CN106811519A - A kind of oesophagus cancer susceptibility gene detection and genotyping kit and its application - Google Patents
A kind of oesophagus cancer susceptibility gene detection and genotyping kit and its application Download PDFInfo
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Abstract
The invention discloses a kind of oesophagus cancer susceptibility gene detection and genotyping kit and its application, the oesophagus cancer susceptibility gene detection and genotyping kit, to PED4D, PLCE1, RUNX1, tri- are detected with the pleomorphism site of esophageal cancer related gene, will appreciate that the individual inherent cause in terms of the cancer of the esophagus, the relative risk of Incidence of Esophageal Cancer is assessed, there is positive meaning for cancer of the esophagus early diagnosis, early treatment, early intervention.The diagnosis of the cancer of the esophagus can also be aided in simultaneously, instruct the rational use of medicines, aid forecasting offspring's risk etc..
Description
Technical field
The present invention relates to a kind of inspection technology field, specifically a kind of oesophagus cancer susceptibility gene detection and genotyping kit and its
Using.
Background technology
With the completion of the Human Genome Project (Human Genome Project, HGP), a large amount of disease related genes
It was found that, promote genome medical model of the traditional biological medical model to predictability, preventability, individuation and property of participation
Transformation, is future development prevention, diagnosis, such as cancer, cardiovascular and cerebrovascular disease, diabetes, the nerve of the treatment long-standing problem mankind
New way is opened with the great Complex Diseases such as mental illness, also for the industrialization of genetic science provides good opportunity.
Whole-genome association (Genome Wide Association Studies, GWAS) is to apply human gene
Millions of SNP (single nucleotide polymorphism, SNP) is carried out for mark in group
Case one compares association analysis, i.e., case group and control group DNA are collected in certain group of people, carries out SNPs chip scannings, uses
Association analysis is compared is had that there was no significant difference by a certain gene frequency of inspection SNPs marks between case group and control group, sieve
Choosing makes a variation with the gene order of disease association, make the SNPs whether the judgement with disease association, the illness rate of predictive disease and
Risk.Structure is not required to before the method research any it is assumed that being no longer limited to candidate gene or candidates region as association
Analysis object, and it is directed to all SNPs in genome, the degree of association of Genotyping, analysis SNPs and disease is carried out at random, can
In full-length genome level, the association study of development multicenter, large sample, the gene verified repeatedly and disease, comprehensive disease in time
The correlated inheritance gene that disease occurs, develops and treats.
Inherent cause is had now been found that, or its interaction between environmental factor take part in almost all of human diseases
Generating process.Some complicated diseases are frequently not to be caused by term single gene mutation, and it occurs development and polygenes multidigit
Point variation is relevant, and the variation in these sites may be related to environmental factor, and these sites can improve or reduce individuality and suffer from certain
The relative risk of disease, is referred to as this kind of susceptibility loci of disease.According to substantial amounts of GWAS results, it has been found that and for proving
The susceptibility loci of serial complex disease has been widely used in field of gene detection.
The cancer of the esophagus is common tumor in digestive tract, and according to global cancer statistics in 2008, the cancer of the esophagus ranked the whole world ten
One of big most common cancer, its fatal rate occupies the 6th of malignant tumour, and most of generations in development in the world
Middle country.The whole world there are about 300,000 people and dies from the cancer of the esophagus every year.China is one of Esophageal Cancer area, GLBOCAN in the world
Statistics show that China in 2008 newly sends out cancer of the esophagus case and has 25.8 ten thousand and die from patient with esophageal carcinoma number 210,000.Food
The typical symptom of pipe cancer is progressive dyscatabrosis, and the food of difficult dry throat, was then semiliquid diet, last water and saliva before this
Can not swallow, due to diagnosing, relatively later and therapeutic effect is poor, and the cancer of the esophagus is one of fatal rate highest tumour in China.
