CN103397103B

CN103397103B - A kind of method and test kit detecting SOCS family gene label single nucleotide polymorphism site

Info

Publication number: CN103397103B
Application number: CN201310376421.XA
Authority: CN
Inventors: 张安强; 蒋建新; 顾玮; 岳彩黎; 王海燕; 杜鹃
Original assignee: Third Affiliated Hospital of TMMU
Current assignee: Third Affiliated Hospital of TMMU
Priority date: 2013-08-26
Filing date: 2013-08-26
Publication date: 2016-04-20
Anticipated expiration: 2033-08-26
Also published as: CN103397103A

Abstract

The invention belongs to biology field, be specifically related to be applicable to single, double heavy PCR detects SOCS family gene label single nucleotide polymorphism site test kit and method in conjunction with pyrosequencing techniques, described method is specially: according to confirmed SOCS family gene label single nucleotide polymorphism site, design specificity amplification primer and sequencing primer, wherein, one in described specificity amplification primer with biotin labeling; With the DNA of sample to be measured for template, carry out PCR reaction with specificity amplification primer, obtain pcr amplification thing; Adopt alkaline denaturation to be separated into strand gained pcr amplification thing, get wherein after biotin labeling strand pcr amplification product purifying, carry out hybridization with described sequencing primer, and analytical results; SOCS target sequence selected by the present invention, and apply test kit of the present invention and can realize quick, easy, accurate, efficient, practical, economic detection SOCS label single nucleotide polymorphism genotype, the needs of Clinical Laboratory real work can be met, be beneficial to the generation of SOCS gene test predictive disease, developing risk.

Description

A kind of method and test kit detecting SOCS family gene label single nucleotide polymorphism site

Technical field

The invention belongs to biology field, be specifically related to be applicable to single, double heavy PCR detects SOCS family gene Tag SNP site test kit and method in conjunction with pyrosequencing techniques.

Background technology

Suppressors of cytokine signaling (SOCS) is as the cytokine signaling negativity Molecular regulator of cell inner expression, Signal Transduction path is started by specific cell surface receptor in cytokine, the growth of mediated immunity cell, differentiation, play a significant role during natural and Acquired immune response reacts.But excessive release or the suppression of the not normal cytokine caused of cytokine inhibitory factor regulation and control may cause the various diseases such as transformation reactions, autoimmune disorder, inflammation or tumour.Therefore the change of SOCS molecule in various diseases occurs, occur with disease, develop and prognosis closely related, but report less to the gene pleiomorphism of these molecules and genetic association research of disease development at present.

Single nucleotide polymorphism (SNP) refers to DNA sequence polymorphism in the colony that on karyomit(e), single nucleotide diversity causes, have that density is high, inheritance stability, be easy to the features such as automated analysis, be considered to the third generation genetic marker after restriction fragment length polymorphism, microsatellite polymorphism, can be used for the aspect such as the assignment of genes gene mapping, polymorphism analysis, is the common counter of study of disease genetic predisposition, medical diagnosis on disease, drug screening etc.Having developed more than 20 plants SNP detection method at present, as direct sequencing, probe hybridization method, flight mass spectrum analytical method, restriction fragment length polymorphism analysis method etc., but because of the expense of these methods, accuracy and the shortcoming being suitable for the aspects such as scale, limit to gene pleiomorphism and disease occurs, to develop and the genetic association of prognosis is studied.And pyrosequencing techniques is the DNA sequence analysis technology of a new generation based on enzyme cascade, under the synergy of archaeal dna polymerase, ATP sulfurylase, luciferase and apyrase 4 kinds of enzymes, each dNTP polymerization on DNA single chain is discharged coupling with first order fluorescence signal get up, by detecting release and the intensity of fluorescence, reach the object of the real time measure DNA sequence dna.This technology has the advantage of its uniqueness, without the need to electrophoresis, DNA fragmentation without the need to fluorescent mark; Have easy and simple to handle, testing cost is low, required sample size is little, quick, accurate, high-throughput, sensitivity high, meets Big Clinical Samples testing requirement.

