CN102559851B - Specific primers and liquid phase chip for SNP (Single Nucleotide Polymorphism) detection of STK39 (Serine/Threonine Kinase) gene - Google Patents
Specific primers and liquid phase chip for SNP (Single Nucleotide Polymorphism) detection of STK39 (Serine/Threonine Kinase) gene Download PDFInfo
- Publication number
- CN102559851B CN102559851B CN201010590874.9A CN201010590874A CN102559851B CN 102559851 B CN102559851 B CN 102559851B CN 201010590874 A CN201010590874 A CN 201010590874A CN 102559851 B CN102559851 B CN 102559851B
- Authority
- CN
- China
- Prior art keywords
- seq
- primer
- tag sequence
- snp site
- comprised
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 30
- 239000007791 liquid phase Substances 0.000 title claims abstract description 29
- 239000002773 nucleotide Substances 0.000 title claims abstract description 8
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 8
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 6
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 title abstract description 7
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 title abstract description 5
- 241000183024 Populus tremula Species 0.000 claims description 33
- 101000701497 Homo sapiens STE20/SPS1-related proline-alanine-rich protein kinase Proteins 0.000 claims description 32
- 230000008859 change Effects 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 5
- 230000000295 complement effect Effects 0.000 claims description 4
- 239000003086 colorant Substances 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 abstract description 7
- 206010064571 Gene mutation Diseases 0.000 abstract description 3
- 230000003321 amplification Effects 0.000 abstract description 3
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 3
- 108700028369 Alleles Proteins 0.000 abstract 1
- 239000004005 microsphere Substances 0.000 abstract 1
- 102100030491 STE20/SPS1-related proline-alanine-rich protein kinase Human genes 0.000 description 13
- 239000012530 fluid Substances 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 11
- 238000013461 design Methods 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 10
- 238000000034 method Methods 0.000 description 10
- 239000000523 sample Substances 0.000 description 10
- 238000009396 hybridization Methods 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 239000011550 stock solution Substances 0.000 description 5
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000013016 damping Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 230000000869 mutational effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000007403 mPCR Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- VLEIUWBSEKKKFX-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O VLEIUWBSEKKKFX-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 108010066124 Protein S Proteins 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 102000029301 Protein S Human genes 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000001631 hypertensive effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
- -1 polyoxyethylene Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses specific primers and a liquid phase chip for SNP (Single Nucleotide Polymorphism) detection of a STK39 (Serine/Threonine Kinase) gene. The liquid phase chip mainly comprises an ASPE (Allele Specific Primer Extension) primer composed of a tag sequence at a 5' side and specific primers aiming at target gene mutation at a 3' side. The specific primers are respectively: SEQ ID NO.7 and SEQ ID NO.8 aiming at the T107C SNP site, SEQ ID NO.9 and SEQ ID NO.10 aiming at the G232A SNP site, and/or SEQ ID NO.11 and SEQ ID NO.12 aiming at the A190C SNP site; a microsphere coated with an anti-tag sequence; and an amplification primer. The coincidence rate of the detection result of the liquid phase chip for SNP detection of the STK39 gene provided by the invention and a sequencing method is as high as 100%. The prepared liquid phase chip has an excellent signal-noise ratio and can realize parallel detection of multiple polymorphism sites.
Description
Technical field
The present invention bends in biology field, relates to medical science and biotechnology, concrete a kind of STK39 gene SNP detection specific primer and the liquid-phase chip of relating to.
Background technology
Serine/threonine kinase gene (serine/threonine kinase, STK39) is positioned on No. 2 karyomit(e)s of the mankind, comprises 18 exons, the about 300kb of total length.
