CN108034723A - The probe and primer that taqman sonde methods carry out the method for TP53 tumor susceptibility gene examinations and use - Google Patents
The probe and primer that taqman sonde methods carry out the method for TP53 tumor susceptibility gene examinations and use Download PDFInfo
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- CN108034723A CN108034723A CN201711371170.0A CN201711371170A CN108034723A CN 108034723 A CN108034723 A CN 108034723A CN 201711371170 A CN201711371170 A CN 201711371170A CN 108034723 A CN108034723 A CN 108034723A
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Abstract
The present invention provides a kind of method that taqman sonde methods carry out TP53 tumor susceptibility gene examinations, include the following steps：Detection method of the present invention comprises the following steps：(1) normal population buccal swab genome extracts；(2) TP53 primers and probe are designed；(3) PCR reactions are carried out to genome using the primer and probe of TP53；(4) fluorescence reading is carried out after PCR, and carries out Genotyping；(5) comprehensive 4 probe in detecting as a result, carrying out comprehensive assessment to the tumor susceptibility of individual.The taqman primers and probe of the present invention has good parting effect, can accurately determine very much the genotype of individual.
Description
Technical field
The present invention relates to tumor susceptibility gene examination and the method for quantitative fluorescent PCR, more particularly to a kind of taqman probes
The method that method carries out TP53 tumor susceptibility gene examinations.
Background technology
Cancer is the threat of 21st century human health maximum, although environment has cancer weight with habits and customs
Influence, but cancer is a kind of genopathy in essence, and inherent cause adds our neurological susceptibilities to cancer.It is popular to say just
It is single nucleotide polymorphism (single nucleotide polymorphism, SNP), it is referred mainly in genomic level,
The DNA sequence polymorphism as caused by making a variation single nucleotide acid.It is a kind of very good, effective molecular labeling.
The P53 albumen of TP53 gene translations is the important regulatory factor of cell growth, propagation and injury repair.TP53 genes
It is a kind of tumor suppressor gene, the low expression in normal cell, the high expression in malignant tumour, it is positioned at No. 17 chromosome of the mankind
Galianconism on, coding and expression TP53 albumen, be the regulatory factor in cell growth cycle, the regulation and control, DNA with the cell cycle are repaiied
The important biological functions such as multiple, cell differentiation, Apoptosis are associated.Wild type TP53 albumen can inhibit with DNA damage
Divide with the cell of chromosome aberration, so that distortion transfer is prevented to daughter cell, the tumor inhibition effect with wide spectrum.Phase
Then the generation with tumour, development have substantial connection for the mutation (missing) of anti-TP53 genes, therefore TP53 is known as gene bodyguard.Therefore
We select target of the TP53 genes as tumor susceptibility gene examination.
The main means of tumor susceptibility gene examination at present are generation sequencing, two generations were sequenced and quantitative fluorescent PCR.A generation and two generations
Although it is higher that accuracy is sequenced, with high costs, take longer.Taqman sonde methods detection SNP site has quick, accurate
The series of advantages such as really, cost is low.The probe of 2 kinds of different fluorescent markers is added in PCR reaction systems, they can be respectively with 2
A allele matches completely.Under normal circumstances, it is glimmering since probe 5 ' holds fluorophor and 3 ' end quenching groups close to together
Light is quenched.Effective with PCR carries out, and the probe matched completely with template is progressively circumscribed by Taq archaeal dna polymerases 5 ' → 3 '
Enzymatic activity is cut, and causes the fluorophor that probe 5 ' is held and the quenching group at 3 ' ends to separate, quenching effect releases, reporter fluorescence
Group is activated；And the probe that cannot be matched completely with template, show that another pair allele cannot effectively be cut, therefore detect
Less than fluorescence signal, SNP site detection can be achieved in the change that fluorescent value is detected by corresponding instrument, as shown in Figure 1.
The content of the invention
In order to solve the problems, such as that this area exists, the present invention provides a kind of taqman sonde methods to carry out TP53 tumor susceptibility genes
The method of examination and used probe and primer.
