CN101805790A - Method for simultaneously detecting polymorphism of 32 SNP loci on 24 sports-related genes - Google Patents
Method for simultaneously detecting polymorphism of 32 SNP loci on 24 sports-related genes Download PDFInfo
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Abstract
The invention provides a method for simultaneously detecting polymorphism of 32 SNP loci on 24 sports-related genes, which realizes the simultaneous detection of 32 mononucleotide polymorphic loci in one reaction system through multiplex PCR, liquid chips and nucleotide hybridization. Compared with the conventional sequence detection and other detection methods, the invention has the advantages of high throughput, high speed, high degree of automation, high accuracy and sensitivity and the like.
Description
Technical field
The present invention relates to a kind of method that detects 32 SNP loci polymorphisms on 24 sports-related genes simultaneously, belong to the Medical Molecular Biology field.
Background technology
As far back as 1997, (Xie Feng such as Collins, Xu Feng, Chinese littleleaf box minister in ancient times etc., single nucleotide polymorphism and hepatocellular carcinoma genetic predisposition, liver, 2006,11 (6): the hypothesis that 413-415) has just proposed " common disease, common variation ", think that the susceptibility of common disease is because some site among the crowd, particularly cause in the common variation of gene coding region or control region.Single nucleotide polymorphism (single nucleotide polymorphism, SNP) be meant the dna sequence polymorphism that causes by single nucleotide diversity on the genomic level, occurrence frequency in the crowd is greater than 1%, comprises manifestation such as the insertion of conversion, transversion and single base of single base or disappearance.It is modal a kind of in human heritable variation, accounts for more than 90% of all known polymorphisms, is between the ethnic group, one of the physical basis of heredity of interindividual variation.
SNP is used widely as a kind of genetic marker, mainly come from following characteristic: (1) density height: SNP is estimated as 500～1000bp in the human genome mean density, distribution in whole genome reaches more than 3,000,000, genetic distance is 2～3cm, its density exceeds several magnitude than microsatellite marker, can any inside of waiting to study gene or near a series of marks are provided.(2) representativeness: some SNP may transcribe, translate, aspect such as splicing and rna stability plays a significant role, and some SNP that is positioned at gene inside might directly influence protein structure or expression level, so they may represent some influencing factor in the disease genetic mechanism.(3) genetic stability: SNP derives from the sudden change that takes place before thousands of year, along with the mankind multiply heredity stably so far from generation to generation, compares with the polymorphic mark of tumor-necrosis factor glycoproteins such as little satellite, and SNP has higher genetic stability.(4) the easy property measured: SNP is two equipotential polymorphisms usually, claims biallelic marker again, can carry out gene type by simply "+/-" during detection, and need not to carry out the analysis of gene fragment length, so the check and analysis of SNP can realize automatization.Possess above advantage, SNP is considered to the third generation molecular genetic marker after first-generation restriction fragment length polymorphism and s-generation microsatellite polymorphism.
At present existing several different methods can be used for SNP and detects, as little sequencing, allele specific oligonucleotide hybridization, special connection, DNA chip and the TaqMan system etc. of oligonucleotide.But, just can carry out other detection then no matter any method at first must be carried out the amplification of target sequence.Traditional SNP detection method is to adopt some existing mature technologies, as dna sequencing, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP), allele specific oligonucleotide oligonucleotide hybridization (ASO) etc.Though these technology can be finished the detection to SNP to a certain extent,, therefore, also differ greatly apart from target quick, efficient, automatization because they must detect by gel electrophoresis.
The DNA chip technology is a kind of mutant dna sequence testing tool newly developed in recent years.The DNA die size is similar to the cpu chip on the computer, with glass, silicon, polypropylene etc. as carrier substrate, the chip upper berth the invisible dna fiber of one deck naked eyes " carpet ", promptly have the probe of specific base sequence.Testing gene is cut into fragment different in size after extracting, behind fluorescence chemical material mark, be expelled on the slide glass that is embedded with chip.Because the degree of DNA and probe hybridization is relevant with fluorescence intensity,, can measure the variation of detected sequence according to the fluorescence power therefore by laser scanning.