The main biochemical function of STK39 is that coding produces a kind of specified protein SPAK, participates in the process of regulation and control renal excretion salt.Investigator has carried out the functional study of cell levels to STK39, find that STK39 and WNK kinases and the positively charged ion-chlorion albumen that cotransports interacts.WNK kinases can activate the Protein S PAK of STK39 coding, can be further after the latter activates and positively charged ion-chlorion protein-interacting that cotransports, thus control the transhipment of sodium-chlor and the secretion of kidney sodium-chlor in osmotic pressure capacity regulating.STK39 is if there is variation, and the ability of body discharges salt will decline, and suffer from hypertensive probability increases thereupon.
The STK39 gene mutation site of target detect of the present invention, it is as shown in the table:
| Sequence number | The content of AGT site mutation | Write a Chinese character in simplified form |
| 1 | , there is T → C sudden change in the 107th Nucleotide of SEQ ID NO.25 | T107C |
| 2 | , there is G → A sudden change in the 232nd Nucleotide of SEQ ID NO.26 | G232A |
| 3 | , there is A → C sudden change in the 190th Nucleotide of SEQ ID NO.27 | A190C |
The product that detects at present STK39 is generally the RT-PCR of PCR-based technology, quantitative fluorescent PCR, traditional solid phase chip technology, direct Sequencing etc., there is separately the shortcomings such as sensitivity is low, sample easily pollutes, false positive rate height in these technology, owing to detecting the limitation of flux, can not meet the needs of practical application simultaneously.
Summary of the invention
One of object of the present invention is to provide a kind of STK39 gene SNP detection liquid-phase chip, and this liquid-phase chip can be used for detecting wild-type and the saltant type of STK39 gene three kinds of common genotype T107C, G232A and A190C.
A STK39 gene SNP detection liquid-phase chip, mainly includes:
(A). the ASPE primer of the wild-type designing respectively for every kind of SNP site and saltant type: the Auele Specific Primer for goal gene sudden change forms by the tag sequence of 5 ' end and 3 ' end for every kind of ASPE primer, and the Auele Specific Primer of described wild-type and saltant type is respectively: for the SEQ ID NO.7 in T107C SNP site and SEQ ID NO.8, for the SEQ ID NO.9 in G232A SNP site and SEQ ID NO.10 and/or for SEQ ID NO.11 and the SEQ ID NO.12 in A190C SNP site; Described tag sequence is selected from SEQ ID NO.1-6;
(B). there is anti-tag sequence microballoon coated, that there is different colours coding, in the middle of described anti-tag sequence is connected with microballoon, be also provided with spacerarm sequence; Described anti-tag sequence is selected from the sequence in SEQ ID NO.13～SEQ ID NO.18, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). for amplifying, need primer detection, that there is the target sequence in corresponding SNP site.
Preferably, described amplimer is: for the SEQ ID NO.19 in T107C SNP site and SEQ ID NO.20, for the SEQ ID NO.21 in G232A SNP site and SEQ ID NO.22 and/or for SEQ IDNO.23 and the SEQ ID NO.24 in A190C SNP site
Preferably, described ASPE primer is: the sequence being comprised of SEQ ID NO.1 and SEQ ID NO.7 and the sequence being comprised of SEQ ID NO.2 and SEQ ID NO.8, the sequence being comprised of SEQ ID NO.3 and SEQ ID NO.9 and the sequence being comprised of SEQ ID NO.4 and SEQID NO.10 and/or the sequence being comprised of SEQ ID NO.5 and SEQ IDNO.11 and the sequence being comprised of SEQ ID NO.6 and SEQID NO.12.
Major advantage of the present invention is:
1. the identical rate of the detected result of STK39 gene SNP detection liquid-phase chip provided by the present invention and sequencing, up to 100%, detects the needed time well below conventional sequencing technologies, and realistic especially application needs.In the Auele Specific Primer very a large amount of, through lot of experiments, reaction checking, obtain the liquid-phase chip system of optimum combination, prepared liquid-phase chip has extraordinary signal-noise ratio, and does not substantially have cross reaction between designed probe and anti-tag sequence, the choosing and the combination of tag sequence label and concrete ASPE primer of tag sequence label, anti-tag sequence label, can avoid cross reaction, realize the parallel detection in a plurality of SNP site.