Summary of the invention

An object of the present invention is the detection method providing SOCS family gene label single nucleotide polymorphism site, and it can simple and direct, intuitive and accurately detect and sentence read result SOCS tagged single-nucleotide polymorphic genotype.

The present invention sets up and optimizes single, double heavy PCR-and sees that phosphoric acid sequencing technologies detects the method in 12 Tag SNP sites of 7 SOCS genes, to it at the genetic characteristics of healthy population and and disease occurs, the genetic association that develops is studied.

For achieving the above object, technical scheme of the present invention is:

1. one kind is detected the method in SOCS family gene label single nucleotide polymorphism site, specifically comprise the following steps: the 1) design of Auele Specific Primer, namely according to confirmed SOCS family gene label single nucleotide polymorphism site, as the data announced in database in embodiment one.Design specificity amplification primer and sequencing primer, wherein, one in described specificity amplification primer with biotin labeling; 2) pcr amplification, namely with the DNA of sample to be measured for template, carry out PCR reaction with the specificity amplification primer described in step 1), obtain pcr amplification thing; 3) Manganic pyrophosphate complex initiation, by step 2) gained pcr amplification thing adopt alkaline denaturation make it be separated into strand, get wherein by after biotin labeled strand pcr amplification product purifying, carry out hybridization with sequencing primer described in step 1), and analytical results is to judge whether the target list nucleic acid of described sample to be measured possesses polymorphism.The innovative point of the technical program is mainly adopting Manganic pyrophosphate complex initiation method to detect the single nucleotide polymorphism of SOCS family gene first.

Further, a kind of described method detecting SOCS family gene label single nucleotide polymorphism site, in step 1), described SOCS family gene label single nucleotide polymorphism site comprises:

CISrs2239751, CISrs414171, SOCS2rs10777530, SOCS3rs8064821, SOCS4rs1209087, SOCS5rs3829835, SOCS5rs17771255, SOCS5rs3768720, SOCS6rs9646604, SOCS6rs3809954, SOCS6rs1351887 and SOCS7rs3748726 site; Above-mentioned site obtains by the following method: utilize HapMap(http: //www.hapmap.org) snp database (HapMapDataRel28PhaseII+III, Aug10, onNCBIB36assembly, dbSNPb126) BeiJing, China's Chinese Han Population (CHB) SNP data within the scope of each gene (CIS, SOCS2-7) and upstream and downstream 3kb thereof are downloaded: 1) frame retrieval input gene name checks that each gene is located at HapMap, and upstream and downstream respectively extends 3kb; 2) select in the option of " Reports & Analysis ": DownloadSNPgenotypedata.3) select BeiJing, China's Chinese Han Population (ChineseHanBeijing, CHB) in " config ", obtain all SNP site in BeiJing, China's each gene of Chinese Han Population SOCS family and elongated area thereof.

