CN102304564A - Specific primers and liquid phase chip for single nucleotide polymorphism (SNP) detection in 15 q 25 section of chromosome - Google Patents
Specific primers and liquid phase chip for single nucleotide polymorphism (SNP) detection in 15 q 25 section of chromosome Download PDFInfo
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Abstract
The invention discloses liquid phase chip and specific primers for single nucleotide polymorphism (SNP) detection in a 15 q 25 section of a chromosome. The liquid phase chip comprises allele specific primer extension (ASPE) primers consisting of tag sequences at the 5' end and specific primers aiming at SNP loci of a target gene at the 3' end, microspheres which are wrapped with specific anti-tag sequences and have different color codes and an amplification primer, wherein the specific primers are represented by SEQ ID No. 11 and SEQ ID No. 12 of A102 G SNP, SEQ ID No. 13 and SEQ ID No. 14 of C77T SNP, SEQ ID No. 15 and SEQ ID No. 16 of C63T SNP, SEQ ID No. 17 and SEQ ID No. 18 of A202C SNP and/or SEQ ID No. 19 and SEQ ID No. 20 of G139C SNP in the 15 q 25 section of the chromosome; and the tap sequence is selected from SEQ ID No.1 to SEQ ID No. 10. The coincidence rate of a detection result of the liquid phase chip and a sequencing method is up to 100 percent. The prepared liquid phase chip for the SNP detection in the 15 q 25 section of the chromosome has a high signal noise ratio, and the parallel detection of multiple SNP loci can be realized.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, the concrete Auele Specific Primer and the liquid-phase chip that relate to a kind of karyomit(e) 15q25 section SNP detection.
Background technology
LOC123688 is the gene that is positioned at a function on the karyomit(e) 15q25 section; It mainly is responsible for the cell receptor of coding Nicotine; Research at present shows; Nicotine is through these acceptors; Can promote the development of lung cancer; Promote cancer cell multiplication, survival, move, intrude into adjacent tissue, even the blood supply of development tumour, so this gene mononucleotide polymorphism (SNP) is relevant with development with the generation of lung cancer.
In addition, also the occurrence risk with lung cancer is relevant to be positioned at the candidate gene CHRNA3 of coding Nicotine acetylcholine receptor of karyomit(e) 15q25 section, and the transgenation on this gene is IIIB phase/IV phase patients with lung cancer independent prognostic factor that influence can not be performed the operation.
The karyomit(e) 15q25 block mutation site of target detect of the present invention, it is as shown in the table:
| Sequence number | The content of site mutation | Write a Chinese character in simplified form |
| 1 | A → G sudden change takes place in the 102nd Nucleotide of SEQ ID NO.41 | A102G |
| 2 | C → T sudden change takes place in the 77th Nucleotide of SEQ ID NO.42 | C77T |
| 3 | C → T sudden change takes place in the 63rd Nucleotide of SEQ ID NO.43 | C63T |
| 4 | A → C sudden change takes place in the 202nd Nucleotide of SEQ ID NO.44 | A202C |
| 5 | G → C sudden change takes place in the 139th Nucleotide of SEQ ID NO.45 | G139C |
At present; The method that the LOC123688 and the CHRNA3 gene pleiomorphism of karyomit(e) 15q25 section detected, analyzes is a lot; As: traditional solid phase chip, PCR-RFLP method, TaqMan technology, directly order-checking or the like, wherein the most frequently used method has quantitative fluorescent PCR and PCR-RFLP method.Easy and simple to handle, advantages such as the result quick, quantification that quantitative fluorescent PCR has, still, this technology exists sample to be prone to pollute, and is prone to take place cross reaction, the shortcoming that false positive rate is high; And the PCR-RFLP method is based on the change of the restriction enzyme enzyme recognition site that transgenation causes; As losing or produce novel site in the site; Through a certain particular segment of pcr amplification; Use the digestion with restriction enzyme amplified production again; The segmental size of electrophoresis observation; This method is used to detect the transgenation that restriction enzyme site changes, and can directly judge genotype, but this method can not be used for not producing the detection in Gene Mutation of new restriction enzyme site.Once more, above-mentioned these methods except that traditional solid phase chip all exist the limitation that detects flux, can only detect a kind of mutation type at every turn, can not satisfy the needs of practical application.