In our research, what single nucleotide polymorphism detected employing is liquid-phase chip technology, and instrument is the GenomeLab of Beckman company
TMSNPstream
The classical single base primers elongation technology of this technology utilization in conjunction with multiple PCR technique and fluorescent mark technology, is introduced 384 hole liquid-phase chip hybridization techniques simultaneously, principle is simple, reliable results, accuracy has surpassed dna sequencing, and each SNP gene type assay only needs the dna sample of 170ng.Major advantage has: 1. higher, flux more flexibly: 384 hole hybridization hybrid chips, every hole can be detected 48 SNP sites simultaneously.Detect flux every day and can reach 4,608-800,000 SNP site.In each hole on 384 orifice plates all predetermined fixed oligonucleotide fragment and 4 contrasting markings (Tag) of 48 known uniquenesses of sequence.Each Tag fragment on the plate can both be extended a complementation in the primer with 48 SNP that contain the Tag complementary sequence, adds the oligonucleotide fragment of 4 contrasts, to guarantee result's accuracy.After single base primers extension is finished, to be transferred on the micro-array chip plate that has contained the specific oligonucleotides sequence, because the Tag chip technology can be set hybridization conditions in strict accordance with known Tag sequence, all hybridizations all are dynamic solution hybridizations, and the high degree of specificity of favorable uniformity and primer makes the Tag chip have accuracy, hybridization efficiency and the circulation ratio higher than other SNP examination technology.2. real multiple SNP detects: every pair of PCR primer and a SNP extend primer all to be finished by the automatic primer-design software of online software www.Autoprimer.com.This software can finish 48 pairs of PCR primers simultaneously and 48 SNP extend the success of primer design to guarantee that multiple SNP detects.The SNP that each design is finished extends primer and is connected with specific Tag sequence label automatically, and this sequence and the sequence oligonucleotide probe complementation that is planted in advance in each 384 orifice plate are so that carry out the accurate detection in SNP site.3. lower cost, higher efficient: the reaction system of 5 μ l, 48 heavy PCR reactions have not only reduced the expense of PCR reaction, have also improved efficient, and its final result had both guaranteed unanimity, can make the cost of each SNP gene type drop to minimum again.4. DNA consumption still less, the DNA of more accurate gene type: 2ng can be used for the detection in 48 SNP sites, and accuracy rate is more than 99%.Because the base of single extension has fluorescent mark, detected result can be read and be analyzed by the Two Colour Fluorescence on the fluorescence chip scanner.Because SNP site quantity in genome is many, distributional stability, and be a kind of biallelic marker, be easy to use the technology platform of high-throughput, automatization to detect.This technology is bound to play an important role in the correlative study of SNP and disease.
Summary of the invention
(1) technical problem that will solve
The purpose of this invention is to provide a kind of method that detects 32 SNP loci polymorphisms on 24 sports-related genes simultaneously.These genes and site are ACE (rs4461142), CKMM (rs1133190), ADRB2 (rs1042718, rs1042713), ADRA2A (rs3750625), GDF8 (rs3791783), CNTF (rs2515362), HLA-A (rs3823342), KCNA4 (rs1323860), CASQ1 (rs3747621), CFTR (rs4148724), PPARGC1A (rs768695), AMPD1 (rs2268701), ACTN3 (rs540874), BDKRB2 (rs2069578), IGF-1 (rs972936, rs7956547), UCP2 (rs660339), LEPR (rs12037879, rs12029311), FABP2 (rs11935130), LEP (rs12706832, rs11761556), NOS3 (rs3918188, rs7830), MYLK (rs820325, rs1350152), CNTFR (rs6476455), COL1A1 (rs2586488, rs2696247) and VDR (rs4334089, rs4760648).In the testing process, multiplex PCR is a process very rambunctious, it exists between primer, between primer and product, and product between influencing each other, reducing this influence is very crucial problem.The online primer-design software autoprimer that we adopt BECKMAN company to provide designs the primer in 32 sites simultaneously, it can consider between 32 pairs of primers simultaneously and primer and product between interaction, make design result more reliable.Experimental result confirms that also our multi-primers system effect is remarkable, is rare multiplex PCR system.
(2) technical scheme
The ultimate principle of the method for the invention is: single base primers elongation technology that utilization is classical, in conjunction with multiple PCR technique and fluorescent mark technology, introduce 384 hole liquid-phase chip hybridization techniques simultaneously, and principle is simple, reliable results, accuracy has surpassed dna sequencing.
Method of the present invention, it comprises the steps:
(1) extract genomic dna and adopt Promega company genomic dna test kit to extract peripheral blood DNA, detect DNA purity and concentration with DU800 then ,-80 ℃ of refrigerators are preserved.
