CN102010900B - Liquid chip and specific primer for detecting SNP of GPIIIa gene and liquid chip and specific primer for detecting SNP of GPIIIa and COX-1 genes - Google Patents
Liquid chip and specific primer for detecting SNP of GPIIIa gene and liquid chip and specific primer for detecting SNP of GPIIIa and COX-1 genes Download PDFInfo
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Abstract
The invention discloses a liquid chip and specific primer for detecting SNP of a GPIIIa gene. The liquid chip mainly comprises a wild type ASPE primer and a mutant type ASPE primer which are respectively designed aiming at the SNP loci of the GPIIIa gene, microspheres which are respectively coated with specific anti-tag sequences and have different colors of codes and an amplification primer for amplifying the GPIIIa gene target sequence containing the T196C SNP loci, wherein each ASPE primer is formed by the tag sequence at the 5, terminal and the specific primer, aiming at the SNP loci of the target gene, at the 3, terminal; and the anti-tag sequences are selected from SEQ ID NO.17 to SEQ ID NO.24 and correspondingly complement and are paired with the tag sequences selected from each ASPE primer. The invention also provides a liquid chip and specific primer for detecting SNP of COX-1 and GPIIIa genes. Besides the corresponding composition of the GPIIIa gene, the liquid chip also comprises ASPE primer pair aiming at the SNP loci of the COX-1 gene, microspheres coated with specific anti-tag sequences and an amplification primer. The liquid chip has very good signal to noise ratio, can avoid cross reaction and can realize parallel detection of a plurality of SNP loci.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete a kind of COX-1 and GPIIIa gene SNP detection liquid-phase chip and the Auele Specific Primer of relating to.
Background technology
Platelet membrane glycoprotein (Glycoprotein IIIa, GPIIIa) is main thrombocyte integrin and activator, and it is a kind of sugar-protein compound of cross-film.Its effect is as acceptor, and the mediation Fibrinogen is incorporated into platelet surface and platelet aggregation subsequently.GPIIIa comprises human platelet antigen 1 (human platelet antigen, HPA) or PIA, there is the pleomorphism site of being combined with Fibrinogen in this gene, the L-LEU-33 that the GPIIIa gene is observed in case control study of the U.S. is replaced by proline(Pro), be the T196C sudden change (Leu33Pro of HPA-1, rs5918, be PLA1/A2 alloantigen system) and the emergency treatment thrombosis between relevant, its advance notice meaning on the known coronary heart disease risk factors such as hypertension, smoking, hypercholesterolemia or diabetes.
Cyclooxygenase (Cyclooxygenase, COX) is that arachidonic acid changes into rate-limiting enzyme important in the eicosanoids approach, as: synthetic (Prostaglandins, the PGs) of prostaglandin(PG).There are two kinds of isozymes in cyclooxygenase; be COX-1 and COX-2; COX-1 (Cyclooxygenase 1) belongs to house keeping enzyme; have another name called prostaglandin endoperoxides G/H synthetic enzyme (prostaglandin endoperoxide G/H synthase; PTGS1); in each tissue of body expression is arranged all, the prostaglandin(PG) that it produces participates in the normal physiological process of body and defencive function, all is stably express in most of normal cells.Activity stabilized generally speaking, under stress situation or when being subjected to some hormone, factors stimulated growth, its activity can slightly increase by 2 times～4 times.COX-1 albumen is comprised of 576 amino acid, and its encoding gene is positioned at karyomit(e) 9, and big or small approximately 22kb contains 11 exons.There are some researches show, carry the C50T of uneven complete linkage and the patient of A842G single nucleotide mutation, acetylsalicylic acid plays larger restraining effect than normal genotype in the PGH2 building-up process.In addition, the G123A loci polymorphism of COX-1 and aspirin resistance (Aspirin Resistance, AR) are relevant.Wherein, C50T (rs3842787) is positioned on the exon2 of COX-1, and A842G (rs10306114) occurs in the promoter region of COX-1, and G123A (rs3842788) occurs on the exon3 of COX-1.
