CN106048021A - Primer combination for detecting mutation of genes P53 in micro-tissues and application of primer combination - Google Patents

Primer combination for detecting mutation of genes P53 in micro-tissues and application of primer combination Download PDF

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CN106048021A
CN106048021A CN201610418005.5A CN201610418005A CN106048021A CN 106048021 A CN106048021 A CN 106048021A CN 201610418005 A CN201610418005 A CN 201610418005A CN 106048021 A CN106048021 A CN 106048021A
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primer
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mutation
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CN106048021B (en
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盛苗苗
罗瑛
唐文如
李珊珊
王芳
赵月光
张继虹
贾舒婷
吴晓明
刘静
周若宇
旦菊花
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Kunming University of Science and Technology
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Abstract

The invention discloses a primer combination for detecting mutation of genes P53 in micro-tissues. The primer combination comprises pre-amplification primer groups, primer groups for detecting mutation of NO.5 exons, primer groups for detecting mutation of NO.6 exons, primer groups for detecting mutation of NO.7 exons and primer groups for detecting mutation of NO.8 exons. The primer combination has the advantages that a method aims to pre-amplifying micro-DNA (deoxyribonucleic acid) to obtain high-concentration DNA templates, and the DNA templates and direct sequencing are combined with one another to detect mutation conditions of the genes P53 in samples; the primer combination is applied to preparing detection reagents for detecting mutation of the genes P53, the concentration of DNA in the samples can be reduced and reaches 100 pg, all mutation of the NO.5, NO.6, NO.7 and NO.8 exons of the genes P53 can be simultaneously detected, and the detection method is high in sensitivity and specificity, low in cost and applicable to detecting mutation of genes P53 of clinical tumor patients and has excellent clinical application value.

Description

Combine and application for detecting the primer of P53 gene mutation in microcomponent
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of in microcomponentP53Detection in Gene Mutation Primer combination and in tumor medication selection and the application of medical diagnosis on disease related fields.
Background technology
P53Gene is to find so far and the highest gene of human tumor dependency, it has been reported that even have more than 50% swollen Tumor patient existsp53The change of gene.Mankind's p53 gene is positioned at No. 17 the short arm of a chromosome 1 districts 3.1 and carries (17q13.1), total length 16-20kb, containing 11 exons and 10 introns, is divided into wild type (wt-p53) and saltant type (mt-p53) (Reich and Levine, 1982;Lane, 1984).Wt-p53 is as antioncogene, the change of its function or disappearance and a large amount of variety classeses Human tumor cell have substantial connection.Wt-p53 can integrate the signal of various different cell emergency, by transcribing or These signals are made the differential responses including cell growth inhibition or apoptosis by non-transcribed approach, monitor cellular genome Integrity；Mt-p53 then can not complete these functions, even becomes proto-oncogene (Haupt S, et al, 2016).
The forfeiture of P53 normal function, topmost mode is gene mutation, by tumor (hepatocarcinoma, breast carcinoma, bladder cancer, Gastric cancer, colon cancer, carcinoma of prostate, soft tissue sarcoma, ovarian cancer, cerebroma, lymphocytic tumours, esophageal carcinoma, pulmonary carcinoma, osteogenic sarcoma Deng) in substantial amounts of Analysis of Mutants, it was demonstrated that major part sudden change concentrate 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, additionally, sudden change Body can also change the spherical conformation of P53.Variety classes tumor is different, as colon cancer has similar epidemiology with breast carcinoma (distinguishing with including and risk factor), butP53Mutational spectrum is not consistent.The conversion of colon cancer G:CA:T accounts for 79%, and most CpG, Dinucleotide site, more than 50% transition mutations occurs to be positioned at numeral 175,248,273 at the CpGC of the 3rd～5 domains)；At breast In adenocarcinoma, only the conversion of discovery 13% is in CpG site.Additionally, G-T transversion accounts for 1/4 in breast carcinoma, but divide rare in colon cancer T. Lymphoma and leukemic P53, mutational formats is similar to colon cancer, i.e. major part sports the conversion in CPG site, G → T transversion Relatively low, A:T → G:C is higher at A:T site mutation.In nonsmall-cell lung cancer, G:C → T:A is the most universal, and esophageal carcinoma transversion rate is very Height, unlike pulmonary carcinoma, G:C and A:T site have as mutation rate.East China area 50% is G → C, the G of 249 cancer numerals → T transversion, and South Africa hepatocarcinoma 80% is G → T transversion.In osteosarcomaP53Mutation rate is 75%, is concentrated mainly on 5～9 exons.
