CN109468388A - A kind of primer sets and detection kit of amplification and locomitivity related gene - Google Patents
A kind of primer sets and detection kit of amplification and locomitivity related gene Download PDFInfo
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Abstract
The present invention provides the primer sets and detection kit of a kind of amplification and locomitivity related gene, are related to the applied technical field of SNP.Primer sets of the present invention include: specific primer and Single base extension primer；The nucleotide sequence of the specific primer is as shown in NO.1~52 SEQ ID；The nucleotide sequence of the Single base extension primer is as shown in NO.53~78 SEQ ID.The amplimer and extension primer that the present invention designs can detect 26 SNP sites in the same reaction, and guide suitable exercise for developing locomitivity detection kit.In terms of exercise guidance, genetic test of the invention can be appreciated that the abilities such as endurance, explosive force, the strength of human body, provide the reference frame of science for suitable exercise.The present invention will move relevant gene association detection, and one-time detection goes out all projects, be that detection gene covering surface is most comprehensive in the product of locomitivity genetic test so far.
Description
Technical field
The invention belongs to the applied technical fields of SNP, and in particular to a kind of primer of amplification and locomitivity related gene
Group and detection kit.
Background technique
Locomitivity refers to the ability that people are engaged in various body movements, including the activity that sustains life it is indispensable walk,
It the basic capacities such as runs, jump, throwing, climbing, climbing, rolling, turning over and participating in athletic training or peculair motion ability that match has, being human body
The general performance of form, quality, function, technical ability and mental power etc..Someone research point out, become elite heredity and
The caused effect of the training day after tomorrow, the former accounts for 2/3, and the latter accounts for 1/3.Molecule genetics research repeatedly confirms to transport with the mankind in recent years
The relevant such as muscle strength of kinetic force, endurance, equilibrant force, harmony, flexibility, aerobic sport ability, anaerobic sport capacity,
Training response and the occurrence and development of fatigue etc. have important Biological background.It is roughly the same in environmental condition and training method
In the case where, Different Individual in terms of locomitivity, physical efficiency, perception and training all there is apparent difference,
Wherein the most fundamental reason is that different individual inheritance backgrounds is different, that is, there is the difference of movement talent.It is examined by gene
Survey can understand relevant to the movement connate ability of individual, thus for targeted selection and cultivating people of ability provide scientific reference according to
According to.
The superiority and inferiority of one individual sports heredodiathesis determines the upper limit of its locomitivity and the degree of trainability.SNP is being visited
Rope human health and cancer diagnosis play key player, so far it has been found that the gene of a chromosome and 18 mitochondrias more than 200
Polymorphic site and the muscle strength of the mankind, physical activity level, endurance level are related to Training Method.Past has developed
The method of a variety of detection gene pleiomorphisms out, for the ability of predicted motion.Wherein than being more comprehensively exactly patent of invention
(CN201711219812.5), 5 gene mononucleotide polymorphism sites relevant to movement are covered, which disclose one
For kind for detecting the primer sets of movement gene SNP, the method used is capillary electrophoresis, but detection gene is not comprehensive, DNA mould
Plate input amount is big, and time-consuming.Patent of invention (CN201310115932.6) discloses a kind of prediction excellent ice-snow sportsmen spring
The molecular biology method of potential, this method only detect ACTN3 and ACE2 gene, and detection gene is not comprehensive, when site increases,
It is complicated for operation.
In conclusion multiple site SNP can be detected simultaneously by finding one kind, operation is fast and convenient, as a result accurately, specificity
Good reaction system and detection method has great importance.
Summary of the invention
In view of this, the purpose of the present invention is to provide the primer sets and detection of a kind of amplification and locomitivity related gene
Kit, can be simultaneously to muscle types, motion-promotion force, movement enthusiasm, explosive force, endurance, pain tolerance, soft tissue injury
Protection, aerobic capacity, Oxidation-free casting, recovery, obesity, lung capacity and the relevant gene of strength are detected,
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides the primer sets of a kind of amplification and locomitivity related gene, and the primer sets include: specificity
Primer and Single base extension primer；The nucleotide sequence of the specific primer is as shown in NO.1~52 SEQ ID；The list alkali
The nucleotide sequence of base extension primer is as shown in NO.53~78 SEQ ID.