Summary of the invention
One of the object of the invention provides the liquid-phase chip that karyomit(e) 15q25 section SNP detects.This liquid-phase chip can be used for detecting wild-type and the mutant of karyomit(e) 15q25 section five kinds of common SNP site A102G, C77T, C63T, A202C and G139C.
For realizing above-mentioned purpose, the present invention has taked following technical scheme:
The liquid-phase chip that a kind of karyomit(e) 15q25 section SNP detects mainly includes:
(A) the ASPE primer of wild-type that designs respectively to the SNP site of every kind of gene and mutant is right, and every ASPE primer is made up of the Auele Specific Primer that the tag sequence and the 3 ' end of 5 ' end is directed against goal gene SNP site; Said Auele Specific Primer is: to SEQ ID NO.15 and SEQ ID NO.16, the SEQ ID NO.17 of A202C SNP and SEQ ID NO.19 and the SEQ ID NO.20 of SEQ ID NO.18 and/or G139C SNP of the SEQ ID NO.13 of the SEQ ID NO.11 of karyomit(e) 15q25 section A102G SNP and SEQ ID NO.12, C77T SNP and SEQ ID NO.14, C63T SNP; Said tag sequence is selected from the sequence among SEQ ID NO.1～SEQ ID NO.10;
(B) be coated with microballoon special anti-tag sequence, that have the different colours coding respectively; Said anti-tag sequence is selected from the sequence among SEQ ID NO.21～SEQ ID NO.30; And said anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing, and said anti-tag sequence also is provided with the spacerarm sequence in the middle of being connected with microballoon;
(C) be used to the increase amplimer of target sequence in A102G, C77T, C63T, A202C and/or G139C SNP site with karyomit(e) 15q25 section.
Preferably, said amplimer is: to SEQ ID NO.35 and SEQ ID NO.36, the SEQ ID NO.37 of A202C SNP and SEQ ID NO.39 and the SEQ ID NO.40 of SEQ ID NO.38 and/or G139C SNP of the SEQ ID NO.33 of the SEQ ID NO.31 of karyomit(e) 15q25 section A102G SNP and SEQ ID NO.32, C77T SNP and SEQ ID NO.34, C63T SNP.
Preferably, said ASPE primer is to being: the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.11 to karyomit(e) 15q25 section A102G SNP reaches the sequence of being made up of SEQ ID NO.2 and SEQ ID NO.12; The sequence of being made up of SEQ ID NO.3 and SEQ ID NO.13 to C77T SNP reaches the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.14; The sequence of being made up of SEQ ID NO.5 and SEQ ID NO.15 to C63T SNP reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.16; The sequence of being made up of SEQ ID NO.7 and SEQ ID NO.17 to A202C SNP reaches the sequence of being made up of SEQ ID NO.8 and SEQ ID NO.18; And/or the sequence of being made up of SEQ ID NO.9 and SEQ ID NO.19 that is directed against G139C SNP reaches the sequence of being made up of SEQ ID NO.10 and SEQ ID NO.20.
Preferably, said spacerarm sequence is 5-10 T.
The present invention also provides a kind of karyomit(e) 15q25 section SNP detection specificity primer, includes to the SEQ ID NO.11 of karyomit(e) 15q25 section A102G SNP and SEQ ID NO.12, to the SEQ ID NO.13 of C77T SNP and SEQ ID NO.14, to the SEQ ID NO.15 of C63T SNP and SEQ ID NO.16, to the SEQ ID NO.17 of A202C SNP and SEQ ID NO.18 and/or to SEQ ID NO.19 and the SEQ ID NO.20 of G139C SNP.