PEDs is divided into by 11 family according to amino acid sequence homology and zymolyte specificity respectively, altogether by 20
Individual genomic constitution.Each PEDs family includes 1-4 different gene.PED4 is made up of tetra- subfamilies of A, B, C, D
(Kostic M M,Erdogan S,Rena G,et al.Altered Expr ession of PDE1 and PDE4
Cyclic Nucleotide Phosphodiesterase Isoforms in 7-oxo-prostacyclin-
preconditioned Rat Heart[J].Journal of Molecula r&Cellular Cardiology,1997,29
(11):3135-46.).PED4D full length genes about 1.6Mb, be positioned on the 5q12 of human genome (Song Q, Cole J W,
O'Connell J R,et al.Phosph odiesterase 4D polymorphisms and the risk of
cerebral infarction in a biracial population:the Stroke Prevention in Young
Women Study.[J].H uman Molecular Genetics,2006,15(16):2468-78.).Opened according to different
Sub-area is different with the alternative splicing after translation, and causing the isomers of PED4D has many kinds.Isomers are in different groups
The different signal reaction of middle regulation is knitted, to play different functions in vital movement.To the research hair of mankind's PED4D genes
Existing, the promoter region of its PED4D5 hypotype contains CCAAT- enhancers binding site and cAMP reaction original papers (cAMP
Responsive element, CRE) binding site (Le J I, Shepherd M, V an H G, et al.Cyclic AMP-
dependent transcriptional up-regulation of ph osphodiesterase 4D5 in human
airway smooth muscle cells.Identificatio n and characterization of a novel
PDE4D5promoter.[J].Journal of Biol ogical Chemistry,2002,277(39):35980-9.).Its
Middle PDE4 families can specific for hydrolysis cA MP, and can in different time and intracellular regulation cAMP concentration change so that
Cause different (Iancu R V, Jones S W, the Harvey R of the signal pathway for mediating in the cell
D.Compartmentation of cAMP Signaling in Cardiac Myocytes:A Computational
Study[J].Biophy sical Journal,2007,92(9):3317-31.).In PDE4 gross activities the content of PDE4D compared with
Height, wherein, PDE4D constitutes about the 60-70% of gross activity in normal cell, and when body is inflamed, PDE4D accounts for PDE4
Gross activity it is higher, about more than 80%.
PLCE1 genes are initially that Shibatohge etc. has found in Caenorhabditis elegans body, and its molecular size range is
210KD, therefore it is named as PLC210 (Shibatohge M, Kariya K, Liao Y, et al.Iden tification of
PLC210,a Caenorhabditis elegans phospholipase C,as a pu tative effector of
Ras.[J].Journal of Biological Chemistry,1998,273(11):6218-22.).PLCE1 except comprising
There is PLC families apokoinou construction domain, it is also specific to include a GTP exchange factors region-CDC25 outside XY, PH, C2 and EF
Domain, two C-terminal Ras binding regions-RA1 and RA2 (Wing M R, Bourdon D M, Harden T K.PLC-
epsilon:a shared e ffector protein in Ras-,Rho-,and G alpha beta gamma-
mediated signalin g.[J].Molecular Interventions,2003,3(5):273-280.).Due to PLCE1
Having one can tie as the connected RA of the CDC25 domains of the Guanine nucleotide exchange factor (GEF) of Ras superfamily GTP enzymes and two
Structure domain, therefore PLCE1 is different from other isodynamic enzymes, can be interacted with small G-protein.Abnet is found that PLCE1 is esophageal squamous cell carcinoma
Tumor susceptibility gene (Abnet C C, Freedman N D, Hu N, et al.A shared susceptibility locus in
PLCE1 at 10q23 for gastric adenocarcinoma an d esophageal squamous cell
carcinoma[J].Nature Genetics,2010,42(9):764-7.), and it was found that PLCE1 rs2274223 sites are
The susceptibility loci of the cancer of the esophagus, can increase the onset risk of the cancer of the esophagus.Li research discoveries, the p53 tables after silence PLCE1 in the cancer of the esophagus
Up to significantly raised (Li Y, An J, Huang S, et al.PLCE1 suppresses p53 expression in
esophageal cancer cells.[J].Cancer Investigation,2014,32(6):236-240.).In oesophagus
In cancerous cell line EC9706, after silence PLCE1, the apoptosis rate of esophageal cancer cell increases, and cycle GAP-associated protein GAP cycl in
D1 is to reduce, and apoptosis-related protein caspase-3 is elevated (Zhao L, Wei Z B, Yang C Q, et
al.Effects of PLCE1 gene silencing by RNA interference on cell c ycling and
apoptosis in esophageal carcinoma cells.[J].Asian Pacific Journal of Cancer
Prevention Apjcp,2014,15(13):5437-42.).Therefore, PLCE1 can be as a kind of new of esophagus cancer diagnosis
Tumor marker.