The nucleotide sequence of the upstream amplification primer in described CISrs2239751 site is as shown in SEQIDNO:1, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:2, and sequencing primer is as shown in SEQIDNO:3; The nucleotide sequence of the upstream amplification primer in described CISrs414171 site is as shown in SEQIDNO:4, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:5, and sequencing primer is as shown in SEQIDNO:6; The nucleotide sequence of the upstream amplification primer in described SOCS2rs10777530 site is as shown in SEQIDNO:7, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:8, and sequencing primer is as shown in SEQIDNO:9; The nucleotide sequence of the upstream amplification primer in described SOCS3rs8064821 site is as shown in SEQIDNO:10, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:11, and sequencing primer is as shown in SEQIDNO:12; The nucleotide sequence of the upstream amplification primer in described SOCS4rs1209087 site is as shown in SEQIDNO:13, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:14, and sequencing primer is as shown in SEQIDNO:15; The nucleotide sequence of the upstream amplification primer in described SOCS5rs3829835 site is as shown in SEQIDNO:16, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:17, and sequencing primer is as shown in SEQIDNO:18; The nucleotide sequence of the upstream amplification primer in described SOCS5rs17771255 site is as shown in SEQIDNO:19, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:20, and sequencing primer is as shown in SEQIDNO:21; The nucleotide sequence of the upstream amplification primer in described SOCS5rs3768720 site is as shown in SEQIDNO:22, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:23, and sequencing primer is as shown in SEQIDNO:24; The nucleotide sequence of the upstream amplification primer in described SOCS6rs9646604 site is as shown in SEQIDNO:25, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:26, and sequencing primer is as shown in SEQIDNO:27; The nucleotide sequence of the upstream amplification primer in described SOCS6rs3809954 site is as shown in SEQIDNO:28, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:29, and sequencing primer is as shown in SEQIDNO:30; The nucleotide sequence of the upstream amplification primer in described SOCS6rs1351887 site is as shown in SEQIDNO:31, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:32, and sequencing primer is as shown in SEQIDNO:33; The nucleotide sequence of the upstream amplification primer in described SOCS7rs3748726 site is as shown in SEQIDNO:34, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:35, and sequencing primer is as shown in SEQIDNO:36.

Refer to following table:

In upper table, " bio " shows the amplimer be labeled.

Further, a kind of described method detecting SOCS family gene label single nucleotide polymorphism site, described step 2) in, described pcr amplification is double PCR amplification, SOCS2rs10777530 and

SOCS6rs9646604 combines, and/or SOCS3rs8064821 and SOCS5rs17771255 composition, and/or SOCS5rs3829835 and SOCS6rs1351887 combination, and/or CISrs414171 and SOCS7rs3748726 combination.

Further, a kind of described method detecting SOCS family gene label single nucleotide polymorphism site, described step 2) in, described pcr amplification is substance pcr amplification, CISrs2239751 site, SOCS4rs1209087 site, the pcr amplification in SOCS5rs3768720 site and SOCS6rs3809954 site is substance pcr amplification.

Two of object of the present invention is to provide a kind of test kit, quick, easy, accurate, efficient, practical, economic detection SOCS tagged single-nucleotide polymorphic genotype can be realized, the needs of Clinical Laboratory real work can be met, be beneficial to the generation of SOCS gene test predictive disease, developing risk.

For achieving the above object, technical scheme of the present invention is:

For detecting the test kit in SOCS family gene label single nucleotide polymorphism site, described test kit comprises the specificity amplification primer in SOCS family gene label single nucleotide polymorphism site, the Pyrosequencing primer in SOCS family gene label single nucleotide polymorphism site, and PCR reaction solution, PCR primer screening liquid, Manganic pyrophosphate complex initiation reaction solution.

Further, the described test kit for detecting SOCS family gene label single nucleotide polymorphism site, described specificity amplification primer is as shown in SEQIDNO:1-3, and/or as shown in SEQIDNO:4-6, and/or as shown in SEQIDNO:7-9, and/or as shown in SEQIDNO:10-12, and/or as shown in SEQIDNO:13-15, and/or as shown in SEQIDNO:16-18, and/or as shown in SEQIDNO:19-21, and/or as shown in SEQIDNO:22-24, and/or as shown in SEQIDNO:25-27, and/or as shown in SEQIDNO:28-30, and/or as shown in SEQIDNO:31-33, and/or as shown in SEQIDNO:34-36.

Three of object of the present invention is to provide SOCS family gene label single nucleotide polymorphism site, and it can be used for judging pyemia risk.

For achieving the above object, technical scheme of the present invention is:

SOCS family gene label single nucleotide polymorphism site, its nucleotide sequence is as shown in SEQIDNO:37-44.