2. the ASPE primer specificity primer of the present invention design can the sensitive mutational site of identifying specifically target detect, accurately distinguishes the genotype of various types; In same reaction system, between different Auele Specific Primers, substantially there is not cross reaction between Auele Specific Primer and the pcr amplification product of non-target detect, detection specificity is good, and cross reacting rate is lower than 3%; Except detecting Single locus sudden change situation, also the polymorphism situation in a plurality of mutational sites of parallel detection simultaneously, detects effect consistent.
3. detection method step of the present invention is simple, three kinds of SNPs detect and can complete the amplification of three target sequences that contain SNP site by a step multiplex PCR, the many uncertain factors that exist in the complex operations processes such as repeated multiple times PCR have been avoided, thereby can greatly improve Detection accuracy, embodied accurate while qualitative and quantitative analysis feature.
It is not high that 4 the present invention have not only overcome traditional solid phase chip susceptibility, and the poor defect of repeatability of detected result is improved existing liquid-phase chip technology simultaneously, makes prepared microballoon can be applicable to different test items, has very strong expansion.The fluorescent signal value detecting improves greatly, thereby the sensitivity detecting is further enhanced, and signal to noise ratio strengthens, and detected result more accurately and reliably.
Embodiment
Embodiment 1STK39 gene SNP detection liquid-phase chip, mainly includes:
One, ASPE primer
Wild-type and saltant type for three kinds of common genotype T107C, G232A and the A190C of STK39 gene, design respectively specific primer sequence.ASPE primer is comprised of " Tag sequence+specific primer sequence ".ASPE primer sequence is as shown in the table:
The ASPE primer sequence (Tag sequence+specific primer sequence) of table 1STK395 gene
Every ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence for anti-tag sequence on corresponding microballoon, and 3 ' end is saltant type or the special primer fragment (as shown in Table 1 above) of wild-type.All ASPE primers are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with respectively the stock solution of 100pmol/mL with 10mmol/L Tris Buffer.
Two, the coated microballoon of anti-tag sequence
According to designed ASPE Auele Specific Primer fragment, select tag sequence, reduce to greatest extent between the anti-tag sequence of each microballoon and secondary structure that tag and ASPE Auele Specific Primer fragment may form, on 6 kinds of microballoons numberings of selection and microballoon, corresponding anti-tag sequence is as shown in table 2:
Corresponding anti-tag sequence on table 2 microballoon numbering and microballoon
6 kinds of microballoons selecting are purchased from U.S. Luminex company, by anti-tag sequence coated with microballoon on.Between anti-tag sequence and microballoon, be connected with the spacerarm sequence of 5-10 T, before each anti-tag sequence, add the spacerarm sequence of the preceding paragraph 5-10 T, anti-tag sequence is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.By synthetic anti-tag sequence sterilizing ddH
2o is made into the stock solution of 100nmol/mL.Described spacerarm is for for spaced apart by anti-tag and microballoon table picture or anti-tag is placed in to the sequence of hydrophilic environments.By the spacerarm sequence of suitable length is set between anti-tag sequence and microballoon, can reduce sterically hinderedly, improve the efficiency of hybridization and the specificity of hybridization.Common spacerarm sequence comprises poly dT, i.e. poly (dT), oligomerization four polyoxyethylene glycol and (CH
2)
nspacerarm (n>=3), as (CH
2)
12, (CH
2)
18deng.In addition, if exist poly (dA) to disturb, can also use poly (TTG) as spacerarm.Spacerarm of the present invention is preferably 5-10 T, and the coated process of microballoon is as follows:
Get respectively 5 * 10
6the carboxylated microballoon of individual above-mentioned numbering (purchased from Luminex company) is suspended in the MES solution of 50ul 0.1mol/L (pH4.5), adds the synthetic anti-tag molecule (100nmol/mL) of 10ul.EDC[1-Ethyl-(3-dimethylaminopropyl) carbodiimide of preparation 10ng/ml] (purchased from Pierce Chemical company) working fluid.Toward the EDC working fluid that adds 2.5ul in microballoon suspension, constant-temperature incubation 30 minutes, then add the EDC working fluid of 2.5ul, then constant-temperature incubation 30 minutes.After reaction finishes, the Tween-20 with 0.02% washs once, then washs once with 0.1% SDS liquid.The Tris-EDTA solution [10mmol/L Tris (pH8.0)] that the microballoon that is coated with anti-tag sequence after washing is resuspended in to 100ul, in 1mmol/LEDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains mutational site
For 3 kinds of common genotype T107C, G232A and the A190C of STK39 gene, design of amplification primers, to (in Table 3), amplifies respectively 3 target sequences that contain SNP site respectively.