Preferably, the primer sets are designed according to following gene: VEGFA, PPARA, BDNF, PAPSS2, intergenic,
MSTN、AGT、ACVR1B、ADRB2、ACTN3、ADRB2、OPRM1、CILP、COL1A1、IL6、AKT1、AMPD1、NOS3、NAT2、
ANKK1, FTO, BMP6, Intergenic, PRDM11, CNTF and ACE.
Preferably, the SNP site of the gene include: rs2010963rs4253778, rs6265, rs10887741,
rs8097348、rs1805086、rs699、rs2854464、rs1042714、rs1815739、rs1042713、rs563649、
rs2073711、rs1800012、rs1800795、rs1130214、rs17602729、rs2070744、rs1208、
Rs1800497, rs9939609, rs6923462, rs4237643, rs2863171, rs1800169 and rs4343.
The present invention also provides a kind of kits for detecting locomitivity related gene, including above-mentioned primer sets.
The present invention provides a kind of amplification and the primer sets and detection kit of locomitivity related gene, by using with
Relevant gene association is moved, specific primer and Single base extension primer are designed, all sites are placed on same anti-
Ying Zhong is expanded by subtracting system multiplex PCR, is detected using flight time mass spectrum, primary to detect 26 sites simultaneously, is reduced
Operation complexity reduces cost, saves the time.
Specific embodiment
The present invention provides the primer sets of a kind of amplification and locomitivity related gene, and the primer sets include: specificity
Primer and Single base extension primer；The nucleotide sequence of the specific primer is as shown in NO.1~52 SEQ ID；The list alkali
The nucleotide sequence of base extension primer is as shown in NO.53~78 SEQ ID.
Primer sets of the present invention are designed according to following gene: VEGFA, PPARA, BDNF, PAPSS2, intergenic,
MSTN、AGT、ACVR1B、ADRB2、ACTN3、ADRB2、OPRM1、CILP、COL1A1、IL6、AKT1、AMPD1、NOS3、NAT2、
ANKK1, FTO, BMP6, Intergenic, PRDM11, CNTF and ACE.The SNP site of the gene includes:
rs2010963rs4253778、rs6265、rs10887741、rs8097348、rs1805086、rs699、rs2854464、
rs1042714、rs1815739、rs1042713、rs563649、rs2073711、rs1800012、rs1800795、
rs1130214、rs17602729、rs2070744、rs1208、rs1800497、rs9939609、rs6923462、
Rs4237643, rs2863171, rs1800169 and rs4343.Specific corresponding relationship is as shown in Table 1 and Table 2, positive in table 2
Primer indicates that reverse primer is indicated with F with R, and Single base extension primer is indicated with D:
1 locomitivity related gene of table and SNP site
2 primer sequence of table
It is interpreted with each SNP site diversity of sports-related genes as shown in table 3:
3 locomitivity related gene SNP site parting meaning of table is interpreted
The present invention also provides a kind of kits for detecting locomitivity related gene, including above-mentioned primer sets.
The present invention application the kit when, preferably using human gene group DNA as template DNA, using multiple PCR technique into
Row amplification.Human gene group DNA's preferred acquisition of the present invention is from oral cavity, acquisition side of the present invention to the oral cavity genomic DNA
There is no particular determinations for method, utilize the conventional method of this field.
Multiplex PCR of the present invention, preferably includes: carrying out PCR using forward primer, reverse primer and template DNA, obtains
PCR product；Alkaline phosphatase treatment is carried out to PCR product, Single base extension is carried out again later, is prolonged using purifying resin single base
Product after stretching, upper machine testing.
The system of PCR of the present invention is preferably 4 μ L systems, comprising: DNA profiling 2 μ L, primer mix 0.67 μ L, 10 ×
Buffer 0.33 μ L, MgCl2(25mM) 0.27 μ L, dNTP (25mM) 0.07 μ L, ddH20.53 μ L, PCR enzyme (5U/ μ L) 0.13 of O
μL.There is no particular determinations to each component source in the PCR system by the present invention.The program of PCR of the present invention is preferred are as follows:
95 DEG C of initial denaturation 2min；(95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min) 45 circulations；72 DEG C of 5min, 4 DEG C of holdings.