(2) design primer bibliographical information Zinc metallopeptidase Zace1 1 (ACE), creatine kinase (CKMM), insulin-like growth factor-i (IGF-1), beta 2 adrenoreceptor (ADRB2), ciliary neurotrophic factor (CNTF), the isogenic single nucleotide polymorphism of adewosine monophosphate desaminase 1 gene (AMPD1) (the Rankinen T.Bray MS that may be closely related with people's motor capacity, Hagberg JM, et al.The Human Gene Map for Performance and Health-RelatedFitness Phenotypes:The 2005 Update.Med Sci Sports Exerc, .2006,38 (11): 1863-1888.).Consult the identification number in our SNP interested site on these genes from websites such as Hapmap, as rs4362, rs4461142, rs1133190 and rs1042718 etc., 32 such rs number numerals are made the Excel form, the software autoprimer (http://www.autoprimer.com/) that Input Online uses, the design primer, designed the back and three primers have been arranged at each SNP site, it is respectively upstream primer, downstream primer and single-basic extension primer (hereinafter to be referred as extending primer) (seeing Table 1), it is synthetic that sequence is transferred to company, the PAGE glue purification ,-20 ℃ store for future use.
Table 1 the present invention is directed to 32 primers that the SNP site is designed of 24 genes
(3) prepare before the genomic dna experiment all samples DNA to be diluted to about 10ng/ μ L.
(4) dilution primer PCR primer dilution: will synthesize good upstream primer and downstream primer and be diluted to 240 μ mol/L respectively, and respectively get 5 μ L then to join in the new centrifuge tube, as new multiple PCR primer pond, every primer final concentration is 2.5 μ M.
Extend the primer dilution: will synthesize all good single-basic extension primers and be diluted to 240 μ mol/L respectively, and respectively get 10 μ L then to join in the new centrifuge tube, and mix, as new extension primer pond, every is extended the primer final concentration is 5 μ M.
(5) every 384 plate consumption of PCR reaction is as follows:
Primer (each 2.5 μ M) 45 μ L
Deoxyribonucleotide (each 10mM) 20 μ L
10 * PCR damping fluid II, 225 μ L
MgCl
2(25mM) 450μL
Gold medal enzyme 5U/mL 45 μ L
Distilled water 565 μ L
Add up to 1350 μ L
Add the PCR mix that 3 μ L prepare in the every hole of 384 plates, will dilute good DNA again and join in the corresponding hole, every hole adds 2 μ L.Carry out following response procedures then
①95℃ 15min
②
3. 4 ℃ of preservations
This step is actual to be one 32 heavy PCR reaction, amplifies near the sequence in 32 SNP sites simultaneously.
(6) PCR reaction back purifying
According to the system in the following table, the preparation purified reagent:
Exonuclease I (10U/ μ L) 90 μ L
Shrimp alkaline phosphotase (1U/ μ L) 448 μ L
10 * shrimp alkaline phosphotase damping fluid, 135 μ L
Distilled water 667 μ L
Add up to 1350 μ L
With reacted 384 plates of PCR, add the purified reagent that configures again, every hole 3 μ L carry out following response procedures then
①37℃ 30min
②96℃ 10min
3. 4 ℃ of preservations
This step is to remove short nucleotide fragments such as primer residual in the reaction system.
(7) single base extension
According to the system configurations SNPware primer extension reaction mixture shown in the following table:
Extend diluent (available from BECKMAN company) 1692.5 μ L
Extend primer 13.5 μ L
20 * extension fluorescent reagent (available from BECKMAN company), 90.0 μ L
Distilled water 1336.5 μ L
Archaeal dna polymerase 9.4 μ L
Add up to 3150.0 μ L
In each good hole of purifying, add the primer extension reaction mixture that 7 μ L configure, carry out following response procedures then
①96℃ 3min
②
3. 4 ℃ of preservations
This step is that the single-basic extension primer in each SNP site combines with SNP site upstream sequence, extending to the SNP site then stops, because every single-basic extension primer has just added when synthetic and the hybridization plate on the complementary bonded label of known array, so in subsequent experimental, can discern the information in each site.
(8) hybridization
The hybridization plate is one 384 orifice plate, can carry out the experiment of 384 samples simultaneously in the above.System behind the primer extension reaction is added hybridization solution (50 μ L hybridizing reagents are added in the 850 μ L hybridization buffers), be added to then on the SNPware hybridization plate, put into the box of a sealing, be placed in the incubator and hatch.The incubator temperature is made as 42 ℃, incubation time be 2h (+/-15min).(9) after hybridization finishes, clean the hybridization plate and use GenomeLab
TMSNPstream
Instrument scanning is with the software kit analytical data and derive the gained result.
Method of the present invention is used to detect the polymorphism in 32 relevant SNP sites of motion.