The purpose of the present invention is realized by following scheme：
A kind of method that taqman sonde methods carry out TP53 tumor susceptibility gene examinations, includes the following steps：
(1) buccal swab genome extracts；
(2) TP53 primers and probe are determined；
(3) PCR reactions are carried out to the genome of step (1) using the primer and probe of TP53；
(4) fluorescence reading is carried out after PCR, and carries out Genotyping；
(5) comprehensive probe in detecting as a result, carrying out comprehensive assessment to the tumor susceptibility of individual.
Preferably, the extracting method in the step (1) is to scrape Oral Mucosal Cells using buccal swab, is used
100ul buccal swabs preserve liquid and preserve, and are discharged through 400ml cell pyrolysis liquids and 40ul, 20ug/ul protease k cell lysis
Genomic DNA, rear selective precipitation remove removing protein, and pure genomic DNA is dissolved in spare in 50ul TE buffer；Institute
State.
Preferably, the primer in the step (2) include TP53 genes on rs78378222, rs12951053,
The primer in rs2287498, rs2078486SNP site.
Preferably, the primer of each SNP site is as shown in the table：
Preferably, the probe of each SNP site is as shown in the table：
Preferably, the PCR reaction systems of the step (3) are 10 μ l：
Preferably, the PCR conditions are：
95 DEG C of 15min, 95 DEG C of 20sec, 60 DEG C of 1min, 40 circulations, 72 DEG C of 5min.
Another aspect of the invention is the primer and probe that TP53 tumor susceptibility gene examinations are carried out for Taqman sonde methods, institute
State the primer in rs78378222, rs12951053, rs2287498, rs2078486SNP site that primer is included on TP53 genes.
Preferably, the primer of each SNP site is as shown in the table：
Preferably, the probe of each SNP site is as shown in the table：
Method provided by the invention using Taqman MGB probe in detecting people's TP53 gene pleiomorphisms, can high throughput detection
Its SNP site, as a result easy interpretation.384 samples can be detected every time, and need not carry out the subsequent analysis of PCR product,
While saving testing cost, detection cycle is substantially reduced, improves detection efficiency.The primer and probe that the present invention uses
High specificity, will not be disturbed be subject to other genes, reduce the risk that PCR product pollution causes false positive, and result is sentenced
Reading is easier.It can realize that high throughput is detected to the SNP site of TP53 genes, have in tumor susceptibility gene detection is carried out to people potential
Application value.
Brief description of the drawings
Fig. 1 is the technical schematic diagram of Taqman probe in detecting SNP；
Fig. 2 is the testing result that there are 3 genotype GG, GT and TT in rs78378222SNP sites, and wherein GG genotype is being schemed
The upper left side of shape, GT is centrally located, and TT is located at lower right；
Fig. 3 is that rs12951053SNP sites have 3 frequency of genotypes AA, the testing result of AC, CC, and wherein AA genotype is located at
The upper left side of figure, AC genotype is centrally located, and CC genotype is located at lower right.
Embodiment
Below in conjunction with the accompanying drawings, detailed explanation is carried out to the present invention.
Following embodiments are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
According to conventional laboratory conditions, as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW,
Molecular cloning:A laboratory manual, 2001), or the condition according to manufacturer's specification suggestion.
Embodiment 1
Utilize the method for Taqman MGB probe in detecting people's TP53rs78378222 single nucleotide polymorphisms
1st, experiment material
The healthy population chosen by the sequencing of a TP53 generation is detection object.
2nd, the extraction of genomic DNA preserves liquid using buccal swab scraping Oral Mucosal Cells first by buccal swab
Preserve, cell pyrolysis liquid cell lysis discharges genomic DNA, and then albumen precipitation liquid selective precipitation removes removing protein, finally
Pure genomic DNA is laid equal stress on by isopropanol precipitating is dissolved in DNA lysates.The extraction of genomic DNA is not limited only to such a
Method, the genomic DNA extracted using other methods is also in the research range of this experiment.
3rd, Taqman MGB sonde methods carry out Genotyping, using rs78378222 sites primer and probe to examining
Survey.
Primer and probe design is carried out for the nucleotide in No. 17 7571752 sites of chromosome.
