CN106048021B - Primer for detecting P53 gene mutation in microcomponent combines and its application - Google Patents
Primer for detecting P53 gene mutation in microcomponent combines and its application Download PDFInfo
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Abstract
The invention discloses one kind for detecting in microcomponentP53The primer of gene mutation combines comprising expands primer sets, the primer sets for detecting the mutation of 5 exons, the primer sets for detecting the mutation of 6 exons, the primer sets for detecting the mutation of 7 exons, the primer sets for detecting the mutation of 8 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 sampleP53The catastrophe of gene；Primer combination is applied to preparation detectionP53In the detection reagent of gene mutation, the DNA concentration of sample can detect down to 100pg, and can detect simultaneouslyP53All mutation of gene the 5th, 6,7 and 8 exons, detection method high sensitivity provided by the invention, high specificity is at low cost, is suitable for clinical tumor patientP53The 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 microcomponentP53Detection in Gene Mutation
Primer combination and its tumour medication selection and medical diagnosis on disease related fields application.
Background technique
P53Gene be so far discovery with the highest gene of human tumor correlation, it has been reported that even have 50% or more swell
Tumor patient existsp53The change of gene.Mankind's p53 gene is located at No. 17 1 areas 3.1 of the short arm of a chromosome with (17q13.1), overall length
16-20kb contains 11 exons and 10 intrones, is divided into wild type (wt-p53) and saltant type (mt-p53) (Reich and
Levine, 1982;Lane, 1984).Wt-p53 is as tumor suppressor gene, the change of function or missing and a large amount of variety classes
Human tumor cell have substantial connection.Wt-p53 can integrate the signal of a variety of different cell emergencies, by transcription or
Non-transcribed approach makes the differential responses including cell growth inhibition or apoptosis to these signals, monitors cellular genome
Integrality；Mt-p53 cannot then complete these functions, or even become proto-oncogene (Haupt S, et al, 2016).
The forfeiture of P53 normal function, most important mode are gene mutations, by tumour (liver cancer, breast cancer, bladder cancer,
Gastric cancer, colon cancer, prostate cancer, soft tissue sarcoma, oophoroma, brain tumor, lymphocytic tumours, cancer of the esophagus, lung cancer, osteogenic sarcoma
Deng) in a large amount of Analysis of Mutants, it was demonstrated that most of mutation concentrates on P53 the 5th, 6,7, (Ferraiuolo on 8 exons
M, et al, 2016;Kamp WM, et al, 2016).Mutant loses the binding ability of specific site, in addition, mutation
Body can also change the spherical conformation of P53.Variety classes tumour is different, as colon cancer and breast cancer have similar epidemiology
(including ground is distinguished and risk factor), butP53The spectrum of mutation is not consistent.Colon cancer G:CA:T conversion accounts for 79%, and majority CpG,
Dinucleotides site, 50% or more transition mutations occur the CpGC of the 3rd~5 structural domain be located at numeral 175,248,273)；In cream
In gland cancer, only the conversion of discovery 13% is in the site CpG.In addition, G-T transversion accounts for 1/4 in breast cancer, but rare at colon cancer T points.
The P53 of lymthoma and leukaemia, mutational formats are similar to colon cancer, i.e., most of conversion for sporting the site CPG, G → T transversion
Lower, A:T → G:C is higher in A:T site mutation.G:C → T:A is most universal in non-small cell lung cancer, and cancer of the esophagus transversion rate is very
Height, unlike lung cancer, mutation rate as the site G:C and A:T has.East China area 50% is G → C, the G of 249 cancer numerals
→ T transversion, and South Africa liver cancer 80% is G → T transversion.In osteosarcomaP53Mutation rate is 75%, is concentrated mainly on 5~9 exons.
The unconventionality expression of P53 is related to the transfer of tumour, recurrence and poor prognosis, therefore for tumour patientP53Gene
The Clinical significance of detecting of catastrophe is great.It is directed at presentP53The detection method of mutation has very much, and such as direct sequencing, the method is to sample
This is more demanding, and detection range is limited.Polymorphism analysis method (RFLP) is a kind of side by PCR in conjunction with restriction enzyme cut phase
Method, this experimental implementation is cumbersome, and detection cycle is long, with high costs, and there are false positives caused by first round digestion not exclusively.Taqman
Hydrolysis probes method detects mutation using amplification refractory mutation system (ARMS) in conjunction with fluorescence probe, designs for this method
Primer so that saltant type is expanded, wild type can not be expanded, to enhance jump signal, convenient for detection.But ARMS
The last one base of technical application mismatches the amplification that can not block wild type DNA completely, and there are the risks of false positive, and only
It can be detected for specific position.In addition, such as high performance liquid chromatography, capillary electrophoresis, need special instrument and equipment, and
It is complicated for operation, it is unfavorable for clinically being promoted on a large scale.Amplification of nucleic acid sequences method (NASBA), author's preface sequence replicating method
Though being isothermal duplication method, they are not strong to the specific amplification of aim sequence for (3SR) and strand displacement replica method (SDA).