The present invention is before the alkaline phosphatase treatment, it is also preferable to include the free dNTPs removed in the PCR product,
The method of the removal is to be handled using SAP (shrimp alkaline phosphatase, shrimp alkaline phosphotase).The present invention
The alkaline phosphatase treatment system is preferably 5.33 μ L systems, is specifically included: 4 μ LPCR products, 0.11 μ of SAP × Buffer
0.2 1.02 μ L of μ L, ddH2O of L, SAP Enzyme (1U/ μ L).There is no special limits in source of the present invention to the SAP Mix
It is fixed.The program of alkaline phosphatase treatment of the present invention is preferred are as follows: 37 DEG C of 40min；85℃5min；4 DEG C of maintenances.
Single base extension of the present invention is completed preferably by Sequenom platform matched reagent and operating procedure to test sample
This SNP site genotyping result.Prepare reaction system: by the PCR product after 5.33 μ L alkaline phosphatase enzymatic treatments, extension primer
0.63 0.13 μ L, Termination mix of μ L, Gold × Buffer, 0.13 μ L, Iplex Enzyme of mix, 0.03 μ L,
ddH2O 0.41μL.Reaction system is tamping after preparing using PCR sealed membrane, prevents sample from evaporating, and concussion mixes, centrifugation；It will
384 orifice plates of sealing, which are placed on ABI9700PCR instrument, to react, reaction condition: 94 DEG C of 30s, { 94 DEG C of 5s, (52 DEG C of 5s, 80 DEG C of 5s) 5
A internal circulation } 40 outer loops, 72 DEG C of 3min, 4 DEG C of holdings；Single base extension product is obtained, is centrifuged spare.
The preferred Sequenom platform matched reagent of purifying resin of the present invention and operating procedure are completed.
Upper machine testing of the present invention preferably includes: being shifted using Agena company MassARRAYTMRS1000 point sample instrument pure
Product after change is detected using Agena company MassARRAYTM analyzer to detection chip.
Primer sets relevant to locomitivity provided by the invention and detection kit are carried out below with reference to embodiment detailed
Thin explanation, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Buccal swab DNA is extracted: being automatically extracted kit using hundred Tyke paramagnetic particle method salivas and swab DNA and is carried out saliva
DNA is extracted.30 μ L Proteinase K are added into buccal swab, 56 DEG C of water-bath 30min are mixed by inversion.Tear 96 hole deep holes
Plate plastic packaging film is separately added into the 350 μ L of sample after being incubated in first row and the 7th column, adds 10 μ L nucleic acid settling agents, 300 μ L
Isopropanol is put into instrument for extracting nucleic acid, and stirring rod is inserted into card slot, temperature is arranged are as follows: and 80 DEG C of cracking temperature, 65 DEG C of eluting temperature,
After instrument end of run, DNA solution, quality inspection, 4 DEG C of preservations are collected.
Embodiment 2
PCR amplification: the DNA for the sample to be tested that embodiment 1 is extracted is added separately in 384 orifice plates, carries out multiplex PCR expansion
Increase.It is added in each reacting hole, reaction system according to the reaction system (4 μ L) of multiplexed PCR amplification: 2 μ L of template DNA, primer
0.67 0.33 0.27 0.07 0.53 μ L of μ L, ddH2O of μ L, dNTP (25mM) of μ L, MgCl2 (25mM) of μ L, 10 × Buffer of mix,
PCR enzyme (5U/ μ L) 0.13 μ L.Reaction system is tamping after preparing using PCR sealed membrane, prevents sample from evaporating, and concussion mixes, from
The heart；384 orifice plates of sealing are placed on ABI9700PCR instrument and are reacted, reaction condition: 95 DEG C of initial denaturation 2min；(95 DEG C of 30s, 56
DEG C 30s, 72 DEG C of 1min) 45 circulations；72 DEG C of 5min, 4 DEG C of holdings；Pcr amplification product is obtained, is centrifuged spare.