Table 3 amplifies the primer of the target sequence with SNP site
All primers are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with respectively the stock solution of 100pmol/mL with 10mmol/L TrisBuffer.
Embodiment 2 uses the detection of STK39 gene test liquid-phase chip to sample
The formula of described various solution is as follows:
MES damping fluid (pH5.0) formula (250mL) of 50mM:
2 * Tm hybridization buffer
| Reagent | Source | Final concentration | The consumption of every 250ml |
| 1M Tris-HCl，pH8.0 | Sigma T3038 | 0.2M | 50ml |
| 5M NaCl | Sigma S5150 | 0.4M | 20ml |
| Triton X-100 | Sigma T8787 | 0.16％ | 0.4ml |
After filtration, be stored in 4 ℃.
ExoSAP-IT test kit is purchased from U.S. USB company.
Biotin labeled dCTP is purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample
Methods involving with reference to < < molecular cloning > > about DNA extraction, obtains DNA to be detected.
Two, the pcr amplification of testing sample
Design three pairs of primers, multiplex PCR one step amplifies and contains respectively three kinds of common genotype T107C, G232A of STK39 gene and the A190C target sequence of totally three, and sequence fragment size is respectively 467bp, 400bp and 467bp.Primer sequence (SEQID NO.19-24) is shown in shown in above-mentioned table 3.
First prepare multiple PCR primer working fluid: respectively get respectively the primer stock solution 100ul of SEQ ID NO.19-24 in 1.5ml Eppendorf tube, mix and be multiple PCR primer working fluid.Multi-PRC reaction system is as follows:
2 * damping fluid is (containing Mg
2+) 25ul
DNTP (each 2.5mmol/L) 4ul
Taq enzyme (5U/ul) 0.2ul
Multiple PCR primer working fluid (each 8.3pmol/mL) 6ul
Template DNA (1Ong/ul) 2ul
ddH
2O 12.8ul
Be total to 50ul
Pcr amplification program is: 95 ℃ of 3min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 40s, 30 circulations; 72 ℃ of 10min; 4 ℃ save backup.
Three, the enzyme of PCR product is cut processing
1. get the reacted product of 7.5ul PCR, add 1ul 10 * SAP damping fluid, 1ul SAP enzyme and 0.5ul Exo-I enzyme;
Hatch 15min for 2.37 ℃, hatch 15min for 80 ℃, the enzyme that deactivation is unnecessary.The product that enzyme is cut after processing is directly used in follow-up ASPE primer extension reaction.
Four, site-specific primer extension reaction (ASPE)
Utilize the ASPE primer of above-mentioned design to carry out primer extension reaction, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on reacted product band.