The unconventionality expression of P53 is relevant to the transfer of tumor, recurrence and poor prognosis, therefore for tumour patientP53Gene The Clinical significance of detecting of catastrophe is great.Currently forP53The detection method of sudden change has a lot, and such as direct sequencing, this method is to sample This requirement is higher, and detection range is limited.Polymorphism analysis method (RFLP), is by PCR and the one side that Restriction Enzyme cut is combined Method, this experimental implementation is loaded down with trivial details, and the detection cycle is long, with high costs, there is the false positive that first round enzyme action not exclusively causes.Taqman Hydrolysis probes method, uses amplification refractory mutation system (ARMS) to be combined with fluorescent probe and detects sudden change, design for the method Primer so that saltant type is expanded, and wild type cannot expand, thus enhances jump signal, it is simple to detection.But ARMS Technology applies last base not mate the amplification that can not block wild type DNA completely, there is false-positive risk, and only Can detect for specific position.Additionally, such as high performance liquid chromatography, blood capillary electrophoresis etc., need special instrument and equipment, and Operation complexity, is unfavorable for promoting the most on a large scale.Amplification of nucleic acid sequences method (NASBA), author's preface sequence replicating method (3SR) with strand displacement duplicating method (SDA) though being isothermal duplication method, but they are the strongest to the specific amplification of aim sequence.
While it is true,P53Sudden change detection method still with tumor tissues Samples detection as goldstandard, but the most usually Owing to tissue samples obtains in shortage so that it is detection has certain limitation.Need a kind of efficiently and accurately the most clinically badly complete In ground, face detection microcomponentP53The product of gene mutation and method, in order to service for tumor personalized treatment.
To this end, the present invention utilizes the method that constant-temperature amplification combines with direct Sequencing, establish a set of for microcomponent In sampleP53The techniqueflow of gene all mutation types detection, this detection method is highly sensitive, high specificity, and low cost has It is beneficial to Clinical practice and popularization.
Summary of the invention
It is an object of the invention to provide in a kind of detection microcomponent sample with high specific and sensitivityP53Base Because of the primer combination of sudden change, with tissue DNA for detection object, in conjunction with the pre-amplification technique of constant temperature and regular-PCR technology, by directly Sequencing determine in tumor sample with or withoutP53Gene mutation, can be rightP53The sudden change of gene detects accurately.
The primer combination that the present invention provides can detectP53There is the institute at No. 5, No. 6, No. 7 and 8 exons in gene There is sudden change.
What the present invention provided is used for detectingP53The primer combination of gene mutation includes such as SEQ ID NO:1-SEQ ID Pre-amplification primer sets shown in NO:18, dashing forward for detection 5 exons as shown in SEQ ID NO:19-SEQ ID NO:20 Become primer sets, as shown in SEQ ID NO:21-SEQ ID NO:22 for detection 6 exons sudden change primer sets, as Being used for shown in SEQ ID NO:23-SEQ ID NO:24 detects the primer sets of 7 exons sudden changes, such as SEQ ID NO:25- The primer sets for detection 8 exon sudden change shown in SEQ ID NO:26.
It is another object of the present invention to above-mentioned primer combination application in preparation detectionP53The detectable of gene mutation In.