At present to fluorescence quantitative PCR method, RFLP method and the dna sequencing method etc. of the general PCR-based technology of product of COX-1 and GPIIIa polymorphic detection, exist sensitivity low, the shortcoming that sample easily pollutes, false positive rate is high owing to detecting the limitation of flux, can not satisfy the needs of practical application simultaneously.
Summary of the invention
One of purpose of the present invention provides a kind of GPIIIa gene SNP detection liquid-phase chip, and this liquid-phase chip can be used for detecting wild-type and the mutant of the common genotype T196C of GPIIIa gene.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind of GPIIIa gene SNP detection liquid-phase chip mainly includes:
(A) for the SNP site of GPIIIa gene, the wild-type that designs respectively and the ASPE primer pair of mutant: every kind of ASPE primer holds the Auele Specific Primer for goal gene SNP site to form by the tag sequence and 3 ' of 5 ' end, and described Auele Specific Primer is: for SEQ ID NO.15 and the SEQ ID NO.16 in GPIIIa gene T196C SNP site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.8;
(B) be coated with respectively microballoon special anti-tag sequence, that have the different colours coding, described anti-tag sequence is selected from the sequence among SEQ ID NO.17～SEQ ID NO.24, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing, and described anti-tag sequence also is provided with the spacerarm sequence in the middle of being connected with microballoon;
(C) for the amplimer that amplifies the GPIIIa gene target sequence with T196C SNP site.
Preferably, described amplimer is the SEQ ID NO.31～SEQ IDNO.32 for GPIIIa gene T196C SNP site.
More preferably, described ASPE primer pair is: for the sequence that is comprised of SEQ ID NO.7 and SEQID NO.15 in GPIIIa gene T196C SNP site and the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.16.
The Auele Specific Primer that the present invention also provides the GPIIIa gene SNP to detect includes: for SEQ ID NO.15 and the SEQ ID NO.16 of wild-type and the mutant in T196C SNP site.
Another object of the present invention provides a kind of COX-1 and GPIIIa gene SNP detection liquid-phase chip, and this liquid-phase chip can carry out synchronous detection to COX-1 and GPIIIa gene.
Concrete technical scheme is as follows:
A kind of GPIIIa and COX-1 gene SNP detection liquid-phase chip mainly include:
(A) for the SNP site of COX-1 gene and GPIIIa gene, the respectively wild-type of design and the ASPE primer pair of mutant: every kind of ASPE primer is comprised of the tag sequence of 5 ' end and 3 ' the end Auele Specific Primer for the goal gene mutational site, and described Auele Specific Primer is: for the SEQ ID NO.9 in COX-1 gene C 50T SNP site and SEQ ID NO.10, for the SEQ ID NO.11 in COX-1 Gene A 842G SNP site and SEQ ID NO.12 and/or for SEQ ID NO.13 and the SEQ ID NO.14 in COX-1 gene G123A SNP site; And
SEQ ID NO.15 and SEQ ID NO.16 for GPIIIa gene T196C SNP site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.8;
(B) be coated with respectively microballoon special anti-tag sequence, that have the different colours coding, described anti-tag sequence is selected from the sequence among SEQ ID NO.17～SEQ ID NO.24, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing, and described anti-tag sequence also is provided with the spacerarm sequence in the middle of being connected with microballoon;
(C) be used for the amplimer that amplification has the COX-1 gene target sequence in C50T, A842G and/or G123A SNP site; And
Be used for the amplimer that amplification has the GPIIIa gene target sequence in T196C SNP site.
Preferably, described amplimer is for the SEQ ID NO.25 in COX-1 gene C 50T SNP site～SEQ ID NO.26, for the SEQ ID NO.27 in COX-1 Gene A 842G SNP site～SEQ ID NO.28, and/or for the SEQ ID NO.29 in COX-1 gene G123A SNP site～SEQ ID NO.30; And for the SEQID NO.31 in GPIIIa gene T196C SNP site～SEQ ID NO.32.