First the ASPE primer working fluid that preparation mixes: get respectively three kinds of common genotype T107C, G232A of STK39 gene to be detected and the corresponding wild-type of A190C and saltant type ASPE primer stock solution 10ul in 1.5ml Eppendorf tube, add 10mmol/L Tris Buffer to mend to 200ul, mix and be ASPE mix primer working fluid.The system of ASPE reaction is as follows:
10 * damping fluid 2ul
MgCl
2(50mmol/L) 0.5ul
Biotin-dCTP(400umol/L) 0.25ul
DATP, dGTP, dTTP mixed solution (each 100umol/L) 1ul
Tsp enzyme (5U/ul) 0.25ul
ASPE primer working fluid (each 500nmol/L) 1ul mixing
Enzyme is cut the pcr amplification product 5ul of processing
ddH
2O 10ul
Be total to 20ul
Response procedures is: 96 ℃ of 2min; 94 ℃ of 30s, 54 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ save backup.
Five, hybridization
1. according to the ASPE primer of design, (microballoon concentration is 2.5 * 10 to the corresponding 6 kinds of microballoons of every group selection
5individual/ml).
2. get respectively the microballoon of every kind of numbering of 1ul in the Eppendorf tube of 1.5ml;
3. microballoon is in >=centrifugal the 1-2min of 10000g;
4. supernatant discarded, microballoon is resuspended in 2 * Tm hybridization buffer of 100ul, and vortex mixes;
5. get the above-mentioned microballoon suspension of 25ul in the 96 corresponding holes of hole filter plate, control wells adds the ddH of 25ul
2o;
6. get the ASPE reaction solution of 5-25ul in corresponding hole, use ddH
2o complements to 50ul;
7. with masking foil, encase 96 orifice plates with lucifuge, 95 ℃ of 60s, 37 ℃ of 15min are hatched hybridization;
8. the microballoon after hybridization is in >=centrifugal the 2-5min of 3000g;
9. remove supernatant, microballoon is resuspended in 1 * Tm hybridization buffer of 75ul;
10. microballoon is in >=centrifugal the 2-5min of 3000g;
11. are resuspended in microballoon in 1 * Tm hybridization buffer of 75ul, and adding 15ul concentration is the SA-PE (SA-PE) of 10ug/ml;
Hatch 15min for 12.37 ℃, on Luminex instrument, detect.
Six, result detects and data analysis
Reaction after product detects by Luminex serial analysis instrument.Detected result is as shown in table 4, table 5 and table 6.
Fluorescent value (MFI) and data processing are had to following requirement:
1. each site need have at least an allelotrope MFI to be greater than 300 and be greater than 10 * PCR negative control MFI;
2.NET MFI=sample MFI-PCR negative control MFI (NET MFI be less than 0 with 0 expression);
3. meet the data of above two conditions, by following formula, calculate sudden change ratio:
Sudden change ratio=saltant type NET MFI ÷ (saltant type NET MFI+ wild-type NET MFI)
4. the sudden change ratio definite threshold (cut-off value) to each detection site rule of thumb, to divide wild-type homozygote, heterozygote and saltant type homozygote.
Use present method to detect 20 increments STK39 gene SNP site originally, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Arranging of threshold value (cut-off value) is as follows: sudden change ratio range is considered as wild-type homozygote at 0%-20%; 30%-70% is considered as heterozygote; 80%-100% is considered as anomaly homozygote.With sequencing, detect with liquid-phase chip result and compare, calculate the identical rate of classifying method detected result provided by the present invention.Present method detects 20 increments STK39 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.Visible STK39 gene SNP detection liquid-phase chip provided by the present invention can detect the SNP type of STK39 exactly, and result is reliable and stable.
