaction solution and 2.5ul gene DNA in being carried out amplification reaction on quantitative fluorescent PCR；
Amplification condition are as follows:
Initial denaturation: it 95 DEG C, 10 minutes, recycles 1 time；
Denaturation: it 94 DEG C, 15 seconds, recycles 45 times；
Annealing extends, fluorescence signal acquisition: 60 DEG C, 45 seconds, recycling 45 times；
Instrument is cooling: 25 DEG C, 1 minute, recycling 1 time；
S2.2, pcr amplification product obtained in step S2.1 is digested:
Take 5 μ L that 2 μ L SAP enzyme mixations are added the pcr amplification product of CT≤30, concussion mixes, after of short duration centrifugation, in PCR instrument
On successively carry out 37 DEG C of 60 minutes and 80 DEG C of 15 minutes enzymatic hydrolysis.
3. c-kit detection method of gene mutation according to claim 1, which is characterized in that the detailed process of step S3 are as follows:
S3.1, sequencing PCR reaction:
It takes 1.5 μ L of enzymolysis product, 1 μ L of sequencing reagent and 18 μ L of sequencing primer to be mixed with pipette tips, PCR amplification is carried out after centrifugation, is obtained
To Sanger segment；Amplification condition are as follows:
Initial denaturation: it 96 DEG C, 1 minute, recycles 1 time；
Denaturation: it 96 DEG C, 10 seconds, recycles 30 times；
Annealing: it 50 DEG C, 5 seconds, recycles 30 times；
Extend: 50 DEG C, 2 minutes, recycling 30 times；
Instrument is cooling: 25 DEG C, 1 minute, recycling 1 time；
S3.2, Sanger fragment purification:
S3.2.1,50 μ L sodium acetates-alcohol mixture (3M NaAc: nothing is added into Sanger segment obtained in step S3.1
Water-ethanol=1:14), it acutely vibrates, is protected from light standing 15 minutes, 4 DEG C of centrifugation half an hour of 12000g or more inhale and abandon supernatant liquid；
S3.2.2,70 μ L, 70% pre-cooled ethanol is added, acutely vibrates, 4 DEG C of 12000g or more are centrifuged 15 minutes, inhale and abandon upper liquid
Body；
S3.2.3, it allows ethyl alcohol to volatilize at room temperature completely, 12 μ L Hi-Di Formamide dissolving DNAs is added, obtain after purification
Sanger segment.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112553313A (en) * | 2020-12-30 | 2021-03-26 | 武汉康圣达医学检验所有限公司 | Method for denaturing sequencing reaction product for Sanger method |
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| CN105969876A (en) * | 2016-06-15 | 2016-09-28 | 昆明理工大学 | Primer combination for detecting mutation of C-KIT gene in trace tissue and application of primer combination |
| CN107523608A (en) * | 2016-06-22 | 2017-12-29 | 海门中科基因生物科技有限公司 | A kind of kit for detecting the mutation of PKU Disease-causing gene |
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| CN105969876A (en) * | 2016-06-15 | 2016-09-28 | 昆明理工大学 | Primer combination for detecting mutation of C-KIT gene in trace tissue and application of primer combination |
| CN107523608A (en) * | 2016-06-22 | 2017-12-29 | 海门中科基因生物科技有限公司 | A kind of kit for detecting the mutation of PKU Disease-causing gene |
| CN108374044A (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 c-kit gene mutation |
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| 田玉旺 等: ""胃肠道间质瘤中c-kit和PDGFRA基因突变多态性分析"", 《解放军医药杂志》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112553313A (en) * | 2020-12-30 | 2021-03-26 | 武汉康圣达医学检验所有限公司 | Method for denaturing sequencing reaction product for Sanger method |
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Application publication date: 20190118 |