Further, described ASPE primer pair is: for the sequence that is comprised of SEQ IDNO.1 and SEQ ID NO.9 in COX-1 gene C 50T SNP site and the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.10, for the sequence that is formed by SEQ ID NO.3 and SEQ ID NO.11 in COX-1 Gene A 842G SNP site and the sequence that is formed by SEQ ID NO.4 and SEQ ID NO.12, and/or for the sequence that is formed by SEQ ID NO.5 and SEQID NO.13 in COX-1 gene G123A SNP site and the sequence that is formed by SEQ ID NO.6 and SEQ ID NO.14; And for the sequence that is formed by SEQ ID NO.7 and SEQ ID NO.15 in GPIIIa gene T196C SNP site and the sequence that is formed by SEQ ID NO.8 and SEQ ID NO.16.
Preferably, the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon is 5-10 T.
Major advantage of the present invention is:
1. the identical rate of detection method provided by the present invention and sequencing is up to 100%.Prepared COX-1 and GPIIIa gene SNP detection liquid-phase chip have extraordinary signal-noise ratio, and basically there is not cross reaction between designed probe and the anti-tag sequence, choosing and tag sequence label and the specifically combination of 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 type specificity primer of the present invention's design has extraordinary specificity, can accurately distinguish the genotype of various types.
3. detection method step of the present invention is simple, four kinds of SNPs detect and can finish four amplifications with the target sequence in SNP site by a step multiplex PCR, many uncertain factors of existing 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.
4. realistic especially application needs well below sequencing technologies commonly used the needed time of detection method provided by the present invention.
5. not only to have overcome traditional solid phase chip susceptibility not high in the present invention, and the defective that the repeatability of detected result is poor is improved existing liquid-phase chip technology simultaneously, so that prepared microballoon can be applicable to different test items, has very strong expansion.The fluorescent signal value that detects improves greatly, thereby so that the sensitivity that detects is further enhanced, signal to noise ratio strengthens, and detected result more accurately and reliably.
Embodiment
Embodiment 1 GPIIIa and COX-1 gene SNP detection liquid-phase chip mainly include:
One, ASPE primer
SNP site T196C (rs5918) for three kinds of common SNP site C50T (rs3842787), the A842G (rs10306114) of COX-1 gene and G123A (rs3842788), GPIIIa gene designs respectively specific primer sequence.The ASPE primer is comprised of " Tag sequence+specific primer sequence ".The ASPE primer sequence is as shown in the table:
Table 1 ASPE primer sequence (Tag sequence+specific primer sequence)
Every the ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence for anti-tag sequence on the corresponding microballoon, and 3 ' end is mutant or the special primer fragment (as shown in Table 1 above) of wild-type.All ASPE primers are synthetic 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 the 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, corresponding anti-tag sequence is as shown in table 2 on 8 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
Eight kinds of microballoons selecting are available from U.S. Luminex company, with the anti-tag sequence coated with microballoon on.Be connected with the spacerarm sequence of 5-10 T between anti-tag sequence and the microballoon, namely add the spacerarm sequence of the preceding paragraph 5-10 T before each anti-tag sequence, the anti-tag sequence is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.With synthetic anti-tag sequence sterilization ddH
2O is made into the stock solution of 100nmol/ml.Described spacerarm is for being used for anti-tag and microsphere surface is spaced apart or anti-tag is placed 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 efficient of hybridization and the specificity of hybridization.Common spacerarm sequence comprises poly dT, i.e. poly (dT), and oligomerization four polyoxyethylene glycol and (CH2) n spacerarm (n 〉=3) are such as (CH2) 12, (CH2) 18 etc.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 process that microballoon is coated with is as follows:
Get respectively 5 * 10
6The carboxylated microballoon of individual above-mentioned numbering (available 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 (N-(3-Dimethylaminopropyl-N-ethylcarbodiimide) (available from the Pierce Chemical company) working fluid of preparation 10ng/ml.The EDC working fluid that adds 2.5ul in the microballoon suspension, constant-temperature incubation 30 minutes adds the EDC working fluid of 2.5ul again, and constant-temperature incubation is 30 minutes again.After reaction finished, the Tween-20 washing with 0.02% was once washed once with 0.1% SDS liquid again.The microballoon that is coated with the anti-tag sequence after the washing is resuspended in the Tris-EDTA solution [10mmol/L Tris (pH8.0)] of 100ul, among the 1mmol/LEDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains the mutational site
Three kinds of common SNP site C50T, A842G and G123A of COX-1 gene, wherein, C50T and A842G are uneven complete linkage, and C50T is positioned on the exon2 of COX-1, A842G occurs in the promoter region of COX-1, and G123A occurs on the exon3 of COX-1; The SNP site T196C of GPIIIa gene occurs on the GPIIIa gene exon 3; Utilize Primer5.O design four pairs of primers (seeing Table 3), amplify respectively four target sequences that contain the SNP site.