Mankind RUNT associated transcription factors (human runt-related transcription factor, R UNX)
It is the general designation of a class transcription factor, is to constitute identical containing monoamino-acid in its molecular structure the characteristics of they are common
DNA lands, the land is made up of and homologous with the segmentation gene R unt of Drosophila 128 amino acid, is referred to as therefrom
Runt domains (Ito Y.Oncogenic potential of the RU NX gene family:'overview'.[J]
.Oncogene,2004,23(24):4198-4208.)., Human Genome Organization (human genome in 1999
Organization, HUGO) gene of the coding with Runt domain proteins is commonly referred to as RUNXX.There are 3 lactations in Runt families
Animal related gene, wherein Runx1 are located at (Levanon D, Negreanu V, Bernstein on human chromosomal 21q22.3
Y,et al.AML1,AML2,and AML3,the Human Members of the runt domain,Gene-Family:
cDNA S tructure,Expression,and Chromosomal Localization[J].Genomics,1994,23
(2):425-432.).Runx albumen is a transcription factor in TGF-β signal path downstream, is responsible for combining DNA, TGF-β/
Played an important role in BMP signal transductions, the change of its function influences the activity of TGF-signal beta, and then develops in tumour
During play an important role.Runx expression deletions will result directly in the limitation of Smad protein functions, and then influence TGF-β
The bioactivity of signal path, leads oncogenic generation development (Miyazono K, Suzuki H, Imamura
T.Regulation of TGF-beta signal ing and its roles in progression of tumors.
[J].Cancer Science,2003,94(3):230-4.).Research shows that Runx1 gene methylations are that the low transcription of its inactivation is another
One principal element (R eed-Inderbitzin E, Moreno-Miralles I, Vanden-Eynden S K, et
al.RUNX1 ass ociates with histone deacetylases and SUV39H1 to repress
transcriptio n.[J].Oncogene,2006,25(42):5777-86.), its cancer suppressing function is caused to be lost.
Therefore, to PED4D, PLCE1, RUNX1 tri- detected with the pleomorphism site of esophageal cancer related gene, can
The individual inherent cause in terms of the cancer of the esophagus of solution, assesses the relative risk of Incidence of Esophageal Cancer, for cancer of the esophagus early diagnosis, early stage
Treatment, early intervention have positive meaning.The diagnosis of the cancer of the esophagus can also be aided in simultaneously, the rational use of medicines is instructed, after aid forecasting
For risk etc..
The content of the invention
It is an object of the invention to provide a kind of oesophagus cancer susceptibility gene detection and genotyping kit and its application, with solution
State the problem proposed in background technology.
To achieve the above object, the present invention provides following technical scheme：
A kind of oesophagus cancer susceptibility gene detection and genotyping kit, the kit includes that DNA extraction kit, DNA expand
Increase kit and sequencing kit；The DNA extraction kit uses the DP322 type products of Tiangeng biochemical technology Co., Ltd,
The DNA cloning kit mainly includes amplimer, enzyme, PCR Buffer, d NTP Mixture, amplimer, the expansion
Increasing primer is：1)F：5'-GGGGAATGAGACAGAGACCA-3', R：5'-ACCATTCCCAATGTCTGCTC-3',2)F：5'-
GGAAGCAGTGAGGTGCAGAGGT-3', R：5'-TCCACAACTGCAAAACGAAGAAA-3',3)F：5'-
TCTCAGCCCATGGTGAGCATTA-3',R：5'-CCCCCATATGGTATCCCCAAAG-3'；The enzyme is using precious bioengineering
The TaKaRa Taq of Co., LtdT MEnzyme；The sequencing kit is using American AB I companiesTerminator
v3.1Cycle Sequ encing Kit。
As further scheme of the invention：The amplimer is to be set based on gene PDE3D, PLCE1 and RUNX1
Meter synthesis.
A kind of application of oesophagus cancer susceptibility gene detection and genotyping kit, concretely comprises the following steps：
(1) first, the sample returned will be gathered and will extract acquisition sample genome DN A according to DNA extraction kit；
(2) and then, by obtain sample genomic dna expanded using DNA cloning kit, the product after being expanded
Thing, then carries out electrophoresis detection and purifying by the product after amplification, obtains product after purification；
(3) then, sequencing kit is added in product after purification carries out mulberry lattice sequencing reaction；
(4) last, the product after above-mentioned steps mulberry lattice sequencing reaction is terminated carries out ethanol/EDTA purifying, goes up after purification
Sample carries out parting judgement in sequenator after sequence analysis.
As further scheme of the invention：Sample is Oral Mucosal Cells in step (1).