Detect 32 mononucleotide polymorphism sites that the method for 32 SNP loci polymorphisms on 24 sports-related genes detects simultaneously with the present invention, the result of player group is:
ACE:rs4461142 T＞C, CKMM:rs1133190 T＜C, ADRB2:rs1042718 C＞A, rs1042713 A＜G, ADRA2A:rs3750625 C＞A, GDF8:rs3791783 T＜C, CNTF:rs2515362 A＜G, HLA-A:rs3823342T＜C, KCNA4:rs1323860 T＜C, CASQ1:rs3747621 T＜C), CFTR:rs4148724 T＞C, PPARGC1A:rs768695 A＜G, AMPD1:rs2268701 T＜G, ACTN3:rs540874 T＞C, BDKRB2:rs2069578 A＜G, IGF-1:rs972936A＞G, rs7956547 T＜C, UCP2:rs660339 T＞C, LEPR:rs12037879 A＞G, rs12029311 A＞G, FABP2:rs11935130 C＞A, LEP:rs12706832 A＞G, rs11761556 C＜A, NOS3:rs3918188C＞A, rs7830 C＞A, MYLK:rs820325 A＞G, rs1350152 A＞G, CNTFR:rs6476455T＜C, COL1A1:rs2586488 A＞G, rs2696247 A＜G and VDR:rs4334089 A＞G, rs4760648 T＞C.
(3) beneficial effect
Method of the present invention is by optimizing primer, and realization can detect 32 SNP loci polymorphisms that motion is relevant simultaneously, and this method is compared with the detection method of direct order-checking, the efficient height, operation is fast, and cost is low, the result judges intuitively, can be used for the rapid detection of a large amount of samples.
It is synthetic by match hundred victory companies that the present invention tests the primer, deoxynucleotide is available from Japanese TaKaRa company, gold medal amplification enzyme is available from Switzerland Roche company, excision enzyme I is available from New England BioLabs company, shrimp alkaline phosphatase and damping fluid thereof, sterilization distilled water be available from U.S. Promega company, all the other reagent, solution and hybridization plate etc. except that marking the source all available from U.S. Beckman Coulter company.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
The collection of detected object selection and sample:
Detect totally 856 of sportsmen simultaneously with method of the present invention, 495 of men, 361 of woman.The non athlete contrasts 284,161 of men, 123 of woman.The sportsmen is the outstanding person of sports achievement, and they are from provincial sports team or national sports team.The normal control sample is the healthy male in 20-30 year of leaving and taking of PLA General Hospital health examination center and women's blood specimen.
Extract genomic dna and adopt Promega company genomic dna test kit to extract the DNA of the peripheral blood sample that is collected, detect DNA purity and concentration with DU800 then ,-80 ℃ of refrigerators are preserved.
Detect the single nucleotide polymorphism in 32 SNP sites of sample.As a result, successfully obtained the single nucleotide polymorphism in these 32 SNP sites, and analyzed their genotype frequency and gene frequency, found to have tangible single nucleotide polymorphism difference such as table 2 and table 3 really between sportsmen and the control group.
The genotype statistical analysis table of all sportsmen of table 2 and normal control group
The allelotrope statistical analysis table of all sportsmen of table 3 and normal control group
Sequence table
＜110〉Chinese People's Liberation Army General Hospital
＜120〉a kind of method that detects 32 SNP loci polymorphisms on 24 sports-related genes simultaneously
<160>96
<210>1
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs820325SNP site
<400>1
GCACAAAAAT?AGCCCCAAG 19
<210>2
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs820325SNP site
<400>2
TCTCCTGCCT?GCCCCTTC?18
<210>3
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs820325SNP site
<400>3
CGATCACCTC?ACTAGAACAA?GTCATGTACA?CAAGGGTAGG?GCAGA 45
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs972936SNP site
<400>4
TCCATGGAAG?TGTTTTCTGC?20
<210>5
<211>22
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs8972936SNP site
<400>5
AAGGGGTCTC?TTTCTCTTAG?CC?22
<210>6
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the reverse extension primer in amplification rs972936SNP site
<400>6
ACAAGAACTC?CATGACTCAA?TATCTGGCCT?GAACTTCTGC?ATTTC 45
<210>7
<211>21
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs3750625SNP site
<400>7
TTACCTAGCC?CTGGCTAATT?C?21
<210>8
<211>31
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs3750625SNP site
<400>8
ATGTGTGCTA?TCAAAAATAG?TGTATATTTA?C?31
<210>9
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the reverse extension primer in amplification rs3750625SNP site