Major advantage of the present invention is:
1. detection liquid-phase chip provided by the present invention the identical rate of detected result and sequencing up to 100%.And detect the needed time well below sequencing technologies commonly used, meet the practical application needs especially.Because in very a large amount of Auele Specific Primers; Through a large amount of tests; The reaction checking; Obtain the liquid-phase chip system of optimum combination; Prepared karyomit(e) 15q25 section polymorphism detects liquid-phase chip and has extraordinary signal-noise ratio; And there is not cross reaction between institute's designed probe and the anti-tag sequence basically; Choosing of tag sequence label, anti-tag sequence label and combining of tag sequence label and concrete ASPE primer; Can avoid cross reaction, realize the parallel detection in a plurality of SNP site.
2. the present invention has chosen optimum combination through the design experiences of contriver's long-term accumulation and the operation of a large amount of experiments from numerous Auele Specific Primers.The genotype of various types is accurately distinguished in the mutational site that designed ASPE primers Auele Specific Primer of the present invention can sensitive recognition objective specifically detects; In same reaction system, between the different Auele Specific Primers, do not have cross reaction basically between the pcr amplification product of Auele Specific Primer and non-target detect, detection specificity is good, and cross reacting rate is lower than 3%; Except detecting single site mutation situation, also the polymorphism situation in a plurality of mutational sites of parallel detection simultaneously detects the effect unanimity.
3. detection method step of the present invention is simple; Five kinds of pleomorphism sites detect and can accomplish five amplifications that contain the target sequence in SNP site through 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 improve the detection accuracy rate greatly, embodied qualitative, quantitative analysis characteristic of accurate while.
4. not only to have overcome traditional solid phase chip susceptibility not high in the present invention, and the defective of the repeatability difference of detected result is improved existing liquid-phase chip technology simultaneously, makes prepared microballoon can be applicable to the different detection project, has very strong expansion.The fluorescent signal value that detects improves greatly, thereby makes the sensitivity that detects be further enhanced, and signal to noise ratio strengthens, and detected result more accurately and reliably.
5. the liquid-phase chip detection technique among the present invention provides a kind of technical scheme of different designs for the parallel detection that solves a plurality of sites several genes type; And produced significant technique effect; Except detecting single site mutation situation; Also the polymorphism situation in a plurality of mutational sites of parallel detection simultaneously detects the effect unanimity.
Embodiment
Embodiment 1 karyomit(e) 15q25 section polymorphism detects liquid-phase chip, mainly includes:
One, ASPE primer
To wild-type and the mutant of five kinds of common genotype A102G, C77T, C63T, A202C and G139C of karyomit(e) 15q25 section, design specific primers sequence respectively.Wherein, the ASPE primer is made up of " Tag sequence+specific primer sequence ".The ASPE primer sequence is as shown in the table:
The ASPE primer sequence (Tag sequence+specific primer sequence) of table 1 karyomit(e) 15q25 section
Every the ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence to anti-tag sequence on the corresponding microballoon, and 3 ' end is mutant or the special primer fragment (shown in above-mentioned table 1) 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 the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Two, the microballoon that encapsulates of anti-tag sequence
According to institute's designed ASPE Auele Specific Primer fragment; Select the tag sequence; Reduce between the anti-tag sequence of each microballoon to greatest extent and secondary structure that tag and ASPE Auele Specific Primer fragment possibly form, corresponding anti-tag sequence is as shown in table 2 on 10 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
10 kinds of microballoons selecting encapsulate the anti-tag sequence on microballoon available from U.S. Luminex company.Be connected with the spacerarm sequence of 5-10 T between anti-tag sequence and the microballoon, promptly before each anti-tag sequence, add the spacerarm sequence of the preceding paragraph 5-10 T, 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.Said spacerarm is to be used for anti-tag and microsphere surface is spaced apart or anti-tag placed the sequence of hydrophilic environments.Through 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 like (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 encapsulates is following:
Get 5 * 10 respectively
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 10ul synthetic anti-tag molecule (100nmol/ml).EDC (N-(3-Dimethylaminopropyl-N-ethylcarbodiimide) (available from the Pierce Chemical company) working fluid of preparation 10ng/ml.Add the EDC working fluid of 2.5ul in the microballoon suspension, constant temperature was hatched 30 minutes, added the EDC working fluid of 2.5ul again, and constant temperature was hatched 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, and among the 1mmol/L EDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains the mutational site
To five kinds of common SNP site A102G, C77T, C63T, A202C and G139C of karyomit(e) 15q25 section, design of amplification primers amplifies five target sequences that contain the SNP site respectively to (seeing table 3).