As further scheme of the invention：Ethanol/EDTA purifying is specially and reaction is removed from PCR instrument in step (4)
Plate, 3800rpm centrifugations 1min；Plus 2.5 μ l 0.125M EDTA, short centrifugation；Stand 5min；Plus 20 μ l precooling absolute ethyl alcohols, patch
Sealed membrane, vibration is mixed；3800rpm, 4 DEG C of centrifugation 30mi n；It is centrifuged after terminating immediately, is inverted PCR plate low-speed centrifugal for a moment, turns
Speed is no more than 800rpm；Plus 60 μ l75% pre-cooled ethanol, pad pasting, vibration mix；3800rpm, 4 DEG C of centrifugation 15min；Centrifugation knot
After beam immediately, it is inverted PCR plate low-speed centrifugal for a moment, rotating speed is no more than 800rpm；PCR instrument denaturation program processes 5mi n, or lucifuge
30min is stood, volatilize ethanol；Plus 10 μ l Hi-Di formamides, pad pasting, short centrifugation；95 DEG C of denaturation 5min；It is immediately placed in -20 DEG C
Refrigerator.
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention prepares a kind of oesophagus cancer susceptibility gene detection and genotyping kit, to PED4D, PLCE1, RUN X1 tri-
Pleomorphism site with esophageal cancer related gene is detected that will appreciate that the individual inherent cause in terms of the cancer of the esophagus, assessment is eaten
The relative risk of pipe cancer morbidity, has positive meaning for cancer of the esophagus early diagnosis, early treatment, early intervention；Also simultaneously
The diagnosis of the cancer of the esophagus can be aided in, the rational use of medicines, aid forecasting offspring's risk etc. is instructed；This patent product uses genetic test
The goldstandard in field --- mulberry lattice PCR sequencing PCR carries out Genotyping；Mulberry lattice PCR sequencing PCR is to produce end by controlling the synthesis of DNA
Terminate in the oligonucleotide fragment of target sequence specific site；First, the Oligonucleolide primers of synthesis are annealed with single-stranded DNA profiling,
Four kinds of different sequencing reactions are set up, archaeal dna polymerase and four kinds of normal dNTP are all contained in each reaction；In addition,
Also contain a small amount of 2', 3'-ddNTP that there is 3'-H to replace common deoxyribose 3'-OH；If ddNTP is incorporated into the DNA in extending
Chain, follow-up dNTP formation phosphodiester bond will be blocked due to lacking 3'-OH, and the further extension of DNA is terminated；Use four kinds
Different ddNTP, the oligonucleotides of generation is terminated at each A, and the position that C, G or T are occupied on template strand is anti-by four kinds
The oligonucleotides that should be produced is added in sequenator, due to of different sizes, speed just different, the sequencing of swimming of oligonucleotide fragment
The image controller of instrument can sequentially, just detect the sequence letter of new synthesis chain 5' to 3' according to oligonucleotides elapsed time
Breath.
Brief description of the drawings
Fig. 1 is the homozygous CC sequencing and typings result figure of risk of the invention；
Fig. 2 is normal homozygous AA sequencing and typings result figure of the invention；
Fig. 3 is the homozygous GG sequencing and typings result figure of risk of the invention.
Specific embodiment
The technical scheme of this patent is described in more detail with reference to specific embodiment.
A kind of oesophagus cancer susceptibility gene detection and genotyping kit, the kit includes that DNA extraction kit, DNA expand
Increase kit and sequencing kit；The DNA extraction kit uses the DP322 type products of Tiangeng biochemical technology Co., Ltd,
The DNA cloning kit mainly includes amplimer, enzyme, PCR Buffer, d NTP Mixture, amplimer, the expansion
Increasing primer is：1)F：5'-GGGGAATGAGACAGAGACCA-3'(SEQ ID NO.1), R：5'-
ACCATTCCCAATGTCTGCTC-3'(SEQ ID NO.2),2)F：5'-GGAAGCAGTGAGGTGCAGAGGT-3'(SEQ ID
NO.3), R：5'-TCCACAACTGCAAAACGAA GAAA-3'(SEQ ID NO.4),3)F：5'-
TCTCAGCCCATGGTGAGCATTA-3'(SEQ ID NO.5),R：5'-CCCCCATATGGTATCCCCAAAG-3'(SEQ ID
NO.6)；The enzyme uses the TaKaRa Taq of precious bioengineering Co., LtdTMEnzyme；The sequencing kit is public using American AB I
DepartmentTermin ator v3.1Cycle Sequencing Kit。
The amplimer is to be designed synthesis based on gene PDE3D, PLCE1 and RUNX1；Wherein, the mesh of detection
Mark SNP site includes rs10052657, rs2274223 and rs2014300, and this 3 sites are located at gene PDE3D, PLCE1 respectively
And RUNX1.