Table 3 amplifies the primer of the target sequence with SNP site
All primers are synthetic 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.
In like manner, those skilled in the art can according to the method described above, in the situation that do not comprise the COX-1 Gene Partial, make up GPIIIa gene test liquid-phase chip.
Embodiment 2 utilization embodiment 1 described GPIIIa and COX-1 gene test liquid-phase chip are to the detection of sample
The prescription of described various solution is as follows:
MES damping fluid (pH5.0) prescription (250ml) of 50mM:
| Reagent | The source | Final concentration | The consumption of every 250ml |
| MES(2[N-Morpholino] ethanesulfonic acid) | Sigma M-2933 | 0.05M | 2.44g |
| 5M NaOH | Fisher SS256-500 | --- | 5 |
2 * Tm hybridization buffer
| Reagent | The source | Final concentration | The consumption of every 250ml |
| 1MTris-HCl，pH8.0 | SigmaT3038 | 0.2M | 50ml |
| 5M NaCl | Sigma S5150 | 0.4M | 20ml |
| Triton X-100 | Sigma T8787 | 0.16％ | 0.4ml |
Be stored in 4 ℃ after the filtration.
The ExoSAP-IT test kit is available from U.S. USB company.
Biotin labeled dCTP is available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
With reference to " molecular cloning " methods involving about DNA extraction, obtain DNA to be detected.
Two, the pcr amplification of testing sample
Utilize four pairs of primers of Primer5.0 design, one step of multiplex PCR amplifies the exon2 of COX-1 gene, the exon 3 of the exon3 of the promoter region of COX-1 and COX-1 gene, GPIIIa gene is totally four target sequences that contain the SNP site, the product size is respectively 554bp, 202bp, 297bp and 391bp, and primer sequence (SEQ ID NO.25-32) is seen shown in the above-mentioned table 3.
At first prepare the multiple PCR primer working fluid: respectively get respectively the primer stock solution 100ul of SEQ ID NO.25-32 in the 1.5ml Eppendorf tube, mix and be the multiple PCR primer working fluid.The multi-PRC reaction system is as follows:
2 * damping fluid (contains 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 (10ng/ul) 2ul
ddH
2O 12.8ul
Be total to 50ul
The 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;
2.37 ℃ hatch 15min, hatch 15min for 80 ℃, the enzyme that deactivation is unnecessary.The product that enzyme is cut after the 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 the reacted product band.