Beneficial effect of the present invention is: (1) PCR primer two provided by the invention re-constituted amplification object fragment, two target DNA band equalizations that electrophoresis result shows under two re-constituteds are bright, single, illustrate that double assembly PCR amplimer efficiency provided by the invention is high, two pairs of primer amplification efficiency are suitable.(2) as can be seen to sample SOCS tagged single-nucleotide polymorphic gene pleiomorphism Manganic pyrophosphate complex initiation result (accompanying drawing 1), adopt test kit of the present invention and method, can simple and direct, intuitive and accurate SOCS tagged single-nucleotide polymorphic genotype be detected and interpretation.

To sum up, SOCS family gene target sequence selected by the present invention, and apply test kit of the present invention and can realize quick, easy, accurate, efficient, practical, economic detection SOCS tagged single-nucleotide polymorphic genotype, the needs of Clinical Laboratory real work can be met, be beneficial to the generation of SOCS gene test predictive disease, developing risk.

Accompanying drawing explanation

Fig. 1 carries out gene type assay result figure to the detection sample in rs2239751G/T site.

Fig. 2 carries out gene type assay result figure to the detection sample in rs1209087T/C site.

Fig. 3 carries out gene type assay result figure to the detection sample in rs3768720A/C site.

Fig. 4 carries out gene type assay result figure to the detection sample in rs3809954G/A site.

Fig. 5 carries out gene type assay result figure to the detection sample of utilization double PCR to rs10777530C/T and rs9646604A/G site.

Fig. 6 carries out gene type assay result figure to the detection sample of utilization double PCR to rs8064821G/T and rs17771255G/A site.

Fig. 7 carries out gene type assay result figure to the detection sample of utilization double PCR to rs3829835C/T and rs1351887T/A site.

Fig. 8 carries out gene type assay result figure to the detection sample of utilization double PCR to rs3748726T/C and rs414171T/A site.

Embodiment

Illustrated embodiment is to be described content of the present invention better, but is not that content of the present invention is only limitted to illustrated embodiment.So those of ordinary skill in the art carry out nonessential improvement and adjustment according to foregoing invention content to embodiment, still belong to protection scope of the present invention.

The screening in embodiment 1 candidate gene label single nucleotide polymorphism site

Utilize HapMap(http: //www.hapmap.org) snp database (HapMapDataRel28PhaseII+III; Aug10; onNCBIB36assembly; dbSNPb126) BeiJing, China's Chinese Han Population (CHB) SNP data within the scope of each gene (CIS, SOCS2-7) and upstream and downstream 3kb thereof are downloaded: 1) frame retrieval input gene name checks that each gene is located at HapMap, and upstream and downstream respectively extends 3kb; 2) select in the option of " Reports & Analysis ": DownloadSNPgenotypedata.3) select BeiJing, China's Chinese Han Population (ChineseHanBeijing, CHB) in " config ", obtain all SNP site in BeiJing, China's each gene of Chinese Han Population SOCS family and elongated area thereof.

Program human window is opened under TAGster software package catalogue.According to structure bin with select Tag SNP standard (minimum gene frequency>=0.05 and linkage relationship r between SNP ²>=0.8) parameters, can there is the Tag SNP of the bin figure and corresponding bin of survey region in action command " fconvert ", " TAGster ".

NCBI is utilized to download the sequence of each gene promoter (upstream region of gene 3kb) region SNP site.The sequence of wild-type and saltant type is submitted to respectively transcription factor forecast database TFSEARCH ( http:// www.cbrc.jp/research/db/ tFSEARCH.html), obtain the nuclear factor that may combine with it, and compare the impact that SNP site combines transcription factor.

Between comprehensive SNP, linkage strength and bioinformatic analysis finally determine that 7 genes, 12 the Tag SNP sites detected are as table 1.