Nevertheless,P53The 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 it is complete clinically to need a kind of efficiently and accurately
Detect in microcomponent to faceP53The 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 sampleP53The techniqueflow of all mutation type detections of gene, the detection method high sensitivity, high specificity is at low cost, has
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 sensitivityP53Base
Because the primer of mutation combines, using tissue DNA as test object, in conjunction with the pre- amplification technique of constant temperature and regular-PCR technology, by direct
PCR sequencing PCR determine in tumor sample whether there is or notP53Gene mutation, can be rightP53The mutation of gene carries out accurate detection.
Primer combination provided by the invention can detectP53Gene occurs in No. 5, No. 6, No. 7 and the institute of 8 exons
There is mutation.
Provided by the present invention for detectionP53The primer combination of gene mutation includes such as SEQ ID NO:1- SEQ ID
Primer sets, prominent for detecting 5 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, such as being mutated as shown in SEQ ID NO:21- SEQ ID NO:22 for detect 6 exons
Primer sets, such as SEQ ID NO:25- shown in SEQ ID NO:23- SEQ ID NO:24 for detecting the mutation of 7 exons
For detecting the primer sets of 8 exons mutation shown in SEQ ID NO:26.
It is another object of the present invention to detect above-mentioned primer combined application in preparationP53The 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 detectP53The detection of gene mutation tries
In 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 detectingP53The 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-primer26 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 lung cancer patient tissue to template, and according to conventional described in " Molecular Cloning:A Laboratory guide "
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 in 10- on ice
20min；0.5 μ L dNTP (10mM each), 0.2 100 × BSA of μ L and 0.5 μ L phi29 archaeal dna polymerase are added,
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)P53Detection in Gene Mutation
ForP53No. 5, No. 6, No. 7 of gene and the mutation of 8 exons, are respectively adopted such as SEQ ID NO:19- SEQ
Primer sets shown in ID NO:20 detect the mutation of 5 exons；The primer as shown in SEQ ID NO:21- SEQ ID NO:22
Group detection 6 exons mutation, it is prominent that the primer sets as shown in SEQ ID NO:23- SEQ ID NO:24 detect 7 exons
Become, the primer sets as shown in SEQ ID NO:25- SEQ ID NO:26 examine the mutation of 8 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 outP53The 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 100pg
DNA 1000ng/ μ L can be obtained after expanding；The catastrophe of multidigit point can be detected simultaneously.After the method for the present invention DNA cloning
DNA fidelity is good.In short, can be realized using the method for the present invention in microcomponent's sampleP53The catastrophe of gene carries out
Efficiently, accurate detection instructs clinical application, and the prognosis of monitoring tumour to have very clinical tumor early screening
Good practical application value.
Table 1: the nucleotide sequence of primer 1-26
。
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 isP53Gene 5, No. 6, No. 7 and 8 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 isP53Gene 5, No. 6, No. 7 and 8 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 isP53Gene 5, No. 6, No. 7 and 8 exon amplifications；
Fig. 4 is the present invention in the DNA profiling of 1000pgP53Gene 5, No. 6, No. 7 and 8 exon sequencing results
Figure；
Fig. 5 is the present invention in the DNA profiling of 500pgP53Gene 5, No. 6, No. 7 and 8 exon sequencing results
Figure；
Fig. 6 is the present invention in the DNA profiling of 100pgP53Gene 5, No. 6, No. 7 and 8 exon sequencing results
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 1:1000pgP53Detection 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-primer26,
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 P53Detection in Gene Mutation
(1) following PCR reaction system is configured, is expanded respectivelyP53No. 5, No. 6, No. 7 and 8 exons of gene；
；
Wherein for the primer of 5 exons mutation as shown in SEQ ID NO:19- SEQ ID NO:20；For 6 extras
The primer of aobvious son mutation is as shown in SEQ ID NO:21- SEQ ID NO:22；The primer such as SEQ ID of 7 exons mutation
Shown in NO:23- SEQ ID NO:24；The primer of 8 exons mutation is as shown in SEQ ID NO:25- SEQ ID NO:26.
(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 wheelP53The 5th of gene
Number, No. 6, No. 7 and 8 exons there is specific amplification；It is then sent to Sangon Biotech (Shanghai) Co., Ltd.
