CN106834431A - A kind of pancreas cancer susceptibility gene detection and genotyping kit and its application - Google Patents
A kind of pancreas cancer susceptibility gene detection and genotyping kit and its application Download PDFInfo
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Abstract
The invention discloses a kind of pancreas cancer susceptibility gene detection and genotyping kit and its application, the pancreas cancer susceptibility gene detection and genotyping kit, to TFF1, TFRC, DAB2, tri- are detected with the pleomorphism site of human pancreatic cancer-associated genes, will appreciate that the individual inherent cause in terms of cancer of pancreas, the relative risk of assessment cancer of pancreas morbidity, has positive meaning for cancer of pancreas early diagnosis, early treatment, early intervention.The diagnosis of cancer of pancreas 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 pancreas 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.
Cancer of pancreas is a kind of high digestive system tumor of grade of malignancy, the concealment of its early symptom, during discovery mostly be late period,
Resection Rate is low, poor prognosis.With human lives' custom and the change of dietary structure, the incidence of disease of cancer of pancreas is in the world
Inside rise year by year.Cancer of pancreas morbidity is the result that inherent cause and environmental factor interact, and it may be with gene mutation, gene
The genetic predisposition that the factors such as polymorphism, epigenetic cause improves relevant.Some hazards related to cancer of pancreas are such as
Smoking, obesity, drink, chronic pancreatitis, diabetes etc. are increasingly received significant attention.The incidence of disease of cancer of pancreas exists in recent years
In ascendant trend year by year in global range, the whole world in 2010 newly sends out cancer of pancreas case and is expected to have reached 293 541
(Jacques Ferlay,Shin H R,Bray F,et al.Estimates of worldwide burden of cancer
in 2008:GLOBOCAN2008[J].International Journal of Cancer Journal International
Du Cancer,2010,127(12):2893-917.)。
Trefoil factor family (Trefoil Family Factor, TFF) contains a similar structures domain, by six and half
Cystine residue is folded into clover proterties by disulfide bond.TFF2 is first trefoil factor family member being found,
Pig Islet purification insulin course is found.The TFF1 initially discoveries in estrogen inducing mammary cancer cell, are named as people again
Breast cancer related peptide 2 (hpS2), TFF1 trefoil peptide domains contain 42 amino acid residues (Thr6to Pro47), and TFF1 passes through
Cys58 forms intermolecular disulfide bond and forms homodimer or the compound of 25KD is collectively forming with other albumen.Genetic chip
Analysis shows, in human body pancreatic cancer cell TFF1 expression quantity be significantly larger than normal pancreatic cells (Arumugam T, Hwang R,
Todd M,et al.TFF1is over expressed early in pancreatic cancer in response to
cancer-stromal interactions and stimulates cancer cell invasiveness.[J]
.Cancer Research,2007,67.)。
TfR (TFRC) is the required albumen of the absorption and regulation cell growth for participating in iron.In Jie of TFRC
Lead down (Qian Z M, Li H, Sun H, et al.Targeted drug delivery via the transferrin
receptor-mediated endocytosis pathway.[J].Pharmacolog ical Reviews,2003,54
(4):561-87.), iron could be by cellular uptake, the synthesis of DNA, the periodicity propagation of cell, immunological regulation and intracellular electronics
The transmission of respiratory chain could be normally orderly carrying out.Because TFRC has weight to the synthesis of DNA TfRs and cell propagation
The influence wanted, therefore in the research of tumour cell metabolism, TFRC has been a great concern.Research shows, with pancreas just
Often tissue is compared, and the TfR expression of tumour cell increases (Ryschich E, Huszty G, Knaebel H P, et
al.Transferrin receptor is a marker of malignant phenotype in human
pancreatic cance r and in neuroendocrine carcinoma of the pancreas[J]
.European Journal of Cancer,2004,40(9):1418-1422.), this is probably the demand to iron due to them
Increase, because in the cell of quick division, iron is the confactor of the ribonucleotide reductase for participating in DNA synthesis.