A table 112 label single nucleotide polymorphism site information

Embodiment 2 single nucleotide polymorphism site pcr amplification primer and Pyrosequencing primer Design and synthesis

Utilize NCBI ( www.ncbi.nlm.nih.gov) GenBank downloads each Tag SNP site and neighbouring about 500bp genome sequence thereof.Utilize Manganic pyrophosphate complex initiation instrument bundled software " AssayDesign " to design single nucleotide polymorphism site pcr amplification primer and Pyrosequencing primer, select the primer pair that scoring is the highest.The primer designed entrusts the synthesis of Shanghai biotechnology company limited, and carries out a wherein PCR primer 5 ' end biotin modification.12 label single nucleotide polymorphism site amplimers and the sequencing primer of design refer to table 2.

Table 212 label single nucleotide polymorphism site amplimer and sequencing primer

Prepared by embodiment three Whole Blood Genomic DNA

Check Whole Blood Genomic DNA purification kit (producing reagent for Promega company of the U.S.), Manganic pyrophosphate complex initiation reagent quality guaranteed period and guarantee to add ethanol in WashBuffer, and carrying out mark at bottle cap; (2) Virahol and 75% ethanol; (3) 1.5mlEppendorf(EP in autoclaving validity period) pipe and various model pipettor and rifle head.

The trauma patient peripheral blood that EDTA anticoagulant tubes collect is taken out, thawed at room temperature mixing for several times of turning upside down from-80 refrigerators; Pipette 300ul whole blood and 900ulCellLysisSolution respectively and add the 1.5mlEP pipe carrying out corresponding sample labeling, incubated at room 10 minutes, put upside down mixing once every 2-3 minute; 10000g, 4 DEG C are centrifugal 1 minute; Take out EP pipe, observe white precipitate, discard red supernatant; Repetitive operation 2-3 time, until redfree agglomerate in precipitation; Add 300ulNucleiLysisSolution, mixing of fully vibrating, breaks up precipitation completely with 1ml or 200ul pipettor, and room temperature places 10 minutes with abundant lysing cell; Add 100ulProteinPrecipitationSolution, thermal agitation 20 seconds visible particulate precipitations; 12000g, 4 DEG C are centrifugal 3 minutes; Observe supernatant, if unlimpid, increase centrifugation time, if limpid pipettor slow sucking-off supernatant liquor is in new 1.5mlEP pipe, recording volume, adds the pre-cold isopropanol of equal-volume (-20 DEG C of precoolings), and the several that turns upside down gently is separated out to white flock DNA; 12000g, 4 DEG C are centrifugal 3 minutes, and white precipitate at the bottom of visible pipe, abandons supernatant; Add 500ul precooling 75% ethanol (-20 DEG C of precoolings), washing precipitation of softly turning upside down; 12000g, 4 DEG C are centrifugal 2 minutes, abandon supernatant; New filter paper placed by clean experiment table, and back-off EP manages, and blots liquid, air-dry DNA precipitation; Range estimation precipitation size, add 50-100ulDNARehydrationSolution, 65 DEG C of water-baths place 15 minutes, take out the abundant dissolving DNA precipitation of vortex oscillation every 5-6 minute.Measure nucleic acid concentration and purity with Nano-Space ultraviolet spectrophotometer after dissolving completely, nucleic acid concentration is greater than 20ng/ul and is considered as qualified, as concentration is not enough, can adds ethanol and again precipitates DNA and dissolve.In tube wall and pipe lid SD sample number again, preserve DNA sample for subsequent use to-80 DEG C of refrigerators.