The nucleotide sequence of the primer of the gene is as follows：
Forward primer：5'-GCCTGCACTGGCGTTCAC-3'
Reverse primer：5'-TCCCCTCCTTCTCCCTTTTTAT-3'；
The nucleotide sequence of the probe is as follows：
P1-G：5'-FAM-CAGCAAAGTTGTATTG-MGB-3'
P1-T：5'-VIC-CAGCAAAGTTTTATTGTA-MGB-3'；
Primer and probe is synthesized by Qing Ke biotech firms, using the 2X GoldStar MasterMix purchased from Kang Wei companies
(Dye), 7500 real-time fluorescence quantitative PCR systems of PCR instrument model ABI.
Reaction system：Using the genomic DNA of extraction as template, with the primer and probe of above-mentioned synthesis in quantitative fluorescent PCR
It is as shown in the table in system to be expanded (10 μ L reaction systems)：
Specific implementation step is as follows：
(1) error of pipettor and suction nozzle is considered, general each 96 orifice plate is pressed 105 Response calculation systems, prepared
mix。
(2) one blank control NTC of each 96 orifice plate setting, two known GG types TP53 genomic controls of setting, one
Known GT types TP53 genomic controls, a known TT types TP53 genomic control.
(3) using the volley of rifle fire in every 1 μ L DNA samples of Kong Zhongjia.
(4) sealed after the completion of with sealed membrane, plate centrifuge centrifugation.
(5) unlatching 7500 real-time fluorescence quantitative PCR systems of ABI, arrange parameter, reaction condition are as follows：
95 DEG C of 15min, 95 DEG C of 20sec, 60 DEG C of 1min, 40 circulations, 72 DEG C of 5min, 60 DEG C are to read fluorescence
4th, analysis result
Data analysis is carried out using 7500 real time fluorescent quantitative softwares of ABI.According to control amplification, parting produces three
Kind genotype, is shown in Fig. 2.
The first genotype：GG, with GG types to impinging upon on an axis；
Second of genotype：GT, with GT types to impinging upon on an axis；
The third genotype：TT, with TT types to impinging upon on an axis.
Embodiment 2
Utilize the method for Taqman MGB probe in detecting people's TP53rs12951053 single nucleotide polymorphisms
1st, experiment material
The healthy population chosen by the sequencing of a TP53 generation is detection object.
2nd, the extraction of genomic DNA preserves liquid using buccal swab scraping Oral Mucosal Cells first by buccal swab
Preserve, the buccal swab DNA extraction kit that biology is held high using will carries out DNA extractions, and Qubit is quantitative, NanoDrop detections
Whether 260/280 and 260/230 is qualified.Professional will readily appreciate that the extraction of genomic DNA is not limited only to such a method, makes
The genomic DNA extracted with other methods is also in the research range of this experiment.
3rd, Taqman MGB sonde methods carry out Genotyping, using rs12951053 sites primer and probe to examining
Survey.
Primer and probe design is carried out for the nucleotide in No. 17 7577407 sites of chromosome.
The nucleotide sequence of the primer of the gene is as follows：
Forward primer：5'-AAAAGAAAACTGAGTGGGAGCAGTA-3'
Reverse primer：5'-TGCCCCAGCCTCTGCTT-3'；
The nucleotide sequence of the probe is as follows：
P2-A：5'-FAM-TAGTATGGAAGAAATC-MGB-3'
P2-C：5'-VIC-TAGTAGTATGGCAGAAAT-MGB-3'.
Primer and probe is synthesized by Qing Ke biotech firms, using the 2X GoldStar MasterMix purchased from Kang Wei companies
(Dye), 7500 real-time fluorescence quantitative PCR systems of PCR instrument model ABI.
Primer and probe is synthesized by Qing Ke biotech firms, using the 2X GoldStar MasterMix purchased from Kang Wei companies
(Dye), 7500 real-time fluorescence quantitative PCR systems of PCR instrument model ABI.
Reaction system：Using the genomic DNA of extraction as template, with the primer and probe of above-mentioned synthesis in quantitative fluorescent PCR
It is as shown in the table in system to be expanded (10 μ L reaction systems)：
Specific implementation step is as follows：
(1) error of pipettor and suction nozzle is considered, general each 96 orifice plate is pressed 105 Response calculation systems, configured
mix；
(2) each 96 orifice plate sets a blank control NTC, sets two known AA types rs12951053 sites controls,
One known AC types rs12951053 sites control, a known CC types rs12951053 sites control；
(3) using the volley of rifle fire in every 1 μ L DNA samples of Kong Zhongjia；
(4) sealed after the completion of with sealed membrane, plate centrifuge centrifugation；
(5) unlatching 7500 real-time fluorescence quantitative PCR systems of ABI, arrange parameter, reaction condition are as follows：
95 DEG C of 15min, 95 DEG C of 20sec, 60 DEG C of 1min, 40 circulations, 72 DEG C of 5min, 60 DEG C of whens, read fluorescence.