Embodiment 3
SAP digestion: utilizing Sequenom platform matched reagent and operating procedure, completes sample to be tested SNP site parting knot
Fruit.By pcr amplification product obtained in embodiment 2, each reacting hole is added to according to the reaction system (1.33 μ L) that SAP digests
In, reaction system: 0.11 μ L, SAP Enzyme (1U/ μ L) of SAP × Buffer, 0.2 1.02 μ L of μ L, ddH2O.Reaction system is matched
It is tamping after making using PCR sealed membrane, prevents sample from evaporating, concussion mixes, centrifugation；384 orifice plates of sealing are placed in
It is reacted on ABI9700PCR instrument, reaction condition: 37 DEG C of 40min, 85 DEG C of 5min, 4 DEG C of holdings；After obtaining alkaline phosphatase enzymatic treatment
PCR product, be centrifuged it is spare.
Embodiment 4
Single base extension: utilizing Sequenom platform matched reagent and operating procedure, completes sample to be tested SNP site
Genotyping result.By the PCR product after alkaline phosphatase enzymatic treatment obtained in embodiment 3, according to single base extension system
(1.33 μ L) is added in corresponding each reacting hole, reaction system: extension primer mix 0.63 μ L, Gold × Buffer
0.13 μ L, Termination mix, 0.13 μ L, Iplex Enzyme, 0.03 0.41 μ L of μ L, ddH2O.Reaction system prepares
It is tamping afterwards using PCR sealed membrane, prevents sample from evaporating, concussion mixes, centrifugation；384 orifice plates of sealing are placed in ABI9700PCR
It is reacted on instrument, reaction condition: 94 DEG C of 30s, { 94 DEG C of 5s, (52 DEG C of 5s, 80 DEG C of 5s) 5 internal circulations } 40 outer loops, 72
DEG C 3min, 4 DEG C of holdings；Single base extension product is obtained, is centrifuged spare.
Embodiment 5
Purifying resin: utilizing Sequenom platform matched reagent and operating procedure, completes sample to be tested SNP site parting.
By 384 orifice plates of the single base extension product of embodiment 4, after sealed membrane of gently tearing, 16 μ L ddH2O are added in every hole；It takes clean
A4 paper 384 plate of 6MG is placed on it, with the appropriate Purification Resin of small bale-out；With plastic plate, left and right is bulldozed Purification Resin repeatedly,
Compacting, keeps every hole resin content uniform；The inversion of 384 plates is pressed on 384 plate of 6MG, two plates are exchanged, and 6MG plate beats 6MG upper
Back falls into resin in 384 orifice plates equipped with single base extension product；After sealed membrane is sealed, 15rpm turns upside down mixing
30min is sufficiently purified.
Chip point sample: 384 orifice plates after purifying resin are centrifuged, and start MassARRAY Nanodispenser RS1000
Point sample instrument moves to the extension products after purifying resin on 384 hole SpectroCHIP chips；Chip after point sample uses
MALDI-TOF analysis, testing result is using TYPER4.0 software parting and exports result.Experimental result is as shown in table 4:
4 SNP site genetic test result of table
By table 4 as it can be seen that the genotype in 26 sites can be detected correctly, recall rate 100%, genotype distribution meets China
The Hans' genotype distribution frequency.
The locomitivity of examined samples can be analyzed in conjunction with table 3 and table 4, testing result solution is carried out by taking sample 1 as an example
It reads, see the table below (table 5):
5 sample of table, 1 genetic test result is interpreted
As shown in Table 5, sample 1 is compatible muscle, and motion-promotion force, pain tolerance, anaerobic sport capacity are normal, fortune
Dynamic enthusiasm is compared with ordinary person's height, but soft tissue protective capability does not protrude, and aerobic sport ability, lung capacity, strength are poor, needs
Motion mode, diet and nutrient supplement are adjusted with this.Gene tester of the invention is suitable for Chinese han population gene
Type detection and its prediction of locomitivity.