Embodiment four polymerase chain reaction

Prepare district's preparation 50ulPCR reaction system (except DNA profiling) at reagent, each component and addition are as following table:

Component	Volume	Final concentration
			2×PCR Mix	25ul	1×
SNP1 upstream primer	1ul	0.2uM
			SNP1 downstream primer	1ul	0.2uM
SNP2 upstream primer	1ul	0.2uM
			SNP2 downstream primer	1ul	0.2uM
Water	20ul

Prepare district at sample and of short duration centrifugal rear taking-up 1ul(30ng managed to the EP that Whole Blood Genomic DNA is housed) in reaction system, and on PCR reaction tubes, mark sample number into spectrum and each combination number, of short duration centrifugal after the mixing of PCR reaction system vortex; Carry out pcr amplification reaction in reaction zone, amplification instrument be set according to following loop parameter:

Step number	Temperature	Treatment time	Cycle number
				1	95℃	3min
2	95℃	15s
				3	Tm	30s
4	72℃	20s	Go to step2，for45cycle
				5	72℃	5min
6	4℃	Holding

After setting program, selective reaction system 50ul, clicks " start " and starts instrument operation.

3. Manganic pyrophosphate complex initiation single stranded sample purifying

Before purifying, reagent and instrument prepare: before Sample Purification on Single, guarantee that all solution reaches room temperature, open precise temperature control heating container, regulate temperature to reach 80 DEG C.

Single stranded sample separation and purification operates: in PSQ96 plate, add the annealingbuffer that 40 μ l contain each 0.3 μM of sequencing primer of SNP1 and SNP2 in advance, generally add each 10pM sequencing primer 2ul; Vertex is used to mix Sepharosebeads; Transfer in an Eppendorf pipe by needing the Sepharoebeads total amount (every sample 2.5 μ l) used; In Sepharosebead, add bindingbuffer, make average each sample about have the volume of 40 μ l, mixture is mixed; Above mixture is added in PCR primer (50 μ l reaction volume), every sample 40 μ l; PCR primer is mixed 10 minutes at normal temperatures, beads is fully combined with vitamin H, for comparatively long segment, can proper extension mixing time; In Vacuumprepworkstation, in four sample panel, add 180ml high purity water, 70% ethanol, washingbuffer and 120mlDenaturationbuffer successively; Open the pump of vacuumprepworkstation, vacuumpreptool is cleaned 30 seconds in high purity water; Then move on in PCR plate by vacuumpreptool, crawl sepharosebeads(please completes this at beads and operates in PCR primer was in conjunction with latter three minutes, does not allow Beads sink to again at the bottom of pipe); Pick up PCR plate, check whether that most of beads has been attracted on vacuumpreptool; Vacuumpreptool is put into 70% ethanol 5 seconds; Then 5 seconds are moved on in denatureationbuffer; Move on to again in washingbuffer and clean 5-10 second; Suction nozzle is placed on the top of the corresponding plate hole containing sequencing primer, does not contact liquid level, turn off pump; Vacuumpreptool is put into the PSQ96 plate containing sequencing primer, shake, release sepharosebeads(sequencing primer also can finally add); The PSQ96 plate being placed with sample to be placed in accurate temperature control box 80 DEG C of heating 2 minutes, to take out cool to room temperature, can Pyrosequencing reaction be carried out.

Clear after separation and purification: not open vacuum pump and valve, use high purity water cleaning vacuumpreptool, the Beads do not come off on a small quantity is eluted; Vacuum pump and valve is opened again, with about 250ml high purity water cleaning tool after changing high purity water; Turn off vacuum pump and valve, be sidelong by vacuumpreptool, room temperature is dried; Clean the plastic channel of all splendid attire reagent solutions, naturally dry, powered-down, hide plant and instrument and prevent dust.

Manganic pyrophosphate complex initiation

Manganic pyrophosphate complex initiation instrument shifts to an earlier date 90 minutes and opens preheating, and setting working procedure, calculates each using amount of reagent of this experiment and be added into corresponding agent bin; The sample prepared and agent bin are put into instrument correspondence position, clicks " Run " working procedure; After detection completes, close software process status window, preserve sequencing result.