A kind of application of oesophagus cancer susceptibility gene detection and genotyping kit, concretely comprises the following steps：
(1) first, the sample returned will be gathered and will extract acquisition sample genome DN A according to DNA extraction kit；
(2) and then, by obtain sample genomic dna expanded using DNA cloning kit, the product after being expanded
Thing, then carries out electrophoresis detection and purifying by the product after amplification, obtains product after purification；
(3) then, sequencing kit is added in product after purification carries out mulberry lattice sequencing reaction；
(4) last, the product after above-mentioned steps mulberry lattice sequencing reaction is terminated carries out ethanol/EDTA purifying, goes up after purification
Sample carries out parting judgement in sequenator after sequence analysis.
Wherein：
Sample is Oral Mucosal Cells in step (1).
Expanded using DNA cloning kit in step (2), the reaction system of amplification is：
TaKaRa Taq TM(5U/μl)1U
10×PCR Buffer(Mg2+)5μl
dNTP Mixture(2.5mM each)4μl
The μ l of forward primer (10pmol/ μ l) 0.5
The μ l of reverse primer (10pmol/ μ l) 0.5
The μ l of template 1
The μ l of distilled water up to 50,
Amplification program is：
Stage1:Pre degeneration
Repeat:1 time
95℃3 minutes
Stage 2:PCR reaction
Repeat:30 cycles
95℃5 second
55℃30 second
72℃1 minutes
Stage 3:Final extension
72℃5 minutes。
Electrophoresis detection and purifying in step (2), wherein electrophoresis detection are concretely comprised the following steps：Prepare 1% Ago-Gel：Claim
A certain amount of agar Icing Sugar adds 10 × TAE heating of corresponding volume to melt agarose, add 10000 after it melts completely ×
Gold ViewⅠ.After gelling is solid, after taking the PCR primer addition Loadi ng Buffer mixing of 5 μ l, in point to glue hole, 120V
Level pressure, runs 15min or so.Observed in TGreen Trans illuminator the clip size of amplified production, unicity and
Amplification concentration；
Wherein purification system is：
The μ l of reaction solution 5 after PCR amplifications
SAP/CIP(1U/μl)1μl
ExoⅠ(5U/μl)1μl
ddH2The μ l of O 3,
Wherein purification reaction program is：
37℃30 minutes
75℃15 minutes。
Mulberry lattice sequencing reaction in step (3), reaction system is：
The μ l of BigDye mixed liquors 1
The μ l of sequencing primer (3.2pmol/ μ l) 1
The μ l of template (purified product) 1
ddH2The μ l of O 2,
Response procedures are：
Stage1:1 time
95℃2 minutes
Stage 2:25 cycles
95℃10 second
50℃5 second
60℃4 minutes。
Ethanol/EDTA purifying is specially and reaction plate is removed from PCR instrument in step (4), 3800rpm centrifugations 1min；Plus 2.5
μ l 0.125M EDTA, short centrifugation；Stand 5min；Plus 20 μ l precooling absolute ethyl alcohols, membrana oralis is sealed, vibration is mixed；3800rpm, 4
DEG C centrifugation 30min；It is centrifuged after terminating immediately, is inverted PCR plate low-speed centrifugal for a moment, rotating speed is no more than 800rpm；Plus 60 μ l 75%
Pre-cooled ethanol, pad pasting, vibration mix；3800rpm, 4 DEG C of centrifugation 15min；Centrifugation terminate after immediately, be inverted PCR plate low speed from
Lamination is carved, and rotating speed is no more than 800rpm；PCR instrument denaturation program processes 5min, or lucifuge stands 30min, and volatilize ethanol；Plus 10 μ l
Hi-Di formamides, pad pasting, short centrifugation；95 DEG C of denaturation 5min；It is immediately placed in -20 DEG C of refrigerators.
Result detection judges：
Parting criterion is as follows, is shown in Table 1.
Table 1
The wherein risk of the normal homozygous AA and rs2014300 of the risk of rs10052657 homozygous CC, rs2274223
Homozygous GG, sequencing and typing result figure, is shown in Fig. 1-3.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be in other specific forms realized.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires to be limited rather than described above, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each implementation method is only wrapped
Containing an independent technical scheme, this narrating mode of specification is only that for clarity, those skilled in the art should
Specification an as entirety, the technical scheme in each embodiment can also be formed into those skilled in the art through appropriately combined
May be appreciated other embodiment.
