CN106978475A - A kind of Alzheimer's tumor susceptibility gene detection and genotyping kit and its application - Google Patents
A kind of Alzheimer's tumor susceptibility gene detection and genotyping kit and its application Download PDFInfo
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Abstract
The invention discloses a kind of Alzheimer's tumor susceptibility gene detection and genotyping kit and its application, the Alzheimer's tumor susceptibility gene detection and genotyping kit, CNTNAP2, CR1 and LRAT and Alzheimer's related gene pleomorphism site are detected, it will appreciate that inherent cause of the individual in terms of Alzheimer's, the relative risk of Alzheimer's morbidity is assessed, there is positive meaning for Alzheimer's early diagnosis, early treatment, early intervention.The diagnosis of Alzheimer's can also be aided in simultaneously, the rational use of medicines, aid forecasting offspring's risk etc. is instructed.
Description
Technical field
The present invention relates to a kind of inspection technology field, specifically a kind of Alzheimer's tumor susceptibility gene detection and genotyping examination
Agent box and its application.
Background technology
With the Human Genome Project (Human Genome Project, HGP) completion, 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 of the treatment long-standing problem mankind, cardiovascular and cerebrovascular disease, diabetes, nerve
New way is opened with the great Complex Diseases such as mental illness, the also industrialization for 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 this method research any it is assumed that being no longer limited to candidate gene or candidates region as association
Object is analyzed, but for 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, carry out the association study of multicenter, large sample, the gene verified repeatedly and disease, comprehensively timely disease
The correlated inheritance gene that disease occurs, develops and treated.
Have now been found that inherent cause, or its interaction between environmental factor take part in almost all of human diseases
Generating process.Some complicated diseases are frequently not that it occurs development and polygenes multidigit caused by term single gene mutation
Point variation is relevant, and the variation in these sites may be related to environmental factor, and these sites can improve or reduce individual and suffer from certain
The relative risk of disease, is referred to as the susceptibility loci of this kind of disease.According to substantial amounts of GWAS results, it has been found that and one proved
The susceptibility loci of serial complex disease has been widely used in field of gene detection.
Alzheimer's disease (Alzheimer's disease, AD) hides onset, with progressive cognition dysfunction, note
The nervous system degeneration disease for the age correlation for recalling power decline mainly to show, is most common type in senile dementia.Man
Female's both sexes are susceptible to this disease, with women slightly very.The sick illness rate exponentially rises in elderly population, and illness rate is about at 65 years old
1%, 40%-50% is reached during to 95 years old, has turned into the popular illness of ever-increasing threat public health at present.Late hair style AD
(late-onset Alzheimer's disease, LOAD) refers to that age of onset is more than the patient of 65 years old, more without family history.To the greatest extent
The pipe A D cause of disease and pathogenesis not yet understands, but effect of the inherent cause in AD morbidities is increasingly paid close attention to by people.
The disease can be caused by occurring mutation in three genes (APP, PSEN1, PSEN2), and APOE4 allele and the danger of illness
Increase is relevant.With human genome progress of research, it might have more genes related to the disease and be found.At present, should
Tested with genetic screening, the people at highest risk of Alzheimer's can be helped to be better equipped to and tackle what future may occur
Disease.