It is another object of the present invention to that the combination of above-mentioned primer is applied to preparation be used for detectingP53The detection examination of gene mutation In agent box, described reagent constituents also comprises one or more in following conventional constituents: the combination of polymerase, primer, PCR react Buffer, dNTP, BSA, deionized water.
The present invention is used for detectingP53The detection using method of the primer combination of gene mutation, specifically comprises the following steps that
(1) primer dilution
After being diluted respectively by primer1-prime18, mixing prepares Primer Mix(table 1), wherein primer1-primer16 draws Final concentration of 0.05-0.1 μM of thing, final concentration of 1-3 μM of primer17-primer18 primer；primer19-primer26 Primer (table 1) is diluted to 5-10 μM.
(2) expand in advance
Primer Mix, 100-1000pg trace DNA templet of the 10 × reaction buffer of 1 μ L, 2.5 μ L is added in amplification pipe (DNA profiling that the present invention uses derives from lung cancer patient tissue, and according to conventional method described in " Molecular Cloning: A Laboratory guide " Extract tissue DNA), complement to 8.8 μ L with deionized water；Mixing, 98 DEG C of denaturations 5-10min, it is subsequently placed in 10-on ice 20min；Add 0.5 μ L dNTP (10mM each), 0.2 μ L 100 × BSA and 0.5 μ L phi29 archaeal dna polymerase, Overnight, 65 DEG C of heating 10-20min make enzyme inactivate in 30-35 DEG C of amplification.
(3) the above-mentioned pre-amplified production of PCR Purification Kit is used.
(4)P53Detection in Gene Mutation
ForP53No. 5 of gene, No. 6, No. 7 and the sudden change of 8 exons, be respectively adopted such as SEQ ID NO:19-SEQ ID Primer sets shown in NO:20 detects 5 exon sudden changes；Primer sets inspection as shown in SEQ ID NO:21-SEQ ID NO:22 Surveying 6 exon sudden changes, the primer sets as shown in SEQ ID NO:23-SEQ ID NO:24 detects 7 exon sudden changes, as Primer sets shown in SEQ ID NO:25-SEQ ID NO:26 examines 8 exon sudden changes.
It is 25 μ L that configuration PCR reacts total system: wherein Pfu Mix mixed liquor 12.5 μ L, described forward primer (5-10 μM) Volume 0.5-1 μ L, the volume 0.5-1 μ L of reverse primer (5-10 μM), pre-amplified production 1-2 μ L, complement to 25 μ L with water. PCR reaction condition is: 1. 94 DEG C, 5min denaturation；2. 94 DEG C, 30s degeneration；3. 55 DEG C, 30s anneals；4. 72 DEG C, 35s extends； Circulation is 2. to 4. 35 times；5. 72 DEG C, 5min；Order-checking detection.
The detection advantage of the present invention is:The present invention uses the combination of many primers, utilizes constant-temperature amplification and regular-PCR phase In conjunction with method, solve initial DNA content in tissue samples low, it is impossible to carry outP53The limitation of detection in Gene Mutation. The reagent used in the present invention is cheap, can large batch of process sample.The inventive method amplification efficiency is high, such as 100pg DNA available 1000ng/ μ L after amplification；The catastrophe in many sites can be detected simultaneously.After the inventive method DNA cloning DNA fidelity is good.In a word, the inventive method is used can to realize in microcomponent's sampleP53The catastrophe of gene is carried out Efficiently, detecting accurately, it is for clinical tumor early screening, instructs clinical application, and the prognosis of monitoring tumor has very Good actual application value.
Table 1: the nucleotide sequence of primer 1-26
。
Accompanying drawing explanation
Fig. 1 is the DNA profiling amplification that the present invention is directed to 1000pg；Wherein A figure is pre-amplification (repeating for 3 times)；B Figure 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 (repeating for 3 times)；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 (repeating for 3 times)；B figure isP53Base Because of No. 5, No. 6, No. 7 and 8 exon amplifications；
Fig. 4 is in the DNA profiling that the present invention is directed to 1000pgP53Gene 5, No. 6, No. 7 and 8 exon sequencing result figures；
Fig. 5 is in the DNA profiling that the present invention is directed to 500pgP53Gene 5, No. 6, No. 7 and 8 exon sequencing result figures；
Fig. 6 is in the DNA profiling that the present invention is directed to 100pgP53Gene 5, No. 6, No. 7 and 8 exon sequencing result figures.
