CN105969875B - Primer for detecting PIK3CA gene mutation in microcomponent combines and its application - Google Patents
Primer for detecting PIK3CA gene mutation in microcomponent combines and its application Download PDFInfo
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Abstract
The invention discloses one kind for detecting in microcomponentPIK3CAThe 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 20 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 samplePIK3CAThe catastrophe of gene；Primer combination is applied to preparation detectionPIK3CAIn the detection reagent of gene mutation, the DNA concentration of sample can detect down to 100pg, and can detect simultaneouslyPIK3CAAll mutation of gene the 9th and 20 exons, detection method high sensitivity provided by the invention, high specificity is at low cost, is suitable for clinical tumor patientPIK3CAThe 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 microcomponentPIK3CAGene mutation inspection
The primer combination of survey and its application in tumour medication selection and medical diagnosis on disease related fields.
Background technique
Phosphatidylinositol-3-kinase (PI3K) is that a kind of phosphatidylinositols that can make inositol ring third position di is sharp
Enzyme has phosphatidyl inositol kinase and serine-threonine protein kinase enzyme double activity (Fruman, D. A. et al.
2014) multiple biological functions such as cell Proliferation, survival, migration, apoptosis and cell cycle, are adjusted.PIK3CA gene encodes I A
Class PI3K catalytic subunit P110 α, is positioned at 3q26.3, includes 21 exons (Volinia, S. et al. 1994).
KANG S etc. the study found thatPIK3CAGene plays the role of oncogene, about 30% human entity tumor in kinds of tumors
In the presence ofPIK3CAThe mutation of gene, wherein the 9th and the 20th exon in the gene occurs for the mutation of 80%-90%, respectively corresponds
The helical region and kinases area of the enzyme.The change in the region causes PIK3CA to be overexpressed, P110 alpha active increases, and then activates downstream
The signaling molecules such as AKT, lead to the growth and conversion of fibroblast and galactophore epithelial cell, and inhibit Apoptosis, finally lead
Cause tumour that (Vivanco, I. et al. 2002) occurs.
It has now been found that in kinds cancer and existsPIK3CAGene mutation (such as breast cancer, non-small cell lung cancer, colon cancer
Deng), mutation will lead to the continuous activation of PI3K/AKT signal path.PI3K is activated as EGFR downstream signaling molecule, leads
Cause tumour cell to the drug resistants such as EGFR-TKI drug and Cetuximab, Victibix, Lapatinib.ThereforePIK3CADetection in Gene Mutation can be improved the specific aim of clinical therapy of tumor, reduce medical expense, when saving valuable treatment
Between.
It is directed at presentPIK3CAThe 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,PIK3CAThe detection method of mutation is still using tumor tissues Samples detection as goldstandard, but clinically
Usually since tissue samples acquisition is in shortage, make its detection that there is certain limitation.Therefore a kind of efficient standard is clinically needed
Really comprehensively detect in microcomponentPIK3CAThe 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 samplePIK3CAThe techniqueflow of all mutation type detections of gene, the detection method high sensitivity, high specificity, cost
It is low, 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 sensitivityPIK3CAThe 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 direct sequencing determine in tumor sample whether there is or notPIK3CAGene mutation, can be rightPIK3CAThe mutation of gene carries out accurate
Detection.
Primer combination provided by the invention can detectPIK3CAGene occurs in all prominent of the 9th and 20 exons
Become.
Provided by the present invention for detectionPIK3CAThe 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, the primer sets being mutated as shown in SEQ ID NO:21- SEQ ID NO:22 for detecting 20 exons.
It is another object of the present invention to detect above-mentioned primer combined application in preparationPIK3CAThe detection of gene mutation tries
In agent.
It is another object of the present invention to the combination of above-mentioned primer is applied to preparation to be used to detectPIK3CAThe inspection of gene mutation
In test agent box, the reagent constituents also include one or more of following conventional constituents: polymerase, primer combination, PCR
Reaction buffer, dNTP, BSA, deionized water.
The present invention is for detectingPIK3CAThe 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-primer22 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 breast cancer patients 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)PIK3CADetection in Gene Mutation
ForPIK3CAThe 9 of gene, 20 exons mutation, are respectively adopted such as SEQ ID NO:19- SEQ ID NO:20
Shown in primer sets detect 9 exons mutation；The detection of the primer sets as shown in SEQ ID NO:21- SEQ ID NO:22 20
Exon mutation.