CNTNAP2 (Contactin-associated protein-like 2, contactin GAP-associated protein GAP sample albumen 2),
On No. 7 chromosomes, belong to neuronin family member, the memebrane protein of the gene code is main to express in nervous system,
Played a significant role during the great-jump-forward nerve impulse conduction of medullated nerve fibre cell.Logue et al. research
As a result show, CNTNAP2 gene pleiomorphism is AD important genetic risk factors, wherein rs10273775 allele with
Significantly correlated (Logue Mark W et al., the A comprehensive genetic association of LOAD onset risks
study of Alzheimer disease in African Americans.,Arch Neurol 68(12):1569-79)。
CR1 (Complement receptor1, complement receptor 1), also known as CRI or CD35, belong to complement and swash
Modulin family member living, is positioned at No. 1 chromosome " RCA clusters " region.CR1 is widely distributed, can be mediated as FI
The confactor of C3b cracking, accelerates C3 convertase decay, promotes phagocyte to be coated with particle to C3b/C4b and immune compound
Phagocytosis, activation and the sterilization metabolism of thing.Studies have found that trangenic mice overexpression C3 can reduce A β deposition and pathology
Effect.On the contrary, the expression for suppressing C3 convertase then accelerates A β deposition and neuron degeneration.A β by C3b mediate with it is red
The CR1 of cell surface is combined, and finally clears out of the circulatory system by red blood cell.LAMBERT et al. result of study shows,
CR1 gene pleiomorphism is the equipotential base of the important genetic risk factors that Caucasian suffers from AD, rs6656401 and rs3818361
Because being the hazards of delayed AD morbidities.An other genome research shows that rs3818361 is also Europe and America crowd
Occur AD hazards (HAROLD D, ABRAHAM R, HOLLINGWORTH P J.Genome-wide associatied
study identifies variants at CLU and PICALM associated with Alzheimer's
disease.Nat Genet,2009,41:1088-1093)。
LRAT (Lecithin Retinol Acyltransferase, lecithin-retinol acyltransferase), is positioned at
On the endoplasmic reticulum of pigment epithelial cell, its N-terminal is located at cytoplasm side, and C-terminal is located at endoplasmic reticulum tube chamber side, and its function is that catalysis is complete
Vitamin A1 aldehyde carries out esterification generation alltrans retinyl ester with phosphatidyl choline, therefore to internal Vitamin A Metabolism approach
Play an important role.Abraham R et al. research discovery, the rs727153 sites at LRAT transcripting start points upstream 13Kb
LOAD has significant correlation, and by more covering LRAT SNP site, further determined that LRAT is the new of LOAD
Candidate's tumor susceptibility gene (Abraham R, Moskvina V, Sims Ret al.A genome-wide association study
for late-onset Alzheimer's disease using DNA pooling.BMC Med Genomics,2008Sep
29；1:44).
Therefore, CNTNAP2, CR1 and LRAT and Alzheimer's related gene pleomorphism site are detected,
Inherent cause of the individual in terms of Alzheimer's is will appreciate that, the relative risk of Alzheimer's morbidity is assessed, it is right
There is positive meaning in Alzheimer's early diagnosis, early treatment, early intervention.Alzheimer ' can also be aided in simultaneously
The diagnosis of Mo's disease, instructs the rational use of medicines, aid forecasting offspring's risk etc..
The content of the invention
It is an object of the invention to provide a kind of Alzheimer's tumor susceptibility gene detection and genotyping kit and its application,
To solve the problems mentioned in the above background technology.
To achieve the above object, the present invention provides following technical scheme：
A kind of Alzheimer's tumor susceptibility gene detection and genotyping kit, 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 draws including amplimer, enzyme, PCR B uffer, dNTP Mixture, amplification
Thing, the amplimer is：1)F：5'-CCAGAGACAACCA GTGCTCA-3', R：5'-AAATCCACCCTGCTGTATGC-
3',2)F：5'-GGCTTAGGGTATGCTCACC A-3', R：5'-TGAGGGACTGCCTCTGTAGG-3',3)F：5'-
AGATGAAAAGGGGCCAGATT-3',R：5'-TATCCCTGGGCAGTTTTGTC-3'；The enzyme is limited using precious bioengineering
The TaKaRa Taq of companyTMEnzyme；The sequencing kit is using American AB I companiesTerminator
v3.1Cycle Sequ encing Kit。
It is used as further scheme of the invention：The amplimer is to be set based on gene C NTNAP2, CR1 and LRAT
Meter synthesis.
A kind of application of Alzheimer's tumor susceptibility gene detection and genotyping kit, is concretely comprised the following steps：
(1) first, the sample returned will be gathered and extracts acquisition sample genomic dna according to DNA extraction kit；
(2) then, sample genomic dna will be obtained to be expanded using DNA cloning kit, the production 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 and carries out mulberry lattice sequencing reaction；
(4) finally, product after above-mentioned steps mulberry lattice sequencing reaction is terminated carries out ethanol/EDTA purifying, after purification on
Sample carries out parting judgement into sequenator after sequence analysis.
It is used as further scheme of the invention：Sample is Oral Mucosal Cells in step (1).