<400>9
CTAACTAAGC?TACGCCGACA?AGCGGGGGAT?GGGGAGGGCA?GGCAG?45
<210>10
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs540874SNP site
<400>10
ATGGAGATGA?GGCAAGCTC?19
<210>11
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs540874SNP site
<400>11
GGCTGGAGAC?CAAGCCTG?18
<210>12
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs540874SNP site
<400>12
TACCTATGAC?CAGCAAGCAC?GTCATCAGGC?TCCATCATCC?CATTC?45
<210>13
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs660339SNP site
<400>13
ATGGAGATGA?GGCAAGCTC?19
<210>14
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs660339SNP site
<400>14
GGCTGGAGAC?CAAGCCTG?18
<210>15
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs660339SNP site
<400>15
TACCTATGAC?CAGCAAGCAC?GTCATCAGGC?TCCATCATCC?CATTC?45
<210>16
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs4334089SNP site
<400>16
TCTGAGATGG?GCAGGGCT18
<210>17
<211>25
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs4334089SNP site
<400>17
ACTAATAACC?TAGAAGATGG?AGACG?25
<210>18
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs4334089SNP site
<400>18
CAGAATAGCC?ACGCCTAGAT?CTCCACCAGG?CAGCTCCGGT?CCCAT?45
<210>19
<211>22
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs12706832SNP site
<400>19
CTGCTTCAGT?AGCACTCAGA?GG 22
<210>20
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs12706832SNP site
<400>20
ATCAGCTGTC?TGCCTGAGC 19
<210>21
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the reverse extension primer in amplification rs12706832SNP site
<400>21
CAGAACATCC?TCAGAAGCAA?CTGCCATTTT?CTTAGAGTTA?AAAGA 45
<210>22
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs2696247SNP site
<400>22
TTCACCTGGA?GGACCAGC 18
<210>23
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs2696247SNP site
<400>23
TGGTCTCTTC?CAGATTCTAA?ACC 23
<210>24
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs2696247SNP site
<400>24
CAAGCAACGA?CCTACTACAA?ACCAGGGAGA?CCCTGTAGGT?GGGAA 45
<210>25
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs7830SNP site
<400>25
CCTCTGTCCC?TAGATTGTGT 20
<210>26
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs7830SNP site
<400>26
ATTCTGGCAG?GAGCGGCT 18
<210>27
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs7830SNP site
<400>27
AATAAGCTCA?CCACCGTCAA?ACTCCCTTCA?GGCAGTCCTT?TAGTC 45
<210>28
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs3823342SNP site
<400>28
TGCCCAGGGC?TCTGATGT 18
<210>29
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs3823342SNP site
<400>29
AAAGAAACCC?CATAGCACAG 20
<210>30
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the reverse extension primer in amplification rs3823342SNP site
<400>30
GCAAGCCATC?AGCTAATACA?CACCCAACAC?ACATTAGGTC?CTCCA?45
<210>31
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs3918188SNP site
<400>31
CCCAGCTGAA?GCATTTAAAA 20
<210>32
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs3918188SNP site
<400>32
TGAAGGGCAG?CTCGGTGG 18
<210>33
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs3918188SNP site
<400>33
CACGACAAGA?CAACAGATAC?TGGGAGCAAG?GCACACGTAC?AAGGG?45
<210>34
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs11761556SNP site
<400>34
TTTTGTTGGA?AGGTTTGGTG 20
<210>35
<211>31
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs11761556SNP site
<400>35
TACTCTTAAC?TCCTTTAATA?TCAAACTTCT?T 31
<210>36
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs11761556SNP site
<400>36
CGCAGAAGCA?ACTCACTTCT?GGCAGAGAAA?GAAGAGACAG?GAGGG 45
<210>37
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs4461142SNP site
<400>37
TCAGCCAAGT?CCAGATCAG 19
<210>38
<211>21
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs4461142SNP site
<400>38
GAGATGGGAG?TTTTCAGTCC?A 21
<210>39
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs4461142SNP site
<400>39
CCATAACAAC?TTACCAGCCA?CTCTCACTTG?AGACAGGAAG?AGGAC 45
<210>40
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs6476455SNP site
<400>40
GATATGAGCC?CCACAACGT 19
<210>41
<211>28
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs6476455SNP site
<400>41
TACACAAAGA?CTAGGAGTTT?AAAGTTCA 28
<210>42
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs6476455SNP site
<400>42
CAACAAGACA?TAACAACGCA?GTAAGCCCCT?AGACCCTAGA?AGGAG 45
<210>43
<211>19
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs1042718SNP site
<400>43
TGTGTCAGGC?CTTACCTCC 19
<210>44
<211>21
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs1042718SNP site