Manganic pyrophosphate complex initiation interpretation of result

In " SNPRuns " folder, open above-mentioned operating file, select " SNPmode ", click " AnalyzeAll " and gene type assay is carried out to all detection samples.Analytical results refers to Fig. 1-Fig. 8: as shown in Figure 1 can clear interpretation CISrs2239751GG/GT/TT genotype, as shown in Figure 2 can clear interpretation, a SOCS4rs1209087TT/TC/CC genotype in 06/806 property amplimer, as shown in Figure 3 can clear interpretation

SOCS5rs3768720AA/AC/CC genotype, as shown in Figure 4 can clear interpretation

SOCS6rs3809954GG/GA/AA genotype, as shown in Figure 5 can clear interpretation

SOCS2rs10777530AA/AG/GG and SOCS6rs9646604AA/AG/GG genotype, as shown in Figure 6 can clear interpretation SOCS3rs8064821AA/AC/CC and SOCS5rs17771255CC/CT/TT genotype, as shown in Figure 7 can clear interpretation SOCS5rs3829835CC/CT/TT and

SOCS6rs1351887AA/AT/TT genotype, as shown in Figure 8 can clear interpretation

CISrs414171AA/AT/TT and SOCS7rs3748726TT/TC/CC genotype; And in 806 routine severe trauma patients the somatotype success ratio in 12 Tag SNP sites between 99.8%-100%.CISrs2239751 somatotype success ratio 100%(806/806 in 806 routine severe trauma patients samples), SOCS4rs1209087 somatotype success ratio 100%(806/806), SOCS5rs3768720 somatotype success ratio 100%(806/806), SOCS6rs3809954 somatotype success ratio 100%(806/806), SOCS2rs10777530 somatotype success ratio 100%(806/806), SOCS6rs9646604 somatotype success ratio 100%(806/806), SOCS3rs8064821 somatotype success ratio 100%(806/806), SOCS5rs17771255 somatotype success ratio 100%(806/806), SOCS5rs3829835 somatotype success ratio 99.8%(804/806), SOCS6rs1351887 somatotype success ratio 100%(806/806), CISrs414171 somatotype success ratio 100%(806/806) and SOCS7rs3748726 somatotype success ratio 100%(806/806).

What finally illustrate is, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.

Claims

1. detect the method in SOCS family gene label single nucleotide polymorphism site, described method is the method for non-treatment and diagnostic purpose, it is characterized in that, specifically comprises the following steps:

1) design of Auele Specific Primer

According to confirmed SOCS family gene label single nucleotide polymorphism site, design specificity amplification primer and sequencing primer, wherein, one in described specificity amplification primer with biotin labeling; Described SOCS family gene label single nucleotide polymorphism site comprises: CISrs2239751, CISrs414171, SOCS2rs10777530, SOCS3rs8064821, SOCS4rs1209087, SOCS5rs3829835, SOCS5rs17771255, SOCS5rs3768720, SOCS6rs9646604, SOCS6rs3809954, SOCS6rs1351887 and SOCS7rs3748726 site;