Detailed description of the invention
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.Following example are merely to illustrate this Invention rather than restriction the scope of the present invention, the present invention can be done by person skilled in art according to the invention described above content Go out some nonessential improvement and adjustment.In following embodiment, showing if non-specific, agents useful for same is analytical pure, all examinations Agent all can obtain from commercial channel, and percentage ratio is mass percent.In literary composition, the experimental technique of unreceipted actual conditions, generally presses According to normal condition described in " Molecular Cloning: A Laboratory guide ", or the condition proposed by reagent manufacturer is implemented.Unless it is fixed separately The same meaning that justice, all specialties used in literary composition and scientific words and one skilled in the art are familiar with.
In the DNA profiling of embodiment 1:1000pgP53Detection in Gene Mutation
1, experiment material
Phi29 archaeal dna polymerase, 10 × reaction buffer, dNTP (10nM), the DNA mould of 100 × BSA, 1000pg Plate, deionized water, primer1-prime18 (i.e. Primer Mix), 2 × Pfu Mix, primer19-primer26, PCR Instrument, ultra-thin Product Purification Kit (TIANGEN Biotech (Beijing) Co., Ltd. DP203), the agarose gel of 1%, electrophresis apparatus.
2, experimental procedure and result
2.1 expand in advance
(1) primer dilution
After being diluted respectively by primer1-prime18, mixing forms Primer Mix(table 1), wherein primer1-primer16 draws Final concentration of 0.1 μM of thing, final concentration of 2 μMs of primer17-primer18 primer.Configure following system:
；
(2) after mentioned reagent being mixed, 98 DEG C of denaturations 10min, then it is immediately placed in 20min on ice；
(3) following system is added:
；
After being mixed by said mixture, overnight, 65 DEG C of heating 10-20min make enzyme inactivate in 30-35 DEG C of amplification.The glue detection of paving 1% (Figure 1A).Result display uses phi29DNA polymerase good to the DNA profiling expanding effect of 1000pg.
2.2 PCR primer purification
(1) column equilibration step: add the balance liquid BL of 500 μ L, 12,000 rpm (～13,400 × g) in adsorption column CB1 Centrifugal 1 min, outwells the waste liquid in collecting pipe, is placed back in collecting pipe by adsorption column；
(2) estimating the volume of PCR reactant liquor, be added thereto to the combination liquid PB of 5 times of volumes, fully mixing is (without removing paraffin Oil or mineral oil)；
(3) adding in an adsorption column CB1 (adsorption column is put in collecting pipe) by previous step gained solution, room temperature places 2 min, 12,000 rpm (～13,400 × g) centrifugal 30-60s, outwells the waste liquid in collecting pipe, is put in collecting pipe by adsorption column CB1；
(4) the most first check whether before adding 600 μ L rinsing liquid PW(uses in adsorption column CB1 and added dehydrated alcohol), 12, 000 rpm (～13,400 × g) centrifugal 30-60s, outwells the waste liquid in collecting pipe, is put in collecting pipe by adsorption column CB1；
(5) repetitive operation step (4)；
(6) 12,000 rpm (～13,400 × g) centrifugal 2 min, remove rinsing liquid as far as possible, adsorption column is placed in room temperature and places number Minute, dry up hill and dale, to prevent the rinsing liquid of residual from affecting next step experiment；
(7) taking out adsorption column CB1, put in a clean centrifuge tube, to adsorbed film centre position, unsettled dropping 20-50 μ L washes De-buffer EB, room temperature places 2 min.12,000rpm (～13,400 × g) centrifugal 2 min, collect DNA solution.