Carry out sequencing detection.Sequencing result as shown in figure 4,P53The 6th exon of gene occur missense mutation c.580C > T p.
Mutation is not detected in L194F, the 5th, 7 and 8 exon.
In the DNA profiling of embodiment 2:500pgP53Detection 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-primer26,
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 5min 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 P53Detection in Gene Mutation
(1) following PCR reaction system is configured, is expanded respectivelyP53No. 5, No. 6, No. 7 and 8 exons of gene.
；
Wherein for the primer of 5 exons mutation as shown in SEQ ID NO:19- SEQ ID NO:20；For 6 extras
The primer of aobvious son mutation is as shown in SEQ ID NO:21- SEQ ID NO:22；The primer such as SEQ ID of 7 exons mutation
Shown in NO:23- SEQ ID NO:24；The primer of 8 exons mutation is as shown in SEQ ID NO:25- SEQ ID NO:26.
(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 wheelP53The 5th of gene
Number, No. 6, No. 7 and 8 exons there is specific amplification；It is then sent to Sangon Biotech (Shanghai) Co., Ltd.
Carry out sequencing detection.Sequencing result as shown in figure 5,P53The 6th exon of gene occur missense mutation c.580C > T p.
Mutation is not detected in L194F, the 5th, 7 and 8 exon.
In the DNA profiling of embodiment 3:100pgP53Detection 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-primer26,
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 8min 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.
2.3 P53Detection in Gene Mutation
(1) following PCR reaction system is configured, is expanded respectivelyP53No. 5, No. 6, No. 7 and 8 exons of gene.
；
Wherein for the primer of 5 exons mutation as shown in SEQ ID NO:19- SEQ ID NO:20；For 6 extras
The primer of aobvious son mutation is as shown in SEQ ID NO:21- SEQ ID NO:22；The primer such as SEQ ID of 7 exons mutation
Shown in NO:23- SEQ ID NO:24；The primer of 8 exons mutation is as shown in SEQ ID NO:25- SEQ ID NO:26.
(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 wheelP53The 5th of gene
Number, No. 6, No. 7 and 8 exons there is specific amplification；It is then sent to Sangon Biotech (Shanghai) Co., Ltd.
Carry out sequencing detection.Sequencing result as shown in fig. 6,P53The 6th exon of gene occur missense mutation c.580C > T p.
Mutation is not detected in L194F, the 5th, 7 and 8 exon.
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 microcomponentP53The primer of gene mutation combines and its application
<160> 26
<170> PatentIn version 3.3
<210> 1
<211> 15
<212> DNA
<213>artificial sequence
<400> 1
tacctggagc tggag 15
<210> 2
<211> 15
<212> DNA
<213>artificial sequence
<400> 2
gagtagatgg agcct 15
<210> 3
<211> 15
<212> DNA
<213>artificial sequence
<400> 3
ctgcctcttg cttct 15
<210> 4
<211> 15
<212> DNA
<213>artificial sequence
<400> 4
acggaacagc tttga 15
<210> 5
<211> 15
<212> DNA
<213>artificial sequence
<400> 5
gcacagagga agaga 15
<210> 6
<211> 15
<212> DNA
<213>artificial sequence
<400> 6
cagggagcac taagc 15
<210> 7
<211> 15
<212> DNA
<213>artificial sequence
<400> 7
agggtgcagt tatgc 15
<210> 8
<211> 15
<212> DNA
<213>artificial sequence
<400> 8
ctttcctagc actgc 15
<210> 9
<211> 15
<212> DNA
<213>artificial sequence
<400> 9
tctactccca accac 15
<210> 10
<211> 15
<212> DNA
<213>artificial sequence
<400> 10
gcagtaagga aatca 15
<210> 11
<211> 15
<212> DNA
<213>artificial sequence
<400> 11
ccagtagatt accac 15
<210> 12
<211> 15
<212> DNA
<213>artificial sequence
<400> 12
tgtgcgccgg tctct 15
<210> 13
<211> 15
<212> DNA
<213>artificial sequence
<400> 13
gcagctcgtg gtgag 15
<210> 14
<211> 15
<212> DNA
<213>artificial sequence
<400> 14
tccaccgctt cttgt 15
<210> 15
<211> 15
<212> DNA
<213>artificial sequence
<400> 15
aggcaaggaa aggtg 15
<210> 16
<211> 15
<212> DNA
<213>artificial sequence
<400> 16
ctttggctgg ggaga 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> 39
<212> DNA
<213>artificial sequence
<400> 19
agggttttcc cagtcacgtg tttctttgct gccgtcttc 39