4th, analysis result
Data analysis is carried out using 7500 real time fluorescent quantitative softwares of ABI.According to control amplification, parting produces three
Kind genotype, is shown in Fig. 3.
The first genotype：AA, with AA types to impinging upon on an axis；
Second of genotype：AC, with AC types to impinging upon on an axis；
The third genotype：CC, with CC types to impinging upon on an axis.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art is in the technical scope of present disclosure, the change or replacement that can readily occur in,
It should all be included within the scope of the present invention.Therefore, protection scope of the present invention should be with the protection of claims
Subject to scope.
Sequence table
<110>U.S. is because of healthy science and technology（Beijing）Co., Ltd
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tcccctcctt ctcccttttt at 22
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tgccccagcc tctgctt 17
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tgtcccccat cttcctttca 20
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gaaggaggtg tacttgcatt aatgg 25
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cagcaaagtt gtattg 16
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tagtagtatg gcagaaat 18
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aactacagct tctcccag 18
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actacagctt ttcccagc 18
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tgattgttag tgcagatc 18
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tgtgtgattg ttagtgcgga 20
Claims (10)
1. a kind of method that taqman sonde methods carry out TP53 tumor susceptibility gene examinations, it is characterised in that include the following steps：
(1) buccal swab genome extracts；
(2) TP53 primers and probe are determined；
(3) PCR reactions are carried out to the genome of step (1) using the primer and probe of TP53；
(4) fluorescence reading is carried out after PCR, and carries out Genotyping；
(5) comprehensive probe in detecting as a result, carrying out comprehensive assessment to the tumor susceptibility of individual.
2. the method that taqman sonde methods according to claim 1 carry out TP53 tumor susceptibility gene examinations, it is characterised in that institute
It is to scrape Oral Mucosal Cells using buccal swab to state the extracting method in step (1), and preserving liquid using 100ul buccal swabs protects
Deposit, genomic DNA, rear selective precipitation are discharged through 400ml cell pyrolysis liquids and 40ul, 20ug/ul protease k cell lysis
Removing protein is removed, pure genomic DNA is dissolved in spare in 50ul TE buffer；It is described.
3. the method that taqman sonde methods according to claim 1 carry out TP53 tumor susceptibility gene examinations, it is characterised in that institute
The primer stated in step (2) includes rs78378222, rs12951053, rs2287498, rs2078486 on TP53 genes
The primer of SNP site.
4. the method that taqman sonde methods according to claim 3 carry out TP53 tumor susceptibility gene examinations, it is characterised in that institute
The primer for stating each SNP site is as shown in the table：
5. the method that taqman sonde methods according to claim 4 carry out TP53 tumor susceptibility gene examinations, it is characterised in that institute
The probe for stating each SNP site is as shown in the table：
6. the method that taqman sonde methods according to claim 1 carry out TP53 tumor susceptibility gene examinations, it is characterised in that institute
The PCR reaction systems for stating step (3) are 10 μ l：
7. the method that taqman sonde methods according to claim 6 carry out TP53 tumor susceptibility gene examinations, it is characterised in that institute
Stating PCR conditions is：
95 DEG C of 15min, 95 DEG C of 20sec, 60 DEG C of 1min, 40 circulations, 72 DEG C of 5min.
8. the primer and probe of TP53 tumor susceptibility gene examinations is carried out for Taqman sonde methods, it is characterised in that the primer bag
Include the primer of rs78378222, rs12951053, rs2287498, rs2078486 SNP site on TP53 genes.
9. primer and probe according to claim 8, it is characterised in that the primer of each SNP site is as shown in the table：
10. the method that taqman sonde methods according to claim 9 carry out TP53 tumor susceptibility gene examinations, it is characterised in that
The probe of each SNP site is as shown in the table：
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Cited By (1)
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