CN106978476A - A kind of parkinson's syndrome tumor susceptibility gene detection and genotyping kit and its application - Google Patents
A kind of parkinson's syndrome tumor susceptibility gene detection and genotyping kit and its application Download PDFInfo
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Abstract
The invention discloses a kind of parkinson's syndrome tumor susceptibility gene detection and genotyping kit and its application, the parkinson's syndrome tumor susceptibility gene detection and genotyping kit, SNCA, LRRK2, HLA DRA tri- and Parkinson's disease related gene pleomorphism site are detected, it will appreciate that inherent cause of the individual in terms of Parkinson's disease, the relative risk of Parkinson's disease morbidity is assessed, there is positive meaning for op parkinson's disease early diagnosis, early treatment, early intervention.The diagnosis of Parkinson's disease 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 parkinson's syndrome tumor susceptibility gene detection and genotyping reagent
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.
Parkinson's (Parkinson's disease, PD) are the second largest common nerves after Alzheimer disease
System degenerative disease, its pathological characteristic is the degeneration of dopaminergic neurons and survived neuronal of progressive in striatum substantia nigra
Interior albumen aggregation, i.e. Lewy body.PD mainly causes dyskinesia, including bradykinesia, static tremor, myotonia and appearance
Gesture abnormal gait.This disease progression can be slowed down currently without medicine, the medicine such as levodopa is only capable of relief of symptoms.The PD cause of disease is still
It is unclear, but h and E factor is considered as common participation morbidity.Effect of the inherent cause in PD morbidities is affirmative, because
It is found to have more than ten of Disease-causing gene at present in Familial Occurrence PD cases, such as SNCA, Parkin, LRRK2, UCH-
L1, PINK1 etc..The mutation of these Disease-causing genes causes the PD of monogenic inheritance form.But the PD cases of monogenic inheritance form
10% or so of all cases is only accounted for, most cases belong to sporadic PD (Joshua M.Shulman, Philip L.De
Jager,and Mel B.Feany.Parkinson's Disease:Genetics and Pathogenesis[J].Annual
Review of Pathology,2011,6:193-222.)。
Synapse nucleoprotein (alpha-synuclein, SNCA) gene is the first pathogenic base found in familial PD
Cause, SNCA gene mutations cause autosomal dominant PD.Albumen-a-synuclein albumen coded by it is Louis
The chief component of corpusculum.The point mutation of SNCA genes, three times repeat mutation and two times of repetition mutation in familial PD cases
In be found, and carry repetition mutation patient after death brain tissue in wild type SNCA expression increase (Singleton A
B,Farrer M,Johnson J,et al.alpha-Synuclein locus triplication causes
Parkinson's disease.[J].Science,2003,302(5646):841-841.；Chartier-Harlin M C,
Kachergus J,Roumier C,et al.α-synuclein locus duplication as a cause of
familial Parkinson's disease[J].Lancet,2004,364(9440):1167-9.).This repetition suggested that
Neurotoxicity may be produced by overexpression to dopaminergic neuron by being mutated SNCA, and cause familial PD.SNCA genes
A familial PD Disease-causing gene is not only, and is a sporadic PD tumor susceptibility gene, SNCA is in sporadic PD patient
Expression increase (Chiba-Falek O, Lopez G J, Nussbaum R L.Levels of alpha- in brain tissue
synuclein mRNA in sporadic Parkinson disease patients[J].Movement Disorders
Official Journal of the Movement Disorder Society,2006,21(10):1703-8.).It is located at
A SNCA gene transcription start sites upstream 10kb microsatellite dinucleotide repetitive sequence polymorphic-Rep1 of allele length,
(Linnertz C, Saucier L, Ge D, et related to sporadic PD neurological susceptibility are confirmed by multiple researchs
al.Genetic regulation of alpha-synuclein mRNA expression in various human
brain tissues.[J].Plos One,2009,4(10)::e7480.)。
LRRK2 gene mutations are most common inherent causes in autosomal dominant inheritance PD, account for white race crowd man
Race's property PD 6% and sporadic PD 2% (Tian Y Y, Tang C J, Wu J, et al.Parkinson's disease
in China[J].Neurological Sciences Official Journal of the Italian
Neurological Society&of the Italian Society of Clinical Neurophysiology,2011,
32(1):23-30.；Pan F,Dong H,Ding H,et al.SNP rs356219of theα-synuclein(SNCA)
gene is associated with Parkinson's disease in a Chinese Han population.[J]
.Parkinsonism&Related Disorders,2012,18(5):632-634.).The research of domestic or even Asia is more
With gene mutation and PD to associate Journal of Sex Research in the majority, the comprehensive visible LRRK2 of these results of study multiple Asias (including in
State, Japan, South Korea etc.) sporadic PD patient in it is most common, it is concerned at most (Sidhu A, Wersinger C, Vernier
P.α-Synuclein regulation of the dopaminergic transporter:a possible role in
the pathogenesis of Parkinson's disease[J].Febs Letters,2004,565(1-3):1–5.；
Tay S P,Lim G G Y.Parkin and Parkinson's Disease[J].Etiology and
Pathophysiology of Parkinson's Disease,2011,10:47-70.)。
Meanwhile, the research such as Hamza T H is it has also been found that HLA-DRA (Hamza T H, Zabetian related to Parkinson's disease
C P,Tenesa A,et al.Common genetic variation in the HLA region is associated
with late-onset sporadic Parkinson's disease[J].Nature Genetics,2010,42(9):
781-5.)