It is used as further scheme of the invention：Ethanol/EDTA purifying is specially that 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；Centrifuge after terminating immediately, be inverted PCR plate low-speed centrifugal a moment, turn
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, PCR plate low-speed centrifugal a moment is inverted, rotating speed is no more than 800rpm；PCR instrument denaturation program handles 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 Alzheimer's tumor susceptibility gene detection and genotyping kit, to CNTNAP2, CR1 and
LRAT and Alzheimer's related gene pleomorphism site are detected, will appreciate that individual in Alzheimer's side
The inherent cause in face, assesses the relative risk of Alzheimer's morbidity, for Alzheimer's early diagnosis, early stage
Treatment, early intervention have positive meaning；The diagnosis of Alzheimer's can also be aided in simultaneously, the rational use of medicines is instructed, helped
Help prediction offspring's risk etc.；This patent product uses the goldstandard of field of gene detection --- and mulberry lattice PCR sequencing PCR carries out gene
Parting；Mulberry lattice PCR sequencing PCR is that the oligonucleotide fragment for terminating at target sequence specific site is produced by controlling DNA synthesis；It is first
First, the Oligonucleolide primers of synthesis are annealed with single-stranded DNA profiling, set up four kinds of different sequencing reactions, in each reaction
All contain archaeal dna polymerase and four kinds of normal dNTP；In addition, also 3'-H replaces common deoxyribose containing a small amount of
3'-OH 2', 3'-ddNTP；If ddNTP is incorporated into the DNA in extension, follow-up dNTP shapes will be blocked due to lacking 3'-OH
Into phosphodiester bond, DNA further extension is terminated；Using four kinds of different ddNTP, the oligonucleotides of generation is terminated at
The position that each A, C, G or T are occupied on template strand, the oligonucleotides that four kinds of reactions are produced is added in sequenator, due to
Oligonucleotide fragment it is of different sizes, the speed of swimming is just different, what the image controller of sequenator passed through according to oligonucleotides
Time sequencing, just can detect new synthesis chain 5' to 3' sequence information.
Brief description of the drawings
Fig. 1 is heterozygous AG sequencing and typing result figures of the invention；
Fig. 2 is normal homozygous CT sequencing and typings result figure of the invention；
Fig. 3 is heterozygous AG sequencing and typing result figures of the invention.
Embodiment
The technical scheme of this patent is described in more detail with reference to embodiment.
A kind of Alzheimer's tumor susceptibility gene detection and genotyping kit, 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'-CCAGAGACAACCAGTGCTCA-3'(SEQ ID NO.1), R：5'-
AAATCCACCCTGCTGTATGC-3'(SEQ ID NO.2),2)F：5'-GGCTTAGGGTATGCTCACCA-3'(SEQ ID
NO.3), R：5'-TGAGGGACTGCCTCTGTAGG-3'(SEQ ID NO.4),3)F：5'-AGATGAAAAGGGGCCAGATT-
3'(SEQ ID NO.5),R：5'-TATCCCTGGGCAGTTTTGTC-3'(SEQ ID NO.6)；The enzyme is using precious biological work
The TaKaRa Taq of journey Co., LtdTMEnzyme；The sequencing kit is using American AB I companiesTerminat or
v3.1Cycle Sequencing Kit。
The amplimer is to be designed synthesis based on gene C NTNAP2, CR1 and LRAT；Wherein, the target of detection
SNP site include rs10273775, rs3818361 and rs727153, this 3 sites respectively be located at gene C NTNAP2, CR1 and
LRAT。
A kind of application of Alzheimer's tumor susceptibility gene detection and genotyping kit, is concretely comprised the following steps：
(1) first, the sample returned will be gathered and extracts acquisition sample genomic dna according to DNA extraction kit；
(2) then, sample genomic dna will be obtained to be expanded using DNA cloning kit, the production 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 and carries out mulberry lattice sequencing reaction；
(4) finally, product after above-mentioned steps mulberry lattice sequencing reaction is terminated carries out ethanol/EDTA purifying, after purification on
Sample carries out parting judgement into sequenator after sequence analysis.
Wherein：
Sample is Oral Mucosal Cells in step (1).
Expanded in step (2) using DNA cloning kit, 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：The Ago-Gel of preparation 1%：Claim
10 × TAE heating that a certain amount of agar Icing Sugar adds corresponding volume melts agarose, add 10000 after it melts completely ×
Gold ViewⅠ.After gelling is solid, 5 μ l PCR primer is taken to add after Loadi ng Buffer mixing, 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
Expand 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 that 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；Centrifuge after terminating immediately, be inverted PCR plate low-speed centrifugal 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 handles 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.
As a result detection judges：
Parting criterion is as follows, is shown in Table 1.
Table 1
The wherein normal pure type CT and rs727153 of rs10273775 heterozygous AG, rs3818361 heterozygous AG, is surveyed
Sequence genotyping 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 realized in other specific forms.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 is required rather than described above is limited, 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 embodiment is only wrapped
Containing an independent technical scheme, this narrating mode of specification is only that for clarity, those skilled in the art should
Using specification as an entirety, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
It may be appreciated other embodiment.