Claims (5)
1. a kind of oesophagus cancer susceptibility gene detection and genotyping kit, it is characterised in that the kit includes DNA extracts reagents
Box, DNA cloning kit and sequencing kit；The DNA extraction kit uses the DP322 of Tiangeng biochemical technology Co., Ltd
Type product, the DNA cloning kit mainly includes amplimer, enzyme, PCR Buffer, dNTP Mixture, amplimer,
The amplimer is：1)F：5'-GGGGAATGAGACAGAGACCA-3', R：5'-ACCATTCCCAATGTCTGCTC-3',2)
F：5'-GGAAGCAGTGAGGTGCAGAGGT-3', R：5'-TCCACAACTGCAAAACGAAGAAA-3',3)F：5'-
TCTCAGCCCATGGTGAGCATTA-3',R：5'-CCCCCATATGGTATCCCCAAAG-3'；The enzyme is using precious bioengineering
The TaKaRa Taq of Co., LtdTMEnzyme；The sequencing kit is using American AB I companiesTerminator
v3.1Cycle Sequencing Kit。
2. oesophagus cancer susceptibility gene detection and genotyping kit according to claim 1, it is characterised in that the amplimer
It is that synthesis is designed based on gene PDE3D, PLCE1 and RUNX1.
3. a kind of application of oesophagus cancer susceptibility gene detection and genotyping kit as described in claim 1-2 is any, its feature exists
In concretely comprising the following steps：
(1) first, the sample returned will be gathered and will extract acquisition sample genomic dna according to DNA extraction kit；
(2) and then, by obtain sample genomic dna expanded using DNA cloning kit, the product after being expanded, with
The product after amplification is carried out into electrophoresis detection and purifying afterwards, product after purification is obtained；
(3) then, sequencing kit is added in product after purification carries out mulberry lattice sequencing reaction；
(4) last, the product after above-mentioned steps mulberry lattice sequencing reaction is terminated carries out ethanol/EDTA purifying, is loaded to after purification
In sequenator, parting judgement is carried out after sequence analysis.
4. the application of oesophagus cancer susceptibility gene detection and genotyping kit according to claim 3, it is characterised in that step
(1) sample is Oral Mucosal Cells in.
5. the application of oesophagus cancer susceptibility gene detection and genotyping kit according to claim 3, it is characterised in that step
(4) ethanol/EDTA purifying is specially and reaction plate is removed from PCR instrument in, 3800rpm centrifugations 1min；Plus 2.5 μ l0.125M
EDTA, short centrifugation；Stand 5min；Plus 20 μ l precooling absolute ethyl alcohols, membrana oralis is sealed, vibration is mixed；3800rpm, 4 DEG C of centrifugations
30min；It is centrifuged after terminating immediately, is inverted PCR plate low-speed centrifugal for a moment, rotating speed is no more than 800rpm；Plus 60 μ l 75% precooling
Ethanol, pad pasting, vibration is mixed；3800rpm, 4 DEG C of centrifugation 15min；It is centrifuged after terminating immediately, is inverted PCR plate low-speed centrifugal for a moment,
Rotating speed is no more than 800rpm；PCR instrument denaturation program processes 5min, or lucifuge stands 30min, and volatilize ethanol；Plus 10 μ l Hi-Di
Formamide, pad pasting, short centrifugation；95 DEG C of denaturation 5min；It is immediately placed in -20 DEG C of refrigerators.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110870879A (en) * | 2018-09-03 | 2020-03-10 | 石河子大学 | Use of PLCE1 inhibitors and recombinant p53 adenoviruses |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104178555A (en) * | 2013-05-23 | 2014-12-03 | 浙江爱易生物医学科技有限公司 | A kit for detecting esophagus cancer related risky genes |
| CN104894238A (en) * | 2015-05-04 | 2015-09-09 | 广州拓普基因科技有限公司 | Kit for auxiliary evaluation of esophagus cancer genetic risk of people to be detected |
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2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104178555A (en) * | 2013-05-23 | 2014-12-03 | 浙江爱易生物医学科技有限公司 | A kit for detecting esophagus cancer related risky genes |
| CN104894238A (en) * | 2015-05-04 | 2015-09-09 | 广州拓普基因科技有限公司 | Kit for auxiliary evaluation of esophagus cancer genetic risk of people to be detected |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110870879A (en) * | 2018-09-03 | 2020-03-10 | 石河子大学 | Use of PLCE1 inhibitors and recombinant p53 adenoviruses |
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