Therefore, SNCA, LRRK2, HLA-DRA tri- and Parkinson's disease related gene pleomorphism site are examined
Survey, will appreciate that inherent cause of the individual in terms of Parkinson's disease, assess the relative risk of Parkinson's disease morbidity, for handkerchief gold
Sen Shi disease early diagnosis, early treatment, early intervention have positive meaning.The diagnosis of Parkinson's disease can also be aided in simultaneously,
Instruct 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 parkinson's syndrome tumor susceptibility gene detection and genotyping kit and its application, with
The problem of solving to propose in above-mentioned background technology.
To achieve the above object, the present invention provides following technical scheme：
A kind of parkinson's syndrome tumor susceptibility gene detection and genotyping kit, the kit include DNA extraction kit,
DNA cloning kit and sequencing kit；The DNA extraction kit uses the DP322 types of Tiangeng biochemical technology Co., Ltd
Product, the DNA cloning kit mainly includes amplimer, enzyme, PCR Buf fer, dNTP Mixture, amplimer,
The amplimer is：1)F：5'-AGGTAACCACCGTGT GGGTTTG-3', R：5'-
TGATCCTGTCAGGGGTTTGTTTT-3',2)F：5'-ACTTTCCACACTGCTG CACAGG-3', R：5'-
TTCTCATCCAAGTGGGATGTGTTT-3',3)F：5'-CAGATCCTGGGTAGGC GTTTTG-3',R：5'-
TATGCTCAGGCTTGGGCAATTT-3'；The enzyme uses the TaKaRa Taq of precious bioengineering Co., LtdTMEnzyme；It is described to survey
Sequence kit is using American AB I companiesTerminator v3.1Cycle Sequencing Kit。
It is used as further scheme of the invention：The amplimer is to be carried out based on gene HLA-DRA, SNCA and LRR K2
Design synthesis.
A kind of application of parkinson's syndrome 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 genome DN A 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 30mi n；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 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 parkinson's syndrome tumor susceptibility gene detection and genotyping kit, to SNCA, LRRK2, HLA-
DRA tri- and Parkinson's disease related gene pleomorphism site are detected, will appreciate that individual in terms of Parkinson's disease
Inherent cause, assesses the relative risk of Parkinson's disease morbidity, dry for op parkinson's disease early diagnosis, early treatment, early stage
There is positive meaning in advance；The diagnosis of Parkinson's disease can also be aided in simultaneously, the rational use of medicines, aid forecasting offspring illness wind is instructed
Danger etc.；This patent product uses the goldstandard of field of gene detection --- and mulberry lattice PCR sequencing PCR carries out Genotyping；Mulberry lattice PCR sequencing PCR
It is that the oligonucleotide fragment for terminating at target sequence specific site is produced by controlling DNA synthesis；First, the few nucleosides of synthesis
Sour primer is annealed with single-stranded DNA profiling, sets up four kinds of different sequencing reactions, all contains archaeal dna polymerase in each reaction
With four kinds of normal dNTP；In addition, also there are the 2', 3'- that 3'-H replaces common deoxyribose 3'-OH containing a small amount of
ddNTP；If ddNTP is incorporated into the DNA in extension, follow-up dNTP formation phosphodiester bond will be blocked due to lacking 3'-OH,
DNA further extension is terminated；Using four kinds of different ddNTP, the oligonucleotides of generation is terminated at each A, C, G or
The position that T is occupied on template strand, the oligonucleotides that four kinds of reactions are produced is added in sequenator, due to oligonucleotide fragment
Of different sizes, the speed of swimming is just different, and the image controller of sequenator can be with according to oligonucleotides elapsed time order, just