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 outPIK3CAThe limitation of detection in Gene Mutation
Property.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 samplePIK3CAThe 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 1-22
。
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 isPIK3CA9,20 exon amplification of gene.
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 isPIK3CA9,20 exon amplification of gene.
Fig. 3 is the DNA profiling amplification that the present invention is directed to 100pg.A figure is pre- amplification (3 repetition)；B figure isPIK3CA9,20 exon amplification of gene.
Fig. 4 is the present invention in the DNA profiling of 1000pgPIK3CA9,20 exon sequencing result figure of gene.
Fig. 5 is the present invention in the DNA profiling of 500pgPIK3CA9,20 exon sequencing result figure of gene.
Fig. 6 is the present invention in the DNA profiling of 100pgPIK3CA9,20 exon sequencing result figure of gene.
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 1:1000pgPIK3CADetection 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-primer22,
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 10-20min inactivate enzyme for 30-35 DEG C of amplification.The glue of paving 1%
It detects (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 30-60s, 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 30-60s, 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, 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 PIK3CADetection in Gene Mutation
(1) following PCR reaction system is configured, is expanded respectivelyPIK3CANo. 9 of gene and 20 exons；
Wherein for the primer of 9 exons mutation as shown in SEQ ID NO:19- SEQ ID NO:20；For No. 20
The primer of exons mutation is as shown in SEQ ID NO:21- SEQ ID NO:22.
(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 wheelPIK3CAThe of gene
There is specific amplification in 9 and 20 exons；Sangon Biotech (Shanghai) Co., Ltd. is then sent to be sequenced
Detection.Sequencing result as shown in figure 4,PIK3CAThe 9th exon of gene mutate c.1634A > C p.E545A and
C.1658_1659GT > C p.S553fs*7, the 20th exon mutation c.3140A > CG p. H1047R.
In the DNA profiling of embodiment 2:500pgPIK3CADetection 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-primer22,
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 10-20min inactivate enzyme for 30-35 DEG C of amplification.The glue of paving 1%
It detects (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 30-60 sec, outwell the waste liquid in collecting pipe, adsorption column CB1 is put into receipts
In collector.
(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 30-60 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, 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 PIK3CADetection in Gene Mutation
(1) following PCR reaction system is configured, is expanded respectivelyPIK3CA9th and 20 exons of gene.
Wherein for the primer of 9 exons mutation as shown in SEQ ID NO:19- SEQ ID NO:20；For No. 20
The primer of exons mutation is as shown in SEQ ID NO:21- SEQ ID NO:22.
(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 wheelPIK3CAThe of gene
There is specific amplification in 9 and 20 exons；Sangon Biotech (Shanghai) Co., Ltd. is then sent to be sequenced
Detection.Sequencing result as shown in figure 5,PIK3CAThe 9th exon of gene mutate c.1634A > C p.E545A and
C.1658_1659GT > C p.S553fs*7, the 20th exon mutation c.3140A > CG p. H1047R.
In the DNA profiling of embodiment 3:100pgPIK3CADetection 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-primer22,
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 10-20min inactivate enzyme for 30-35 DEG C of amplification.The glue of paving 1%
It detects (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.
2.3PIK3CADetection in Gene Mutation
(1) following PCR reaction system is configured, is expanded respectivelyPIK3CA9th and 20 exons of gene.
；
Wherein for the primer of 9 exons mutation as shown in SEQ ID NO:19- SEQ ID NO:20；For No. 20
The primer of exons mutation is as shown in SEQ ID NO:21- SEQ ID NO:22.
(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 wheelPIK3CAThe of gene
There is specific amplification in 9 and 20 exons；Sangon Biotech (Shanghai) Co., Ltd. is then sent to be sequenced
Detection.Sequencing result as shown in fig. 6,PIK3CAThe 9th exon of gene mutate c.1634A > C p.E545A and
C.1658_1659GT > C p.S553fs*7, the 20th exon mutation c.3140A > CG p. H1047R.