DAB2 genes are one of two kinds of Disabled of class of mammals Drosophila, most early in macrophage colony stimulatory factor
There is phospholipoprotein to act on and be found in 1 (CSF-1) signal transduction pathway.The DAB2 genes of people are located at No. five length of chromosome
On arm (5p12), there are two kinds of shear patterns：Coding p96, p67 albumen.DA B2 mrna length 40kb, by 15 extrons and 14
Individual introne composition, produces mRNA (S heng Z, Smith E R, He J, the et al.Chromosomal of 3.6kb
location of murine Disabled-2gene and structural comparison with its human
ortholog[J].Gene,2001,268(1–2):31-39.；Dong-Hua Yang M.D.Ph.D,Smith E R,Cohen
C,et al.Mole cular events associated with dysplastic morphologic
transformation an d initiation of ovarian tumorigenicity[J].Cancer,2002,94
(9):2380-92.).DAB2 contains three functional areas：The phosphotyrosine land (PTB) at N- ends, phosphatizing area (P ID)
With the C- petiolareas (PRD) containing abundant proline, molecular regulation effect is respectively provided with.Research shows, the expression inhibiting high of DA B2
Growth (Genest D.DOC-2/hDab2, a candidate tumor suppressor gene involved of cancer cell
in the development of gestational trophoblas tic diseases[J].Oncogene,1998,17
(4):419-24.), illustrate that it is a tumor suppressor gene.There is researcher to find simultaneously, rat Dab2 protein subunits p82's
Expression increase can strengthen cell send out ability (Chetrit D, Ziv N, Ehrlich M.Dab2regulates clathrin
assembly and cell spreading.[J].Biochemical Journal,2009,418(3):701-15.), thus it is speculated that
May be relevant with cancer metastasis.
Therefore, to TFF1, TFRC, DAB2 tri- detected with the pleomorphism site of human pancreatic cancer-associated genes, will appreciate that
The individual inherent cause in terms of cancer of pancreas, the relative risk of assessment cancer of pancreas morbidity, for cancer of pancreas early diagnosis, controls in early days
Treat, early intervention has positive meaning.The diagnosis of cancer of pancreas can also be aided in simultaneously, instruct the rational use of medicines, aid forecasting offspring
Risk etc..
The content of the invention
It is an object of the invention to provide a kind of pancreas 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 pancreas 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'-TCCACAGAGCAGCCCATGTTTA-3', R：5'-TGGGCCGTAACAGTGGTATCTG-3',2)F：
5'-AACAAATGGGCCTTGGTTTA-3', R：5'-AAGGGAAAAGGGCTGCTTTA-3',3)F：5'-
TGAAGACTGAAAGCAGGGGAGTG-3',R：5'-GCTTGGATAACCCTGGGGATCA-3'；The enzyme is using precious biology work
The TaKaRa Taq of journey Co., LtdTMEnzyme；The sequencing kit is using American AB I companiesTerminator
v3.1Cycle Seq uencing Kit。
As further scheme of the invention：The amplimer is to be designed based on gene TFF1, TFRC and DAB2
Synthesis.