<400>44
GTCTCATTGG?CATAGCAGTT?G 21
<210>45
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs1042718SNP site
<400>45
GATCCATCAA?CAGACATCAC?CTTGCCCATT?CAGATGCACT?GGTAC 45
<210>46
<211>25
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs4760648SNP site
<400>46
TTGAATAATT?TTCCTAGTGG?AGAAC 25
<210>47
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs4760648SNP site
<400>47
TTTGTCTAAC?ATGGCTACAA?AGAG 24
<210>48
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs4760648SNP site
<400>48
GCAACATAAG?ACCGCTCAAC?AGCTCTGTAT?CATGCACAGA?TGGAA 45
<210>49
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs3791783SNP site
<400>49
AATGCTAAGG?CAGCTCAGAA 20
<210>50
<211>30
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs3791783SNP site
<400>50
TGTATATATT?TCTTTGTGGA?ATACTCCTAA 30
<210>51
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the reverse extension primer in amplification rs3791783SNP site
<400>51
CCACTCAACT?CCACGAATAC?CTCCTAATAA?AATTTCAAGT?TACAT 45
<210>52
<211>25
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs768695SNP site
<400>52
TTTGGTCTTT?ACTGAGTTTT?TTTCA 25
<210>53
<211>25
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs768695SNP site
<400>53
TTGAAGCCAG?TGTAATTTAA?GAATT 25
<210>54
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs768695SNP site
<400>54
ACAATCAACA?TACGAACAGC?AATTACAGTC?TCAATCATAA?AAAAC 45
<210>55
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs4148724SNP site
<400>55
TAAGAGTCTT?GTGTTTTCTT?CCG 23
<210>56
<211>27
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs4148724SNP site
<400>56
TATAAACATG?CTTATGGTAT?AAATGGG?27
<210>57
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs4148724SNP site
<400>57
AACATACAGA?CGCACTCCTC?TACAAACTCT?TCCCCCTTGT?CAACA 45
<210>58
<211>27
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs1350152SNP site
<400>58
AAAAGAAGAA?GAAAAAAAGA?ATGTCTG 27
<210>59
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs1350152SNP site
<400>59
AGTGAGTGAG?TGATTGATTG?AATG 24
<210>60
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs1350152SNP site
<400>60
CAACAATACG?AGCCAGCAAG?CAACAGCTAA?GGCAGGCATC?CAGAA 45
<210>61
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs2515362SNP site
<400>61
TATAAGCCGT?GCCCAACC 18
<210>62
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs2515362SNP site
<400>62
TGAACTTTGG?AAGCTGGG 18
<210>63
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the reverse extension primer in amplification rs2515362SNP site
<400>63
CCAGATCCTC?ACCATGTAAG?ATGTTTAGTA?ATACAGCCTG?GTTTT?45
<210>64
<211>24
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs7956547SNP site
<400>64
AAAGTCTTTA?AGCAAGCTCA?GTGT 24
<210>65
<211>25
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs7956547SNP site
<400>65
ATATCACTTT?ACATTGAGAC?CACCC 25
<210>66
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the reverse extension primer in amplification rs7956547SNP site
<400>66
CCGCCAGTAA?GACCTAGACG?TTCAGAATGC?TCACTCAGTA?TCACT?45
<210>67
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs2268701SNP site
<400>67
TAATGTGTAG?CATTGGTCAA?ACA 23
<210>68
<211>25
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs2268701SNP site
<400>68
AAGTTATGAA?TCAATTCCTG?TCATC 25
<210>69
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs2268701SNP site
<400>69
AGACTTCTAC?GCAAGCACTG?AGATAATACA?TGATATGACT?TAGTA 45
<210>70
<211>29
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs11935130SNP site
<400>70
TTGTGCAATA?TATAATGTAG?ACAAACTTC 29
<210>71
<211>25
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs11935130SNP site
<400>71
AAATGATTTC?TGCTCTATTT?CATTG 25
<210>72
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs11935130SNP site
<400>72
ACAACTCACG?CAAGTACCAT?TGGCTTCTTC?AGTTAGTGAA?GGAGA 45
<210>73
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs2069578SNP site
<400>73
AGAAGAAGGG?CACATTCCTG 20
<210>74
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs2069578SNP site
<400>74
ATTGCTGTCT?GATCCACTGG 20
<210>75
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs2069578SNP site
<400>75
TACAAGCACG?CACTAGACAT?TATCCCAGAT?GAACTTGGAA?GTGAA 45
<210>76
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs1323860SNP site
<400>76
AAGAGCCATG?TGGGCCAT 18
<210>77
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs1323860SNP site
<400>77
AATATTTTCA?AGAGTATGGG?GCA 23
<210>78
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs1323860SNP site