2.3 P53Detection in Gene Mutation
(1) configure following PCR reaction system, expand respectivelyP53No. 5, No. 6, No. 7 and 8 exons of gene；
；
Wherein for 5 exons sudden change primer as shown in SEQ ID NO:19-SEQ ID NO:20；For 6 exons The primer of sudden change is as shown in SEQ ID NO:21-SEQ ID NO:22；The primer such as SEQ ID NO:23-of 7 exon sudden changes Shown in SEQ ID NO:24；The primer of 8 exon sudden changes is as shown in SEQ ID NO:25-SEQ ID NO:26.
(2) reaction condition: 1. 94 DEG C, 5min denaturation；2. 94 DEG C, 30s degeneration；3. 55 DEG C, 30s anneals；4. 72 DEG C, 35s extends；Circulation is 2. to 4. 35 times；5. 72 DEG C, 5min；
(3), after PCR reaction terminates, glue detection (Figure 1B) of paving 1%, result shows that second takes turns PCR pairP53No. 5 of gene, 6 Number, No. 7 and 8 exons specific amplification all occurs；It is then sent to Sangon Biotech (Shanghai) Co., Ltd. carry out Order-checking detection.Sequencing result as shown in Figure 4,P53Gene the 6th exon generation missense mutation is c.580C > T p. L194F, the 5,7 and 8 exons are not detected by sudden change.
In the DNA profiling of embodiment 2:500pgP53Detection in Gene Mutation
1, experiment material
Phi29 archaeal dna polymerase, 10 × reaction buffer, dNTP (10nM), the DNA mould of 100 × BSA, 1000pg Plate, deionized water, primer1-prime18 (i.e. Primer Mix), 2 × Pfu Mix, primer19-primer26, PCR Instrument, ultra-thin Product Purification Kit (TIANGEN Biotech (Beijing) Co., Ltd. DP203), the agarose gel of 1%, electrophresis apparatus.
2, experimental procedure
2.1 expand in advance
(1) primer dilution.After being diluted respectively by primer1-prime18, mixing forms Primer Mix(table 1), wherein Final concentration of 0.1 μM of primer1-primer16 primer, final concentration of 2 μMs of primer17-primer18 primer.Configuration is such as Lower system:
；
(2) after mentioned reagent being mixed, 98 DEG C of denaturations 5min, then it is immediately placed in 20min on ice.
(3) following system is added:
；
After being mixed by said mixture, overnight, 65 DEG C of heating 10-20min make enzyme inactivate in 30-35 DEG C of amplification.The glue detection of paving 1% (Fig. 2 A).Result display uses phi29DNA polymerase good to the DNA profiling expanding effect of 500pg.
2.2 PCR primer purification
(1) column equilibration step: add the balance liquid BL of 500 μ L, 12,000 rpm (~ 13,400 × g) in adsorption column CB1 Centrifugal 1 min, outwells the waste liquid in collecting pipe, is placed back in collecting pipe by adsorption column.
(2) estimating the volume of PCR reactant liquor, be added thereto to the combination liquid PB of 5 times of volumes, fully mixing is (without removing Paraffin oil or mineral oil).
(3) adding in an adsorption column CB1 (adsorption column is put in collecting pipe) by previous step gained solution, room temperature places 2 Min, 12,000 rpm (~ 13,400 × g) are centrifuged 30-60 sec, outwell the waste liquid in collecting pipe, adsorption column CB1 is put into receipts In collector.
(4) the most first check whether before adding 600 μ L rinsing liquid PW(uses in adsorption column CB1 and added dehydrated alcohol), 12,000 rpm (~ 13,400 × g) are centrifuged 30-60 sec, outwell the waste liquid in collecting pipe, adsorption column CB1 is put into collecting pipe In.
(5) repetitive operation step 4.