Claims (5)
1. a kind of Alzheimer's tumor susceptibility gene detection and genotyping kit, it is characterised in that the kit includes DNA
Extracts kit, DNA cloning kit and sequencing kit；The DNA extraction kit uses the limited public affairs of Tiangeng biochemical technology
The DP322 type products of department, the DNA cloning kit mainly include amplimer, enzyme, PCR Buffer, dNTP Mixture,
Amplimer, the amplimer is：1)F：5'-CCAGAGACAACCAGTGCTCA-3', R：5'-
AAATCCACCCTGCTGTATGC-3',2)F：5'-GGCTTAGGGTATGCTCACCA-3', R：5'-
TGAGGGACTGCCTCTGTAGG-3',3)F：5'-AGATGAAAAGGGGCCAGATT-3',R：5'-
TATCCCTGGGCAGTTTTGTC-3'；The enzyme uses the TaKaRa Taq of precious bioengineering Co., LtdTMEnzyme；The sequencing
Kit is using American AB I companiesTerminator v3.1Cycle Sequencing Kit。
2. Alzheimer's tumor susceptibility gene detection and genotyping kit according to claim 1, it is characterised in that described
Amplimer is to be designed synthesis based on gene C NTNAP2, CR1 and LRAT.
3. a kind of application of Alzheimer's tumor susceptibility gene detection and genotyping kit as described in claim 1-2 is any,
Characterized in that, concretely comprising the following steps：
(1) first, the sample returned will be gathered and extracts acquisition sample genomic dna according to DNA extraction kit；
(2) then, sample genomic dna will be obtained to be expanded using DNA cloning kit, the product after being expanded, with
The product after amplification is subjected to electrophoresis detection and purifying afterwards, product after purification is obtained；
(3) then, sequencing kit is added in product after purification and carries out mulberry lattice sequencing reaction；
(4) finally, 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 Alzheimer's tumor susceptibility gene detection and genotyping kit according to claim 3, its feature exists
In sample is Oral Mucosal Cells in step (1).
5. the application of Alzheimer's tumor susceptibility gene detection and genotyping kit according to claim 3, its feature exists
In ethanol/EDTA purifying is specially that 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
Centrifuge 30min；Centrifuge after terminating immediately, be inverted PCR plate low-speed centrifugal a moment, rotating speed is no more than 800rpm；Plus 60 μ l 75%
Pre-cooled ethanol, pad pasting, vibration is mixed；3800rpm, 4 DEG C of centrifugation 15min；Centrifuge after terminating immediately, be inverted PCR plate low-speed centrifugal
For a moment, rotating speed is no more than 800rpm；PCR instrument denaturation program handles 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.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112151113A (en) * | 2020-09-27 | 2020-12-29 | 类承斌 | Early screening method for late Alzheimer disease associated gene variation |
Citations (1)
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| CA2766876A1 (en) * | 2009-07-03 | 2011-01-06 | University College Cardiff Consultants Limited | Variants of clu/apoj, picalm, abca7, cr1 and bin1 gene loci and the ms4a gene cluster for the diagnosis of alzheimer's disease |
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2016
- 2016-08-17 CN CN201610676427.2A patent/CN106978475A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2766876A1 (en) * | 2009-07-03 | 2011-01-06 | University College Cardiff Consultants Limited | Variants of clu/apoj, picalm, abca7, cr1 and bin1 gene loci and the ms4a gene cluster for the diagnosis of alzheimer's disease |
Non-Patent Citations (4)
| Title |
|---|
| DR. MARK W等: ""A Comprehensive Genetic Association Study of Alzheimer Disease in African Americans"", 《ARCH NEUROL》 * |
| NILÜFER ERTEKIN-TANER等: ""Genetics of Alzheimer disease in the pre- and post-GWAS era"", 《ALZHEIMER’S RESEARCH & THERAPY》 * |
| 刘艳芳等: "《临床病毒学检验》", 30 April 2009 * |
| 朱伟锋等: ""碱裂解法快速提取口腔拭子DNA对CHRNA3基因多态性的研究"", 《重庆医学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112151113A (en) * | 2020-09-27 | 2020-12-29 | 类承斌 | Early screening method for late Alzheimer disease associated gene variation |
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Application publication date: 20170725 |