The nucleotide sequence of the upstream amplification primer in described CISrs2239751 site is as shown in SEQIDNO:1, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:2, and sequencing primer is as shown in SEQIDNO:3; The nucleotide sequence of the upstream amplification primer in described CISrs414171 site is as shown in SEQIDNO:4, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:5, and sequencing primer is as shown in SEQIDNO:6; The nucleotide sequence of the upstream amplification primer in described SOCS2rs10777530 site is as shown in SEQIDNO:7, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:8, and sequencing primer is as shown in SEQIDNO:9; The nucleotide sequence of the upstream amplification primer in described SOCS3rs8064821 site is as shown in SEQIDNO:10, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:11, and sequencing primer is as shown in SEQIDNO:12; The nucleotide sequence of the upstream amplification primer in described SOCS4rs1209087 site is as shown in SEQIDNO:13, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:14, and sequencing primer is as shown in SEQIDNO:15; The nucleotide sequence of the upstream amplification primer in described SOCS5rs3829835 site is as shown in SEQIDNO:16, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:17, and sequencing primer is as shown in SEQIDNO:18; The nucleotide sequence of the upstream amplification primer in described SOCS5rs17771255 site is as shown in SEQIDNO:19, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:20, and sequencing primer is as shown in SEQIDNO:21; The nucleotide sequence of the upstream amplification primer in described SOCS5rs3768720 site is as shown in SEQIDNO:22, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:23, and sequencing primer is as shown in SEQIDNO:24; The nucleotide sequence of the upstream amplification primer in described SOCS6rs9646604 site is as shown in SEQIDNO:25, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:26, and sequencing primer is as shown in SEQIDNO:27; The nucleotide sequence of the upstream amplification primer in described SOCS6rs3809954 site is as shown in SEQIDNO:28, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:29, and sequencing primer is as shown in SEQIDNO:30; The nucleotide sequence of the upstream amplification primer in described SOCS6rs1351887 site is as shown in SEQIDNO:31, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:32, and sequencing primer is as shown in SEQIDNO:33; The nucleotide sequence of the upstream amplification primer in described SOCS7rs3748726 site is as shown in SEQIDNO:34, and the nucleotide sequence of downstream amplification primer is as shown in SEQIDNO:35, and sequencing primer is as shown in SEQIDNO:36

2) pcr amplification

With the DNA of sample to be measured for template, carry out PCR reaction with the specificity amplification primer described in step 1), obtain pcr amplification thing;

3) Manganic pyrophosphate complex initiation

By step 2) gained pcr amplification thing adopt alkaline denaturation make it be separated into strand, get wherein by after biotin labeled strand pcr amplification product purifying, carry out hybridization with sequencing primer described in step 1), and analytical results is to judge whether CIS, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6 and SOCS7 target site of described sample to be measured possesses polymorphism.

2. a kind of method detecting SOCS family gene label single nucleotide polymorphism site according to claim 1, it is characterized in that: described step 2) in, double PCR amplification is carried out in the combination in described SOCS2rs10777530 site and described SOCS6rs9646604 site, and/or the composition in described SOCS3rs8064821 site and described SOCS5rs17771255 site carries out double PCR amplification, and/or described SOCS5rs3829835 site and SOCS6rs1351887 Sites Combination carry out double PCR amplification, and/or described CISrs414171 site and described SOCS7rs3748726 Sites Combination carry out double PCR amplification.

3. a kind of method detecting SOCS family gene label single nucleotide polymorphism site according to claim 1, it is characterized in that: described step 2) in, described CISrs2239751 site, described SOCS4rs1209087 site, the pcr amplification in described SOCS5rs3768720 site and described SOCS6rs3809954 site is substance pcr amplification.

4. for detecting the test kit in SOCS family gene label single nucleotide polymorphism site, it is characterized in that: described test kit comprises the Auele Specific Primer in SOCS family gene label single nucleotide polymorphism site, the Pyrosequencing primer in SOCS family gene label single nucleotide polymorphism site, and PCR reaction solution, PCR primer screening liquid, Manganic pyrophosphate complex initiation reaction solution; Described specificity amplification primer is as SEQIDNO:1-2, SEQIDNO:4-5, SEQIDNO:7-8, SEQIDNO:10-11, SEQIDNO:13-14, SEQIDNO:16-17, SEQIDNO:19-20, SEQIDNO:22-23, SEQIDNO:25-26, shown in SEQIDNO:28-29, SEQIDNO:31-32, SEQIDNO:34-35; Described sequencing primer is as shown in SEQIDNO:3, SEQIDNO:6, SEQIDNO:9, SEQIDNO:12, SEQIDNO:15, SEQIDNO:18, SEQIDNO:21, SEQIDNO:24, SEQIDNO:27, SEQIDNO:30, SEQIDNO:33, SEQIDNO:36.

5. the test kit for detecting SOCS family gene label single nucleotide polymorphism site according to claim 4, is characterized in that, described test kit also comprises the vitamin H marking described amplimer.