(6) 12,000 rpm (~ 13,400 × g) are centrifuged 2 min, remove rinsing liquid as far as possible.Adsorption column is placed in room temperature put Put several minutes, dry up hill and dale, to prevent the rinsing liquid of residual from affecting next step experiment.
(7) take out adsorption column CB1, put in a clean centrifuge tube, the unsettled dropping 20-50 to adsorbed film centre position μ L elution buffer EB, room temperature places 2 min.12,000rpm (~ 13,400 × g) are centrifuged 2 min, collect DNA solution.
2.3 P53Detection in Gene Mutation
(1) configure following PCR reaction system, expand respectivelyP53No. 5, No. 6, No. 7 and 8 exons of gene.
；
Wherein for 5 exons sudden change primer as shown in SEQ ID NO:19-SEQ ID NO:20；For 6 exons The primer of sudden change is as shown in SEQ ID NO:21-SEQ ID NO:22；The primer such as SEQ ID NO:23-of 7 exon sudden changes Shown in SEQ ID NO:24；The primer of 8 exon sudden changes is as shown in SEQ ID NO:25-SEQ ID NO:26.
(2) reaction condition: 1. 94 DEG C, 5min denaturation；2. 94 DEG C, 30s degeneration；3. 55 DEG C, 30s anneals；4. 72 DEG C, 35s extends；Circulation is 2. to 4. 35 times；5. 72 DEG C, 5min；
(3), after PCR reaction terminates, glue detection (Fig. 2 B) of paving 1%, result shows that second takes turns PCR pairP53No. 5 of gene, 6 Number, No. 7 and 8 exons specific amplification all occurs；It is then sent to Sangon Biotech (Shanghai) Co., Ltd. carry out Order-checking detection.Sequencing result as it is shown in figure 5,P53Gene the 6th exon generation missense mutation is c.580C > T p. L194F, the 5,7 and 8 exons are not detected by sudden change.
In the DNA profiling of embodiment 3:100pgP53Detection in Gene Mutation
1, experiment material
Phi29 archaeal dna polymerase, 10 × reaction buffer, dNTP (10nM), the DNA mould of 100 × BSA, 1000pg Plate, deionized water, primer1-prime18 (i.e. Primer Mix), 2 × Pfu Mix, primer19-primer26, PCR Instrument, ultra-thin Product Purification Kit (TIANGEN Biotech (Beijing) Co., Ltd. DP203), the agarose gel of 1%, electrophresis apparatus.
2, experimental procedure
2.1 expand in advance
(1) primer dilution.After being diluted respectively by primer1-prime18, mixing forms Primer Mix(table 1), wherein Final concentration of 0.1 μM of primer1-primer16 primer, final concentration of 2 μMs of primer17-primer18 primer.
Configure following system:
；
(2) after mentioned reagent being mixed, 98 DEG C of denaturations 8min, then it is immediately placed in 20min on ice.
(3) following system is added:
After being mixed by said mixture, overnight, 65 DEG C of heating 10-20min make enzyme inactivate in 30-35 DEG C of amplification.The glue detection of paving 1% (Fig. 3 A).Result display uses phi29DNA polymerase good to the DNA profiling expanding effect of 100pg.
2.2 PCR primer purification
(1) column equilibration step: add the balance liquid BL of 500 μ L, 12,000 rpm (~ 13,400 × g) in adsorption column CB1 Centrifugal 1 min, outwells the waste liquid in collecting pipe, is placed back in collecting pipe by adsorption column.
(2) estimating the volume of PCR reactant liquor, be added thereto to the combination liquid PB of 5 times of volumes, fully mixing is (without removing Paraffin oil or mineral oil).
(3) adding in an adsorption column CB1 (adsorption column is put in collecting pipe) by previous step gained solution, room temperature places 2 Min, 12,000 rpm (~ 13,400 × g) are centrifuged 30-60 sec, outwell the waste liquid in collecting pipe, adsorption column CB1 is put into receipts In collector.