The ASPE primer working fluid that mixes of preparation at first: get respectively the corresponding wild-type of T196C of C50T, A842G, G123A and GPIIIa gene of COX-1 gene and mutant ASPE primer stock solution 10ul in the 1.5ml Eppendorf tube, add 10mmol/LTris Buffer and 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 that mixes
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, 58 ℃ 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 optimum microballoon of every group selection
5Individual/ml).Every kind of microballoon is encoded with different colours respectively, simultaneously every kind of microsphere surface is connected with respectively the specific oligonucleotide sequence (anti-tag) of one section 24bp, and these anti-tag sequences can be respectively and the tag sequence specific combination of corresponding ASPE primer 5 ' end;
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, the vortex mixing;
5. get the above-mentioned microballoon suspension of 25ul in the corresponding hole of 96 hole filter plates, 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. encase 96 orifice plates with lucifuge with masking foil, 95 ℃ of 60s, 37 ℃ of 15min are hatched hybridization;
8. the microballoon after the 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. microballoon is resuspended in 1 * Tm hybridization buffer of 75ul, adding 15ul concentration is the SA-PE (SA-PE) of 10ug/ml;
12.37 ℃ hatch 15min, on the Luminex instrument, detect.
Six, the result detects and data analysis
The reaction after product detects by Luminex serial analysis instrument.Detected result is shown in table 4, table 5 and table 6.
Fluorescent value (MFI) and data processing there are following requirement:
1. each site need have an allelotrope MFI at least greater than 300 and greater than 10 * PCR negative control MFI;
2.NET MFI=sample MFI-PCR negative control MFI (NET MFI less than 0 with 0 the expression);
3. satisfy the data of above two conditions, calculate sudden change ratio by following formula:
Sudden change ratio=mutant NET MFI ÷ (mutant NET MFI+ wild-type NET MFI)
4. rule of thumb to the sudden change ratio definite threshold (cut-off value) of each detection site, to divide wild-type homozygote, heterozygote and mutant homozygote.
Use present method to detect 20 increments COX-1 and GPIIIa 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: the sudden change ratio range is considered as the wild-type homozygote at 0%-20%; 30%-70% is considered as heterozygote; 80%-100% is considered as the anomaly homozygote.Compare with the liquid-phase chip result with the sequencing detection, calculate the identical rate of classifying method detected result provided by the present invention.Present method detects this COX-1 and the identical rate of GPIIIa genotype detection result and sequencing result of 20 increments and reaches 100%.As seen COX-1 provided by the present invention and GPIIIaSNP detect the SNP type that liquid-phase chip can detect COX-1 and GPIIIa exactly, and the result is reliable and stable.
Table 4 pattern detection result (MFI)
Table 5 sample COX-1 and GPIIIa transgenation ratio (%)
Table 6 sample COX-1 and GPIIIa gene mutation type analytical results
The liquid-phase chip of the ASPE primer that embodiment 3 is different is to the detection of COX-1 and GPIIIa gene SNP site
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
Detect liquid-phase chip as example take COX-1 gene C 50T and GPIIIa gene T196C site mutation, respectively for the wild-type of C50T and T196C and the specific primer sequence of mutant design ASPE primer 3 ' end, the Tag sequence of ASPE primer 5 ' end then is selected from SEQ ID NO.1-SEQ ID NO.8, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing that is coated on the microballoon is selected from SEQ ID NO.17-SEQ ID NO.24.Specific design is shown in following table (table 7).Synthetic, the coated microballoon of anti-tag sequence of ASPE primer, amplimer, detection method are described like embodiment 1 and embodiment 2.
The design of table 7 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned design preparation, by embodiment 2 described testing processes and method sample 21-40 is detected, detected result is as follows:
Table 8 pattern detection result and Polymorphism Analysis one
Table 9 pattern detection result and Polymorphism Analysis two
Other is for the liquid-phase chip in different SNP sites, and the ASPE primer uses different Tag sequences, and its result is still reliable and stable, and concrete data are omitted.