Table 4 pattern detection result (MFI)
Table 5 sample STK39 transgenation ratio (%)
Table 6 sample STK39 gene mutation type analytical results
| Catalogue number(Cat.No.) | Liquid-phase chip detected result | Sequencing result |
| 1 | Wild-type | Wild-type |
| 2 | Wild-type | Wild-type |
| 3 | 107AG | 107AG |
| 4 | 232AA | 232AA |
| 5 | Wild-type | Wild-type |
| 6 | Wild-type | Wild-type |
| 7 | Wild-type | Wild-type |
| 8 | Wild-type | Wild-type |
| 9 | 107GG | 107GG |
| 10 | 190CC | 190CC |
| 11 | Wild-type | Wild-type |
| 12 | Wild-type | Wild-type |
| 13 | Wild-type | Wild-type |
| 14 | Wild-type | Wild-type |
| 15 | Wild-type | Wild-type |
| 16 | 190CC | 190CC |
| 17 | Wild-type | Wild-type |
| 18 | Wild-type | Wild-type |
| 19 | Wild-type | Wild-type |
| 20 | Wild-type | Wild-type |
The detection of the liquid-phase chip of the ASPE primer that embodiment 3 is different to STK39 gene SNP site
One, the design that prepared by liquid-phase chip (selection of Tag sequence and Anti-Tag sequence)
It is example that the STK39 gene T107C site mutation of take detects liquid-phase chip, respectively for the wild-type of T107C and the specific primer sequence of saltant type design ASPE primer 3 ' end, the Tag sequence of ASPE primer 5 ' end is selected from SEQ ID NO.1-SEQID NO.6, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing being coated on microballoon is selected from SEQ ID NO.13-SEQ ID NO.18.Specific design is as shown in following table (table 7).Synthetic, the anti-tag sequence of ASPE primer are coated with microballoon, amplimer, detection method etc. as described in embodiment 1 and embodiment 2.
Design prepared by table 7 liquid-phase chip
Two, sample detection
The liquid-phase chip that adopts above-mentioned design to prepare, detects sample 21-40 by testing process described in embodiment 2 and method, and detected result is as follows:
Table 8 pattern detection result and Polymorphism Analysis
Other is for the liquid-phase chip in different mutational sites, and ASPE primer uses different Tag sequences, and its result is still reliable and stable, and concrete data are omitted.And ASPE primer is while selecting in embodiment 1 collocation of tag sequence and specific primer sequence, effect better (signal to noise ratio is better), referring to the present embodiment test group 1.Other different tag sequences and specific primer sequence collocation, with coming to the same thing of embodiment 2 and the present embodiment, concrete data are omitted.
Be more than for the illustrating of possible embodiments of the present invention, but this embodiment is not in order to limit the scope of the claims of the present invention, allly do not depart from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the scope of the claims of the present invention.
Claims (4)
1. a STK39 gene SNP detection liquid-phase chip, is characterized in that, mainly includes:
(A). the ASPE primer of the wild-type designing respectively for every kind of SNP site and saltant type: the Auele Specific Primer for goal gene sudden change forms by the tag sequence of 5 ' end and 3 ' end for every kind of ASPE primer, and the Auele Specific Primer of described wild-type and saltant type is respectively: for the SEQ ID NO.7 in T107C SNP site and SEQ ID NO.8, for the SEQ ID NO.9 in G232A SNP site and SEQ ID NO.10 and/or for SEQ ID NO.11 and the SEQ ID NO.12 in A190C SNP site; Described tag sequence is selected from SEQ ID NO.1-6;
(B). there is anti-tag sequence microballoon coated, that there is different colours coding, in the middle of described anti-tag sequence is connected with microballoon, be also provided with spacerarm sequence; Described anti-tag sequence is selected from the sequence in SEQ ID NO.13～SEQ ID NO.18, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). for amplifying the primer need detecting, have the target sequence in corresponding SNP site, described amplimer is: for the SEQ ID NO.19 in T107C SNP site and SEQ ID NO.20, for the SEQ ID NO.21 in G232A SNP site and SEQ ID NO.22 and/or for SEQ ID NO.23 and the SEQ ID NO.24 in A190C SNP site;
Described T107C SNP site is that T → C sudden change occurs the 107th Nucleotide of SEQ ID NO.25,
Described G232A SNP site is that G → A sudden change occurs the 232nd Nucleotide of SEQ ID NO.26,
Described A190C SNP site is that A → C sudden change occurs the 190th Nucleotide of SEQ ID NO.27.