(4) the most first check whether before adding 600 μ L rinsing liquid PW(uses in adsorption column CB1 and added anhydrous second Alcohol), 12,000 rpm (~ 13,400 × g) are centrifuged 30-60 sec, outwell the waste liquid in collecting pipe, are put into by adsorption column CB1 In collecting pipe.
(5) repetitive operation step 4.
(6) 12,000 rpm (~ 13,400 × g) are centrifuged 2 min, remove rinsing liquid as far as possible.Adsorption column is placed in room temperature put Put several minutes, dry up hill and dale, to prevent the rinsing liquid of residual from affecting next step experiment.
(7) take out adsorption column CB1, put in a clean centrifuge tube, the unsettled dropping 20-50 to adsorbed film centre position μ L elution buffer EB, room temperature places 2 min.12,000rpm (~ 13,400 × g) are centrifuged 2 min, collect DNA solution.
2.3 P53Detection in Gene Mutation
(1) configure following PCR reaction system, expand respectivelyP53No. 5, No. 6, No. 7 and 8 exons of gene.
；
Wherein for 5 exons sudden change primer as shown in SEQ ID NO:19-SEQ ID NO:20；For 6 exons The primer of sudden change is as shown in SEQ ID NO:21-SEQ ID NO:22；The primer such as SEQ ID NO:23-of 7 exon sudden changes Shown in SEQ ID NO:24；The primer of 8 exon sudden changes is as shown in SEQ ID NO:25-SEQ ID NO:26.
(2) reaction condition: 1. 94 DEG C, 5min denaturation；2. 94 DEG C, 30s degeneration；3. 55 DEG C, 30s anneals；4. 72 DEG C, 35s extends；Circulation is 2. to 4. 35 times；5. 72 DEG C, 5min；
(3), after PCR reaction terminates, glue detection (Fig. 3 B) of paving 1%, result shows that second takes turns PCR pairP53No. 5 of gene, 6 Number, No. 7 and 8 exons specific amplification all occurs；It is then sent to Sangon Biotech (Shanghai) Co., Ltd. carry out Order-checking detection.Sequencing result as shown in Figure 6,P53Gene the 6th exon generation missense mutation is c.580C > T p. L194F, the 5,7 and 8 exons are not detected by sudden change.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for the skill of this area For art personnel, the present invention can have various modifications and variations.All within the spirit and principles in the present invention, that is made any repaiies Change, equivalent, improvement etc., should be included within the scope of the present invention.
Sequence table
<110>Kunming University of Science and Technology
<120>it is used for detecting in microcomponentP53The primer combination of gene mutation and application thereof
<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. it is used for detecting in microcomponentP53The primer combination of gene mutation, it is characterised in that: include such as SEQ ID NO:1- Pre-amplification primer sets shown in SEQ ID NO:18, being used for as shown in SEQ ID NO:19-SEQ ID NO:20 detect No. 5 The primer sets of exons mutation, being used for as shown in SEQ ID NO:21-SEQ ID NO:22 detect 6 exons sudden changes Primer sets, being used for as shown in SEQ ID NO:23-SEQ ID NO:24 detect the primer sets of 7 exons sudden changes, such as SEQ The primer sets for detection 8 exon sudden change shown in ID NO:25-SEQ ID NO:26.
2. the primer sets described in claim 1 is combined in preparation detectionP53Application in the detectable of gene mutation.
3. the primer sets described in claim 1 is combined in preparation detectionP53Application in the detection kit of gene mutation.
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CN108330167A (en) * 2017-09-13 2018-07-27 昆明理工大学 The new application of p53 gene mutations
CN108676852A (en) * 2018-05-30 2018-10-19 朱运峰 The primer pair group and kit of P53 gene epigenetics modification difference are detected in peripheral blood dissociative 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

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN108676852A (en) * 2018-05-30 2018-10-19 朱运峰 The primer pair group and kit of P53 gene epigenetics modification difference are detected in peripheral blood dissociative DNA
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

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