Embodiment 4 different interval arm liquid-phase chips detect the GPIIIa gene SNP
One, the design (selection of spacerarm) of liquid-phase chip preparation
Take the detection liquid-phase chip of GPIIIa gene T196C site mutation as example, verify the impact that different spacerarm liquid-phase chips detects the GPIIIa gene SNP.For the wild-type of T196C and the specific primer sequence of mutant design ASPE primer 3 ' end, the Tag sequence of ASPE primer 5 ' end then is respectively SEQ ID NO.7 and SEQ ID NO.8, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing that is coated on the microballoon is respectively SEQ ID NO.23 and SEQ ID NO.24.Verify the impact that different spacerarm liquid-phase chips detects the GPIIIa gene SNP, wherein, different spacerarms is (CH2) 12 or 5-10 T, and specific design is shown in following table (table 10).Synthetic, the coated microballoon of anti-tag sequence of ASPE primer, amplimer, detection method are described like embodiment 1 and embodiment 2.
The design of table 10 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned design preparation, by embodiment 2 described testing processes and method sample 41-60 is detected, detected result is as follows:
Table 11 pattern detection result and Polymorphism Analysis
Spacerarm is the liquid-phase chip of (CH2) 12 among the embodiment 4, its detected result is reliable and stable, and (detected result of other spacerarm is also like this, concrete data are omitted), and the hybridization rate of the spacerarm of preferred 5-10 T and hybridization specificity all are better than the liquid-phase chip that spacerarm is (CH2) 12, and the spacerarm of a 5-10 of the present invention T all is better than other spacerarms.
More than be for the specifying of possible embodiments of the present invention, but this embodiment limits claim of the present invention, allly do not break away from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the claim of the present invention.
Sequence table
＜110〉Guangzhou Yishan Biotechnology Co., Ltd.
＜120〉GPIIIa and COX-1 gene SNP detection liquid-phase chip and Auele Specific Primer
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<400>26
gttcccagac ggttagagg 19
<210>27
<211>20
<212>DNA
＜213〉artificial sequence
<400>27
gccttccgat aactgagcac 20
<210>28
<211>20
<212>DNA
＜213〉artificial sequence
<400>28
tcaaatccag actccacagc 20
<210>29
<211>19
<212>DNA
＜213〉artificial sequence
<400>29
gcacccagtg attcaggac 19
<210>30
<211>19
<212>DNA
＜213〉artificial sequence
<400>30
gagcagaagg gctcaagac 19
<210>31
<211>20
<212>DNA
＜213〉artificial sequence
<400>31
atcccaggaa agaccacaac 20
<210>32
<211>19
<212>DNA
＜213〉artificial sequence
<400>32
ctctatgccc acctgcttc 19
Claims (6)
1. a GPIIIa gene SNP detection liquid-phase chip is characterized in that, mainly includes:
(A) for the SNP site of GPIIIa gene, the wild-type that designs respectively and the ASPE primer pair of mutant: every kind of ASPE primer holds the Auele Specific Primer for goal gene SNP site to form by the tag sequence and 3 ' of 5 ' end, and described Auele Specific Primer is: for SEQ ID NO.15 and the SEQ ID NO.16 in GPIIIa gene T196C SNP site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.8;
(B) be coated with respectively microballoon special anti-tag sequence, that have the different colours coding, described anti-tag sequence is selected from the sequence among SEQ ID NO.17～SEQ ID NO.24, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing, and described anti-tag sequence also is provided with the spacerarm sequence in the middle of being connected with microballoon;
(C) for the amplimer that amplifies the GPIIIa gene target sequence with T196C SNP site, described amplimer is for the SEQ ID NO.31 in GPIIIa gene T196C SNP site～SEQ ID NO.32.
2. GPIIIa gene SNP detection liquid-phase chip according to claim 1 is characterized in that,
Described ASPE primer pair is: for the sequence that is comprised of SEQ ID NO.7 and SEQ ID NO.15 in GPIIIa gene T196C SNP site and the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.16.
3. GPIIIa gene SNP detection liquid-phase chip according to claim 1 and 2 is characterized in that, described spacerarm sequence is 5-10 T.