2. STK39 gene SNP detection liquid-phase chip according to claim 1, it is characterized in that, described ASPE primer is: the primer being comprised of SEQ ID NO.1 and SEQ ID NO.7 and the primer being comprised of SEQ ID NO.2 and SEQ ID NO.8, the primer being comprised of SEQ ID NO.3 and SEQ ID NO.9 and the primer being comprised of SEQ ID NO.4 and SEQ ID NO.10 and/or the primer being comprised of SEQ ID NO.5 and SEQ ID NO.11 and the primer being comprised of SEQ ID NO.6 and SEQ ID NO.12.
3. STK39 gene SNP detection liquid-phase chip according to claim 1, is characterized in that, mainly comprises following composition:
(A). described ASPE primer is: the primer being comprised of SEQ ID NO.1 and SEQ ID NO.7 and the primer being comprised of SEQ ID NO.2 and SEQ ID NO.8, the primer being comprised of SEQ ID NO.3 and SEQ ID NO.9 and the primer being comprised of SEQ ID NO.4 and SEQ ID NO.10 and the primer being comprised of SEQ ID NO.5 and SEQ ID NO.11 and the primer being comprised of SEQ ID NO.6 and SEQ ID NO.12;
(B). there is anti-tag sequence microballoon coated, that there is different colours coding, in the middle of described anti-tag sequence is connected with microballoon, be also provided with spacerarm sequence; Described anti-tag sequence is selected from the sequence in SEQ ID NO.13～SEQ ID NO.18, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). described amplimer is: for the SEQ ID NO.19 in T107C SNP site and SEQ ID NO.20, for the SEQ ID NO.21 in G232A SNP site and SEQ ID NO.22 with for SEQ ID NO.23 and the SEQ ID NO.24 in A190C SNP site.
4. according to the STK39 gene SNP detection liquid-phase chip described in claim 1-3 any one, it is characterized in that, described spacerarm is 5-10 T.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010590874.9A CN102559851B (en) | 2010-12-16 | 2010-12-16 | Specific primers and liquid phase chip for SNP (Single Nucleotide Polymorphism) detection of STK39 (Serine/Threonine Kinase) gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010590874.9A CN102559851B (en) | 2010-12-16 | 2010-12-16 | Specific primers and liquid phase chip for SNP (Single Nucleotide Polymorphism) detection of STK39 (Serine/Threonine Kinase) gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102559851A CN102559851A (en) | 2012-07-11 |
| CN102559851B true CN102559851B (en) | 2014-11-26 |
Family
ID=46406455
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010590874.9A Expired - Fee Related CN102559851B (en) | 2010-12-16 | 2010-12-16 | Specific primers and liquid phase chip for SNP (Single Nucleotide Polymorphism) detection of STK39 (Serine/Threonine Kinase) gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102559851B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293831A (en) * | 2014-09-28 | 2015-01-21 | 上海云舜生物技术有限公司 | Method for establishing hypertension mouse model and application of hypertension mouse model |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009128953A2 (en) * | 2008-04-18 | 2009-10-22 | University Of Maryland, Baltimore | Genetic variants in a hypertension susceptibility gene stk39 and uses thereof |
| CN101565749B (en) * | 2009-04-15 | 2012-01-11 | 广州益善生物技术有限公司 | CYP2C19 and ABCB1 gene SNP detection liquid-phase chip and detection method thereof |
| CN101671739B (en) * | 2009-10-19 | 2012-07-25 | 广州益善生物技术有限公司 | Specific sequence, liquid phase chip and method for SNP detection of TPMT gene |
| CN101824466B (en) * | 2009-12-29 | 2012-07-18 | 广州益善生物技术有限公司 | Specific primer, liquid-phase chip and method for SNP detection of CYP2C9 and VKORC1 genes |
-
2010
- 2010-12-16 CN CN201010590874.9A patent/CN102559851B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN102559851A (en) | 2012-07-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102234684B (en) | Specific primer and liquid phase chip for BRAF genetic mutation detection | |