<400>78
TCCAGAATAG?ACAACAGACG?GTGGAGAGAG?GAAAAATAAT?TGAGT 45
<210>79
<211>25
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs12029311SNP site
<400>79
TCATCATTTA?CACCTCAAAT?CTTTT 25
<210>80
<211>25
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs12029311SNP site
<400>80
ACGAGTTAGA?CTAGAATGAT?GGTGA 25
<210>81
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs12029311SNP site
<400>81
CAACAAGTAA?TCCGCAGACT?GTTGTAGCTT?TGAATAATAG?TCTTC 45
<210>82
<211>25
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs12037879SNP site
<400>82
AATTAAAAAC?ACACAAGCAC?ACATA 25
<210>83
<211>23
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs12037879SNP site
<400>83
ATGGAAACAC?ATAAAGTGCA?ATT 23
<210>84
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs12037879SNP site
<400>84
CAGCCATCCA?TTCACTATCT?GCAGAACACA?GTACAGAAAA?TTGCC?45
<210>85
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs1042713SNP site
<400>85
TCACCTGCCA?GACTGCGC 18
<210>86
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs1042713SNP site
<400>86
ACACCTCGTC?CCTTTCCT 18
<210>87
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs1042713SNP site
<400>87
ATCTAACGCA?CCTACGACCT?CAGCGCCTTC?TTGCTGGCAC?CCAAT 45
<210>88
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs3747621SNP site
<400>88
AGGAGGTGGG?TGGGGCAA 18
<210>89
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs3747621SNP site
<400>89
ATGATCCCAG?GCTTCCCA 18
<210>90
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the extension primer in amplification rs3747621SNP site
<400>90
CTCAGACTAC?GAATCCACGT?ATGTGGGGCC?CCGGTGATTA?TCAAA 45
<210>91
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs2586488SNP site
<400>91
GCGGAAGTTC?CATTGGCA 18
<210>92
<211>20
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs2586488SNP site
<400>92
CCGTGGCCCC?GCCGTAAGTA 20
<210>93
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the reverse extension primer in amplification rs2586488SNP site
<400>93
CAGCACTATT?ACCATCACGT?TACCCTGCTG?TGTCCCCCAT?GCCTT 45
<210>94
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the upstream primer in amplification rs1133190SNP site
<400>94
TCCTGATCTC?CCACCCGC 18
<210>95
<211>18
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the downstream primer in amplification rs1133190SNP site
<400>95
TTGATGCTGC?GGCCAGTG 18
<210>96
<211>45
<212>DNA
＜213〉artificial sequence
＜223〉to the description of artificial sequence: the present invention is designed for the reverse extension primer in amplification rs1133190SNP site
<400>96
ATACCTACCA?CGCTACAGCC?GGACGCGGCT?GCTGAGCACG?TAGTT 45
Claims (1)
1. a method that detects 32 SNP loci polymorphisms on 24 sports-related genes simultaneously is characterized in that it comprises the steps:
(1) determines 24 mononucleotide polymorphism sites on the sports-related genes, these genes and site are: ACErs4461142, CKMM rs1133190, ADRB2 rs1042718, rs1042713, ADRA2A rs3750625, GDF8rs3791783, CNTF rs2515362, HLA-A rs3823342, KCNA4 rs1323860, CASQ1 rs3747621, CFTR rs4148724, PPARGC1A rs768695, AMPD1 rs2268701, ACTN3 rs540874, BDKRB2rs2069578, IGF-1 rs972936, rs7956547, UCP2 rs660339, LEPR rs12037879, rs12029311, FABP2 rs11935130, LEP rs12706832, rs11761556, NOS3 rs3918188, rs7830, MYLK rs820325, rs1350152, CNTFR rs6476455, COL1A1 rs2586488, rs2696247 and VDRrs4334089, rs4760648;
(2) PCR primer and the single-basic extension primer that designs each mononucleotide polymorphism site seen the described primer sequence of sequence 1-96, and it is synthetic to transfer to biotech company;
(3) the national elite's of collection anticoagulated whole blood sample extracts genomic dna ,-80 ℃ of freezing preservations.After specimen amount reaches some amount, carry out follow-up experimental procedure;
(4) on the PCR instrument, carry out multiplex PCR with the amplification aim sequence, and carry out multiple single base extension; Then with sample panel on existing probe carry out hybridization; By the gene type system mononucleotide polymorphism site in each sample is carried out gene type, analysis, derived data at last.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010100752 CN101805790A (en) | 2010-01-26 | 2010-01-26 | Method for simultaneously detecting polymorphism of 32 SNP loci on 24 sports-related genes |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010100752 CN101805790A (en) | 2010-01-26 | 2010-01-26 | Method for simultaneously detecting polymorphism of 32 SNP loci on 24 sports-related genes |