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 microcomponentPIK3CAThe primer of gene mutation combines and its application
<160> 22
<170> PatentIn version 3.3
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tggaatgcca gaact 15
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<400> 7
tgaaagctca ctctg 15
<210> 8
<211> 15
<212> DNA
<213>artificial sequence
<400> 8
aggcaaagac cgatt 15
<210> 9
<211> 15
<212> DNA
<213>artificial sequence
<400> 9
tcgaatagct agata 15
<210> 10
<211> 15
<212> DNA
<213>artificial sequence
<400> 10
ttccagagcc aagca 15
<210> 11
<211> 15
<212> DNA
<213>artificial sequence
<400> 11
gtctttcgaa tgtat 15
<210> 12
<211> 15
<212> DNA
<213>artificial sequence
<400> 12
ttcatgaaat actcc 15
<210> 13
<211> 15
<212> DNA
<213>artificial sequence
<400> 13
ggaagatcca atcca 15
<210> 14
<211> 15
<212> DNA
<213>artificial sequence
<400> 14
gctttcattt tctca 15
<210> 15
<211> 15
<212> DNA
<213>artificial sequence
<400> 15
gcctgctgag agtta 15
<210> 16
<211> 15
<212> DNA
<213>artificial sequence
<400> 16
gctgttcatg gattg 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> 42
<212> DNA
<213>artificial sequence
<400> 19
agggttttcc cagtcacgtg ctttttctgt aaatcatctg tg 42
<210> 20
<211> 27
<212> DNA
<213>artificial sequence
<400> 20
aaacagagaa tctccatttt agcactt 27
<210> 21
<211> 41
<212> DNA
<213>artificial sequence
<400> 21
agggttttcc cagtcacggg cttatctagc tattcgacag c 41
<210> 22
<211> 28
<212> DNA
<213>artificial sequence
<400> 22
agagtgagct ttcattttct cagttatc 28
Claims (3)
1. for detecting in microcomponentPIK3CAThe 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 20 exons be mutated
Primer sets composition；
For detectingPIK3CAThe 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-primer22 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)PIK3CADetection in Gene Mutation
ForPIK3CAThe 9 of gene, 20 exons mutation, are respectively adopted as shown in SEQ ID NO:19- SEQ ID NO:20
Primer sets detect 9 exons mutation；The primer sets as shown in SEQ ID NO:21- SEQ ID NO:22 detect 20 extras
Aobvious son mutation；
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 preparationPIK3CAApplication in the detection reagent of gene mutation.
3. primer combination described in claim 1 is detected in preparationPIK3CAApplication in the detection kit of gene mutation.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533958A (en) * | 2010-12-28 | 2012-07-04 | 苏州科贝生物技术有限公司 | Method and kit for detecting human PIK3CA gene mutation |
| CN102628077A (en) * | 2011-12-23 | 2012-08-08 | 厦门艾德生物医药科技有限公司 | Polygene joint detection probe for early diagnosis of tumour, primer and kit |
| CN102634510A (en) * | 2012-04-27 | 2012-08-15 | 昆明理工大学 | Pre-amplification method for trace DNA applied in medicolegal expertise |
| CN102816839A (en) * | 2012-07-06 | 2012-12-12 | 杭州艾迪康医学检验中心有限公司 | Kit for detecting hot spot mutation sites in colorectal cancer PIK3CA gene |
| CN103773837A (en) * | 2013-06-25 | 2014-05-07 | 宁波有成生物医药科技有限公司 | Fluorescent quantitation PCR detection kit and detection method for PIK3CA (phosphatidylinositol3-kinase catalytic alpha) gene mutations |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533958A (en) * | 2010-12-28 | 2012-07-04 | 苏州科贝生物技术有限公司 | Method and kit for detecting human PIK3CA gene mutation |
| CN102628077A (en) * | 2011-12-23 | 2012-08-08 | 厦门艾德生物医药科技有限公司 | Polygene joint detection probe for early diagnosis of tumour, primer and kit |
| CN102634510A (en) * | 2012-04-27 | 2012-08-15 | 昆明理工大学 | Pre-amplification method for trace DNA applied in medicolegal expertise |
| CN102816839A (en) * | 2012-07-06 | 2012-12-12 | 杭州艾迪康医学检验中心有限公司 | Kit for detecting hot spot mutation sites in colorectal cancer PIK3CA gene |
| CN103773837A (en) * | 2013-06-25 | 2014-05-07 | 宁波有成生物医药科技有限公司 | Fluorescent quantitation PCR detection kit and detection method for PIK3CA (phosphatidylinositol3-kinase catalytic alpha) gene mutations |
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;全文 * |
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