| CN102533948B (en) | apoB (apolipoprotein B) gene SNP (Single Nucleotide Polymorphism) detection specific primer and liquid-phase chip | |
| CN103374609B (en) | ABO gene mutation detection specific primers and liquid chip | |
| CN102994621B (en) | PTPN11 gene mutation detection specificity primer and liquid chip thereof | |
| CN102559851B (en) | Specific primers and liquid phase chip for SNP (Single Nucleotide Polymorphism) detection of STK39 (Serine/Threonine Kinase) gene | |
| CN102912002B (en) | ABCB2 gene polymorphism detection specific primers and liquid chip | |
| CN102191337B (en) | Specific primers and liquid phase chip for detecting polymorphism of cyckin-dependent kinase 5 regulatorysubunit-associated protein 1-like 1(CDKAL1) gene | |
| CN103031369B (en) | CDKN2A gene mutation detection specific primer and liquid chip | |
| CN102021237B (en) | Liquid phase chip and specificity primer for SNP detection of CYP4F2 and EPHX1 genes | |
| CN102559850B (en) | ApoA5 genic mutation detection specific primer and liquid phase chip | |
| CN102952866B (en) | Specific primer and liquid chip for detecting polymorphism of DPYD (dihydropyrimidine dehydrogenase) gene | |
| CN102181573B (en) | Specific primers and liquid-phase chip for detection of KITLG gene | |
| CN103374607B (en) | ABCC (ATP (adenosine triphosphate)-binding cassette, sub-family C)1 gene mutation detection specific primers and liquid chip | |
| CN102010898B (en) | EPHX1 (Microsomal epoxide hydrolase, mEH, EPHX1) gene detection specific primer and liquid phase chip | |
| CN103031367A (en) | VHL (Von Hippel Lindau) genetic mutation detection specific primer and liquid phase chip | |
| CN102533954B (en) | CYP17A1 (Cytochrome P450) genetic polymorphism detection specific primer and liquid-phase chip | |
| CN102533950B (en) | Specific primers and liquid-phase chip for SNP (Single Nucleotide Polymorphism) detection of apoC3 gene | |
| CN102888444B (en) | Factor II and Factor V genetic polymorphism detection specific primer and liquid-phase chip | |
| CN102191335B (en) | Specific primer and liquid-phase chip for SNP (single nucleotide polymorphism) detection of PIGU (phosphatidylinositol glycan anchor biosynthesis, class U) genes | |
| CN102952867B (en) | Specific primer and liquid chip for detecting polymorphism of SLC22A6 gene | |
| CN103374608B (en) | MTRR (5-methyltetrahydrofolate-homocysteine methyltransferase reductase) gene mutation detection specific primers and liquid chip | |
| CN102952870B (en) | Specific primer and liquid chip for detecting polymorphism of SLC22A2 gene | |
| CN102952868B (en) | Specific primer and liquid chip for detecting polymorphism of SLC15A2 gene | |
| CN103374611B (en) | LIG3 (DNA (deoxyribonucleic acid) Ligase III) gene mutation detection specific primers and liquid chip | |
| CN102952872B (en) | Specific primer and liquid chip for detecting polymorphism of SLCO1B3 gene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent of invention or patent application | ||
| CB02 | Change of applicant information |
Address after: Five 510663 Guangdong city of Guangzhou province Guangzhou Science City Moon Road No. 80, Guangzhou technology innovation base B, C Applicant after: SUREXAM BIO-TECH Co.,Ltd. Address before: Five 510663 Guangdong city of Guangzhou province Guangzhou Science City Moon Road No. 80, Guangzhou technology innovation base B, C Applicant before: GUANGZHOU SUREXAM BIO-TECH Co.,Ltd. |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: APPLICANT; FROM: GUANGZHOU YISHAN BIOTECHNOLOGY CO., LTD. TO: SUREXAM BIOTECHNOLOGY CO., LTD. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141126 |