4. a GPIIIa and COX-1 gene SNP detection liquid-phase chip is characterized in that, mainly include:
(A) for the SNP site of COX-1 gene and GPIIIa gene, the respectively wild-type of design and the ASPE primer pair of mutant: every kind of ASPE primer is comprised of the tag sequence of 5 ' end and 3 ' the end Auele Specific Primer for the goal gene mutational site, and described Auele Specific Primer is: for the SEQ ID NO.9 in COX-1 gene C 50T SNP site and SEQ ID NO.10, for the SEQ ID NO.11 in COX-1 Gene A 842G SNP site and SEQ ID NO.12 and/or for SEQ ID NO.13 and the SEQ ID NO.14 in COX-1 gene G123A SNP site; And for SEQ ID NO.15 and the SEQ ID NO.16 in GPIIIa gene T196C SNP site;
Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.8;
(B) be coated with respectively microballoon special anti-tag sequence, that have the different colours coding, described anti-tag sequence is selected from the sequence among SEQ ID NO.17～SEQ ID NO.24, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing, and described anti-tag sequence also is provided with the spacerarm sequence in the middle of being connected with microballoon;
(C) be used for the amplimer that amplification has the COX-1 gene target sequence in C50T, A842G and/or G123A SNP site; And the amplimer that has the GPIIIa gene target sequence in T196C SNP site for amplification; Described amplimer is for the SEQ ID NO.25 in COX-1 gene C 50T SNP site～SEQ ID NO.26, for the SEQ ID NO.27 in COX-1 Gene A 842G SNP site～SEQ ID NO.28, and/or for the SEQ ID NO.29 in COX-1 gene G123A SNP site～SEQ ID NO.30; And for the SEQ ID NO.31 in GPIIIa gene T196C SNP site～SEQ ID NO.32.
5. GPIIIa according to claim 4 and COX-1 gene SNP detection liquid-phase chip is characterized in that,
Described ASPE primer pair is: for the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.9 in COX-1 gene C 50T SNP site and the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.10, for the sequence that is formed by SEQ ID NO.3 and SEQ ID NO.11 in COX-1 Gene A 842G SNP site and the sequence that is formed by SEQ ID NO.4 and SEQ ID NO.12, and/or for the sequence that is formed by SEQ ID NO.5 and SEQ ID NO.13 in COX-1 gene G123A SNP site and the sequence that is formed by SEQ ID NO.6 and SEQ ID NO.14; And for the sequence that is formed by SEQ ID NO.7 and SEQ ID NO.15 in GPIIIa gene T196C SNP site and the sequence that is formed by SEQ ID NO.8 and SEQ ID NO.16.
6. according to claim 4 or 5 described GPIIIa and COX-1 gene SNP detection liquid-phase chip, it is characterized in that, described spacerarm sequence is 5-10 T.
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| CN 201010196858 CN102010900B (en) | 2010-06-08 | 2010-06-08 | Liquid chip and specific primer for detecting SNP of GPIIIa gene and liquid chip and specific primer for detecting SNP of GPIIIa and COX-1 genes |
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| WO2002059355A2 (en) * | 2001-01-25 | 2002-08-01 | Tm Bioscience Corporation | Polynucleotides for use as tags and tag complements, manufacture and use thereof |
| CN101487052A (en) * | 2009-02-24 | 2009-07-22 | 广州益善生物技术有限公司 | Liquid phase chip for CYP2C9 and CYP2C19 gene mutation detection and detecting method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2002059355A2 (en) * | 2001-01-25 | 2002-08-01 | Tm Bioscience Corporation | Polynucleotides for use as tags and tag complements, manufacture and use thereof |
| CN101487052A (en) * | 2009-02-24 | 2009-07-22 | 广州益善生物技术有限公司 | Liquid phase chip for CYP2C9 and CYP2C19 gene mutation detection and detecting method thereof |
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| Title |
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| .2008,第66卷(第2期),222页以及224页表一. * |
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