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| Publication Number | Publication Date |
|---|---|
| CN101805790A true CN101805790A (en) | 2010-08-18 |
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| CN 201010100752 Pending CN101805790A (en) | 2010-01-26 | 2010-01-26 | Method for simultaneously detecting polymorphism of 32 SNP loci on 24 sports-related genes |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864223A (en) * | 2012-08-30 | 2013-01-09 | 山东百福基因科技有限公司 | Detection kit for highly specific multiple genes related to movement functions and detection method thereof |
| CN103020490A (en) * | 2011-09-26 | 2013-04-03 | 深圳华大基因科技有限公司 | Quality control locus selection method and device for sequencing of target area |
| CN106086222A (en) * | 2016-08-24 | 2016-11-09 | 厦门美因生物科技有限公司 | Motion detecting and evaluating genes method and system based on qPCR typing method |
| CN107475400A (en) * | 2017-09-07 | 2017-12-15 | 西北农林科技大学 | A kind of method and its dedicated kit of MYLK4 genes auxiliary detection ox growth traits |
| CN107893120A (en) * | 2017-11-28 | 2018-04-10 | 保定佑安生物科技有限公司 | The detection method and application of the primer sets of detection motion gene SNP and its application and product and detection motion gene SNP |
| CN107904302A (en) * | 2017-11-29 | 2018-04-13 | 昆明理工大学 | One group of primer for detecting anticoagulant related gene polymorphism at the same time and application |
| CN109468388A (en) * | 2018-12-25 | 2019-03-15 | 北京北基医学检验实验室有限公司 | A kind of primer sets and detection kit of amplification and locomitivity related gene |
| CN112195229A (en) * | 2020-09-07 | 2021-01-08 | 中国人民解放军火箭军特色医学中心 | Kit for simultaneously detecting multiple SNP sites related to radiosensitivity |
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2010
- 2010-01-26 CN CN 201010100752 patent/CN101805790A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103020490A (en) * | 2011-09-26 | 2013-04-03 | 深圳华大基因科技有限公司 | Quality control locus selection method and device for sequencing of target area |
| CN103020490B (en) * | 2011-09-26 | 2015-11-25 | 深圳华大基因科技服务有限公司 | Quality Control site choosing method and device in the order-checking of target area |
| CN102864223A (en) * | 2012-08-30 | 2013-01-09 | 山东百福基因科技有限公司 | Detection kit for highly specific multiple genes related to movement functions and detection method thereof |
| CN102864223B (en) * | 2012-08-30 | 2015-02-11 | 山东百福基因科技有限公司 | Detection kit for highly specific multiple genes related to movement functions and detection method thereof |
| WO2018036495A1 (en) * | 2016-08-24 | 2018-03-01 | 厦门美因生物科技有限公司 | Qpcr genotyping technique-based athleticism gene detection and evaluation method and system |
| CN106086222A (en) * | 2016-08-24 | 2016-11-09 | 厦门美因生物科技有限公司 | Motion detecting and evaluating genes method and system based on qPCR typing method |
| CN107475400A (en) * | 2017-09-07 | 2017-12-15 | 西北农林科技大学 | A kind of method and its dedicated kit of MYLK4 genes auxiliary detection ox growth traits |
| CN107475400B (en) * | 2017-09-07 | 2020-03-06 | 西北农林科技大学 | Method for auxiliary detection of cattle growth traits through MYLK4 gene and special kit thereof |
| CN107893120A (en) * | 2017-11-28 | 2018-04-10 | 保定佑安生物科技有限公司 | The detection method and application of the primer sets of detection motion gene SNP and its application and product and detection motion gene SNP |
| CN107893120B (en) * | 2017-11-28 | 2021-05-04 | 保定佑安生物科技有限公司 | Primer group for detecting motion gene SNP, application and product thereof, and detection method and application for detecting motion gene SNP |
| CN107904302A (en) * | 2017-11-29 | 2018-04-13 | 昆明理工大学 | One group of primer for detecting anticoagulant related gene polymorphism at the same time and application |
| CN109468388A (en) * | 2018-12-25 | 2019-03-15 | 北京北基医学检验实验室有限公司 | A kind of primer sets and detection kit of amplification and locomitivity related gene |
| CN112195229A (en) * | 2020-09-07 | 2021-01-08 | 中国人民解放军火箭军特色医学中心 | Kit for simultaneously detecting multiple SNP sites related to radiosensitivity |
| CN112195229B (en) * | 2020-09-07 | 2022-07-12 | 中国人民解放军火箭军特色医学中心 | Kit for simultaneously detecting multiple SNP sites related to radiosensitivity |
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Application publication date: 20100818 |