The amplimer and extension primer that the present invention designs can detect 26 SNP sites in the same reaction, be used for
Exploitation locomitivity detection kit and guides suitable exercise.In terms of exercise guidance, genetic test of the invention can be appreciated that people
The abilities such as endurance, explosive force, the strength of class body provide the reference frame of science for suitable exercise.The present invention will move correlation
Gene association detection, it is to detect gene in the product of locomitivity genetic test so far that one-time detection, which goes out all projects,
Covering surface is most comprehensive.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Beijing North base Laboratory of medical test Co., Ltd
<120>a kind of primer sets and detection kit of amplification and locomitivity related gene
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<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
acgttggatg aaagaggtag caagagctcc 30
<210> 42
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
acgttggatg aaagcaggtc actcactttg 30
<210> 43
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
acgttggatg ctgacaaatg gcagcaaaag 30
<210> 44
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
acgttggatg acatgtgtct accccaaagc 30
<210> 45
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
acgttggatg gggtgattta aggacactgg 30
<210> 46
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
acgttggatg actccactga tcagtgcttg 30
<210> 47
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
acgttggatg cctaccagat ctgacgaatg 30
<210> 48
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
acgttggatg tgagttccac gtatttcggg 30
<210> 49
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
acgttggatg ctctatattt ttggttggg 29
<210> 50
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
acgttggatg tgcaagtggt ttatgaggag 30
<210> 51
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
acgttggatg acactgttgt aggagtctcg 30
<210> 52
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
acgttggatg gcaagtggat ggaaaaccc 29
<210> 53
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
ctggctgctc cctga 15
<210> 54
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 54
ctgcccgagg ctgac 15
<210> 55
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 55
tggctagggt taggt 15
<210> 56
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
tcaagctctt ccctggc 17
<210> 57
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 57
cacccagctg attgtca 17
<210> 58
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 58
gcgactgctg tgaattt 17
<210> 59
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 59
ggttgaagaa gtgctga 17
<210> 60
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 60
gcccacacct cgtcccttt 19
<210> 61
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 61
ccttttgctg gcacccaat 19
<210> 62
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 62
atgtgacgtc ctttagcat 19
<210> 63
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 63
gtctcccagg aggtttttg 19
<210> 64
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 64
cccttctggc tcatttccca 20
<210> 65
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 65
gggcaagccc tcatcccgcc c 21
<210> 66
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 66
gccctcctca aagtgctggt c 21
<210> 67
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 67
gcaggatcat catttctagg a 21
<210> 68
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 68
aaatgttatg cattttatgg g 21
<210> 69
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 69
gaaaaaaaat accctcaaca ca 22
<210> 70
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 70
ttcctccaac agctcttcta tca 23
<210> 71
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 71
atagcatgca ggtctataac caa 23
<210> 72
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 72
aggggaaatg aagcttttga atc 23
<210> 73
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 73
gagttcactc actttgcccc tgtc 24
<210> 74
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 74
cagcaaaagt aatgcaatac tcac 24
<210> 75
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 75
ttccggccct gatgcttcac ctggc 25
<210> 76
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 76
cagctctgac gaatgtgatg gccac 25
<210> 77
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 77
tgctaattta ggagaggaat tctag 25
<210> 78
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 78
tctaaaatac aatacaataa agtagtaa 28
Claims (4)
1. a kind of primer sets of amplification and locomitivity related gene, which is characterized in that the primer sets include: specific primer
With Single base extension primer；The nucleotide sequence of the specific primer is as shown in NO.1~52 SEQ ID；The single base is prolonged
Extend object nucleotide sequence as shown in NO.53~78 SEQ ID.
2. primer sets according to claim 1, which is characterized in that the primer sets are designed according to following gene: VEGFA,
PPARA、BDNF、PAPSS2、intergenic、MSTN、AGT、ACVR1B、ADRB2、ACTN3、ADRB2、OPRM1、CILP、
COL1A1, IL6, AKT1, AMPD1, NOS3, NAT2, ANKK1, FTO, BMP6, Intergenic, PRDM11, CNTF and ACE.
3. primer sets according to claim 2, which is characterized in that the SNP site of the gene includes:
rs2010963rs4253778、rs6265、rs10887741、rs8097348、rs1805086、rs699、rs2854464、
rs1042714、rs1815739、rs1042713、rs563649、rs2073711、rs1800012、rs1800795、
rs1130214、rs17602729、rs2070744、rs1208、rs1800497、rs9939609、rs6923462、
Rs4237643, rs2863171, rs1800169 and rs4343.
4. a kind of any one of kit, including claims 1 to 3 for detecting locomitivity related gene primer sets.
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| CN114703292A (en) * | 2022-03-03 | 2022-07-05 | 北京体育大学 | Athlete screening method based on VEGFA gene |
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