<210> 20
<211> 38
<212> DNA
<213>artificial sequence
<400> 20
taatacgact cactataggt ctccagcccc agctgctc 38
<210> 21
<211> 38
<212> DNA
<213>artificial sequence
<400> 21
agggttttcc cagtcacggc tgctcagata gcgatggt 38
<210> 22
<211> 21
<212> DNA
<213>artificial sequence
<400> 22
cagagacccc agttgcaaac c 21
<210> 23
<211> 24
<212> DNA
<213>artificial sequence
<400> 23
tcttgggcct gtgttatctc ctag 24
<210> 24
<211> 39
<212> DNA
<213>artificial sequence
<400> 24
agggttttcc cagtcacggg gatgtgatga gaggtggat 39
<210> 25
<211> 39
<212> DNA
<213>artificial sequence
<400> 25
agggttttcc cagtcacgaa gggtggttgg gagtagatg 39
<210> 26
<211> 41
<212> DNA
<213>artificial sequence
<400> 26
taatacgact cactataggg cttcttgtcc tgcttgctta c 41
Claims (3)
1. for detecting in microcomponentP53The primer of gene mutation combines, it is characterised in that: by such as SEQ ID NO:1- SEQ
Primer sets, aobvious for detecting 5 extras as shown in SEQ ID NO:19- SEQ ID NO:20 are expanded shown in ID NO:18 in advance
The primer sets of son mutation, the primer being mutated as shown in SEQ ID NO:21- SEQ ID NO:22 for detecting 6 exons
Group, the primer sets as shown in SEQ ID NO:23- SEQ ID NO:24 for detecting the mutation of 7 exons, such as SEQ ID
For detecting the primer sets composition of 8 exons mutation shown in NO:25- SEQ ID NO:26；
For detectingP53The 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-primer26 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)P53Detection in Gene Mutation
ForP53No. 5, No. 6, No. 7 of gene and the mutation of 8 exons, are respectively adopted such as SEQ ID NO:19- SEQ ID
Primer sets shown in NO:20 detect the mutation of 5 exons；The inspection of the primer sets as shown in SEQ ID NO:21- SEQ ID NO:22
The mutation of 6 exons is surveyed, the primer sets as shown in SEQ ID NO:23- SEQ ID NO:24 detect the mutation of 7 exons, such as
Primer sets shown in SEQ ID NO:25- SEQ ID NO:26 examine the mutation of 8 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 preparationP53Application in the detection reagent of gene mutation.
3. primer combination described in claim 1 is detected in preparationP53Application in the detection kit of gene mutation.
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| CN107365854A (en) * | 2017-08-16 | 2017-11-21 | 厦门智因互联科技有限公司 | A kind of TP53 gene SNP sites multiple real time fluorescence PCR detection primers, probe and its kit |
| CN108330167A (en) * | 2017-09-13 | 2018-07-27 | 昆明理工大学 | The new application of p53 gene mutations |
| CN108676852B (en) * | 2018-05-30 | 2021-08-27 | 朱运峰 | Primer pair group and kit for detecting epigenetic modification difference of P53 gene in peripheral blood free DNA |
| CN111334582A (en) * | 2020-04-30 | 2020-06-26 | 北京和合医学诊断技术股份有限公司 | Method for synchronously detecting 4 exon gene mutations of P53 gene |
Citations (3)
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| CN101392286A (en) * | 2007-11-19 | 2009-03-25 | 中国科学院上海微系统与信息技术研究所 | Method for directly detecting P53 gene mutation in lung cancer sample based on nano probe |
| CN102634510A (en) * | 2012-04-27 | 2012-08-15 | 昆明理工大学 | Pre-amplification method for trace DNA applied in medicolegal expertise |
| CN103436593A (en) * | 2012-09-11 | 2013-12-11 | 上海赛安生物医药科技有限公司 | Rapid detection method for p53 gene mutation |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101392286A (en) * | 2007-11-19 | 2009-03-25 | 中国科学院上海微系统与信息技术研究所 | Method for directly detecting P53 gene mutation in lung cancer sample based on nano probe |
| CN102634510A (en) * | 2012-04-27 | 2012-08-15 | 昆明理工大学 | Pre-amplification method for trace DNA applied in medicolegal expertise |
| CN103436593A (en) * | 2012-09-11 | 2013-12-11 | 上海赛安生物医药科技有限公司 | Rapid detection method for p53 gene mutation |
Non-Patent Citations (2)
| 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 * |
| 高分辨熔解曲线法检测非小细胞肺癌p53基因的突变;陈志红等;《中国肺癌杂志》;20111031;摘要、第768页表1 * |
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