A kind of application of pancreas 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 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
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 30min；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 5min, 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 pancreas cancer susceptibility gene detection and genotyping kit, to TFF1, TFRC, DAB2 tri- and pancreas
The pleomorphism site of gland cancer related gene detected, will appreciate that the individual inherent cause in terms of cancer of pancreas, assesses cancer of pancreas
The relative risk of morbidity, has positive meaning for cancer of pancreas early diagnosis, early treatment, early intervention；Simultaneously can also be auxiliary
The diagnosis of cancer of pancreas is helped, the rational use of medicines, aid forecasting offspring's risk etc. is instructed；This patent product uses field of gene detection
Goldstandard --- mulberry lattice PCR sequencing PCR carries out Genotyping；Mulberry lattice PCR sequencing PCR is to produce to terminate at by controlling the synthesis of DNA
The oligonucleotide fragment of target sequence specific site；First, the Oligonucleolide primers of synthesis are annealed with single-stranded DNA profiling, are set up
Four kinds of different sequencing reactions, archaeal dna polymerase and four kinds of normal dNTP are all contained in each reaction；In addition, also contain
There is a small amount of 2', 3'-ddNTP for thering is 3'-H to replace common deoxyribose 3'-OH；If ddNTP is incorporated into the DNA in extending,
Follow-up dNTP formation phosphodiester bond will be blocked due to lacking 3'-OH, the further extension of DNA is terminated；Using four kinds not
Same ddNTP, the oligonucleotides of generation is terminated at each A, the position that C, G or T are occupied on template strand, by four kinds of reactions
The oligonucleotides of generation is added in sequenator, of different sizes due to oligonucleotide fragment, and the speed of swimming is just different, sequenator
Image controller the sequence information of new synthesis chain 5' to 3' can sequentially, be just detected according to oligonucleotides elapsed time.
Brief description of the drawings
Fig. 1 is the homozygous AA sequencing and typings result figure of risk of the invention；
Fig. 2 is normal homozygous CC sequencing and typings result figure of the invention；
Fig. 3 is the homozygous AA 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 pancreas 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'-TCCACAACTGCAAAACGAAGAAA-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 TFF1, TFRC and DAB2；Wherein, the target of detection
SNP site include rs1547374, rs4927850 and rs2255280, this 3 sites respectively be located at gene TFF1, TFRC and
DAB2。
A kind of application of pancreas 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 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
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:1time
95℃3minutes
Stage 2:PCR reaction
Repeat:30cycles
95℃5second
55℃30second
72℃1minutes
Stage 3:Final extension
72℃5minutes。
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 Loading 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℃30minutes
75℃15minutes。
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：
Stage1:1time
95℃2minutes
Stage 2:25cycles
95℃10second
50℃5second
60℃4minutes。
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 CC and rs2255280 of the risk of rs1547374 homozygous AA, rs4927850
Homozygous AA, 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 pancreas 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'-TCCACAGAGCAGCCCATGTTTA-3', R：5'-TGGGCCGTAACAGTGGTATCTG-
3',2)F：5'-AACAAATGGGCCTTGGTTTA-3', R：5'-AAGGGAAAAGGGCTGCTTTA-3',3)F：5'-
TGAAGACTGAAAGCAGGGGAGTG-3',R：5'-GCTTGGATAACCCTGGGGATCA-3'；The enzyme is using precious biology work
The TaKaRa Taq of journey Co., LtdTMEnzyme；The sequencing kit is using American AB I companiesTerminat or
v3.1Cycle Sequencing Kit。
2. pancreas 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 TFF1, TFRC and DAB2.
3. a kind of application of pancreas 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 pancreas 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 pancreas 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 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 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 (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110819713A (en) * | 2019-11-21 | 2020-02-21 | 中国医学科学院肿瘤医院 | Application of SNP marker in pancreatic cancer prognosis |
| CN111321223A (en) * | 2019-11-01 | 2020-06-23 | 苏州乾康基因有限公司 | Primer and probe for detecting pancreatic cancer related site polymorphism based on multiple PCR flight nucleic acid mass spectrometry technology and application of primer and probe |
-
2016
- 2016-08-17 CN CN201610677867.XA patent/CN106834431A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111321223A (en) * | 2019-11-01 | 2020-06-23 | 苏州乾康基因有限公司 | Primer and probe for detecting pancreatic cancer related site polymorphism based on multiple PCR flight nucleic acid mass spectrometry technology and application of primer and probe |
| CN110819713A (en) * | 2019-11-21 | 2020-02-21 | 中国医学科学院肿瘤医院 | Application of SNP marker in pancreatic cancer prognosis |
| CN110819713B (en) * | 2019-11-21 | 2020-08-11 | 中国医学科学院肿瘤医院 | Application of SNP marker in pancreatic cancer prognosis |
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