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 heterozygous CT sequencing and typing result figures of the invention；
Fig. 3 is heterozygous CG 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 parkinson's syndrome tumor susceptibility gene detection and genotyping kit, the kit include DNA extraction kit,
DNA cloning kit and sequencing kit；The DNA extraction kit uses the DP322 types of Tiangeng biochemical technology Co., Ltd
Product, the DNA cloning kit mainly includes amplimer, enzyme, PCR Buf fer, dNTP Mixture, amplimer,
The amplimer is：1)F：5'-AGGTAACCACCGTGT GGGTTTG-3'(SEQ ID NO.1), R：5'-
TGATCCTGTCAGGGGTTTGTTTT-3'(SEQ ID N O.2),2)F：5'-ACTTTCCACACTGCTGCACAGG-3'(SEQ
ID NO.3), R：5'-TTCTCAT CCAAGTGGGATGTGTTT-3'(SEQ ID NO.4),3)F：5'-
CAGATCCTGGGTAGGCGTTTTG-3'(SEQ ID NO.5),R：5'-TATGCTCAGGCTTGGGCAATTT-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
DepartmentTerminator v3.1Cycle Sequencing Kit。
The amplimer is to be designed synthesis based on gene HLA-DRA, SNCA and LRRK2；Wherein, the mesh of detection
Marking SNP site includes rs3129882, rs356220 and rs1491942, and this 3 sites are located at gene HLA-DRA, SNCA respectively
And LRRK2.
A kind of application of parkinson's syndrome 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 genome DN A 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 heterozygous CT and rs1491942 of rs3129882 heterozygous AG, rs356220 heterozygous CG, sequencing
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 parkinson's syndrome tumor susceptibility gene detection and genotyping kit, it is characterised in that the kit includes DNA and carried
Take kit, DNA cloning kit and sequencing kit；The DNA extraction kit uses Tiangeng biochemical technology Co., Ltd
DP322 type products, the DNA cloning kit mainly include amplimer, enzyme, PCR Buffer, dNTP Mixture, expand
Increase primer, the amplimer is：1)F：5'-AGGTAACCACCGTGTGGGTTTG-3', R：5'-
TGATCCTGTCAGGGGTTTGTTTT-3',2)F：5'-ACTTTCCACACTGCTGCACAGG-3', R：5'-
TTCTCATCCAAGTGGGATGTGTTT-3',3)F：5'-CAGATCCTGGGTAGGCGTTTTG-3',R：5'-
TATGCTCAGGCTTGGGCAATTT-3'；The enzyme uses the TaKaRa TaqTM enzymes of precious bioengineering Co., Ltd；It is described to survey
Sequence kit is using American AB I companies Terminator v3.1Cycle Sequencing Kit。
2. parkinson's syndrome tumor susceptibility gene detection and genotyping kit according to claim 1, it is characterised in that the expansion
Increase primer to be designed synthesis based on gene HLA-DRA, SNCA and LRRK2.
3. a kind of application of parkinson's syndrome tumor susceptibility gene detection and genotyping kit as described in claim 1-2 is any, its
It is characterised by, concretely comprises the following steps：
(1) first, the sample returned will be gathered and extracts acquisition sample genome DN A 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 parkinson's syndrome tumor susceptibility gene detection and genotyping kit according to claim 3, it is characterised in that
Sample is Oral Mucosal Cells in step (1).
5. the application of parkinson's syndrome tumor susceptibility gene detection and genotyping kit according to claim 3, it is characterised in that
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 (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113046431A (en) * | 2021-03-16 | 2021-06-29 | 杭州惠煜医疗科技有限公司 | Detection method for acquiring genetic Parkinson disease pathogenic gene mutation related information |
| CN114941026A (en) * | 2022-06-06 | 2022-08-26 | 苏州珈安华钰生物科技有限公司 | A molecular marker related to Parkinson's disease, its detection kit and application |
| CN115820743A (en) * | 2022-10-13 | 2023-03-21 | 浙江双糖生物科技有限公司 | Parkinson gene editing pig neural stem cell model, construction method and application |
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