Table 3 amplifies the primer of the target sequence with pleomorphism site
All primers are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with the stock solution of 1OOpmol/mL respectively with 10mmol/L Tris Buffer.
Embodiment 2 utilization karyomit(e) 15q25 section SNP detect the detection of liquid-phase chip to sample
The prescription of said various solution is following:
MES damping fluid (pH5.0) prescription (250ml) of 50mM:
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, obtain DNA to be detected about DNA extraction.
Two, the pcr amplification of testing sample
Design five pairs of primers; Multiplex PCR one step amplifies five kinds of common SNP site A102G, C77T, C63T, A202C and G139C totally five the target sequences that contain karyomit(e) 15q25 section respectively; The product size is respectively 281bp, 408bp, 342bp, 391bp and 374bp, and primer sequence (SEQ ID NO.31-40) is seen shown in the above-mentioned table 3.
At first prepare the multiple PCR primer working fluid: the primer stock solution 100ul that respectively gets SEQ ID NO.31-40 respectively mixes and is the multiple PCR primer working fluid in the 1.5ml Eppendorf tube.The multi-PRC reaction system is following:
The pcr amplification program is: 95 ℃ of 3min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 40s, 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations are subsequent use.
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 directly is used for follow-up ASPE primer extension reaction.
Four, site-specific primer extension reaction (ASPE)
Utilize above-mentioned designed ASPE primers 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.
At first prepare blended ASPE primer working fluid: get site to be detected corresponding wild type and mutant ASPE primer stock solution 10ul respectively in the 1.5ml Eppendorf tube; Add 10mmol/L Tris Buffer and mend, mix and be ASPE mix primer working fluid to 200ul.The system of ASPE reaction is following:
Response procedures is: 96 ℃ of 2min; 94 ℃ of 30s, 54 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ of preservations are subsequent use.
Five, hybridization
1. according to designed ASPE primers, (every kind of microballoon concentration is 2.5 * 10 to the corresponding 10 kinds of microballoons of every group selection
5Individual/ml);
2. the microballoon of getting every kind of numbering of 1ul respectively is 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. the ASPE reaction solution of getting 5-25ul is used ddH in corresponding hole
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 Streptavidin-phycoerythrin (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 is through Luminex serial analysis instrument detecting.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 the sudden change ratio of each detection site is confirmed threshold value (cut-off value), to divide wild-type homozygote, heterozygote and mutant homozygote.
Use present method to detect 20 increments karyomit(e) 15q25 section pleomorphism site originally, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Being provided with as follows of threshold value (cut-off value): 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 20 increments karyomit(e) 15q25 zone detection result and the sequencing result rate of coincideing originally and reaches 100%.Can detect karyomit(e) 15q25 section polymorphic position vertex type exactly it is thus clear that karyomit(e) 15q25 section polymorphism provided by the present invention detects liquid-phase chip, and the result is reliable and stable.
Table 4 pattern detection result (MFI)
Table 5 sample dyeing body 15q25 block mutation ratio (%)
Table 6 sample dyeing body 15q25 block mutation type analysis result
| Catalogue number(Cat.No.) | The liquid-phase chip detected result | Sequencing result |
| 1 | Wild-type | Wild-type |
| 2 | 202AC | 202AC |
| 3 | Wild-type | Wild-type |
| 4 | Wild-type | Wild-type |
| 5 | Wild-type | Wild-type |
| 6 | Wild-type | Wild-type |
| 7 | Wild-type | Wild-type |
| 8 | 102GG | 102GG |
| 9 | Wild-type | Wild-type |
| 10 | 77CT | 77CT |
| 11 | Wild-type | Wild-type |
| 12 | 139CC | 139CC |
| 13 | Wild-type | Wild-type |
| 14 | Wild-type | Wild-type |
| 15 | 202CC | 202CC |
| 16 | Wild-type | Wild-type |
| 17 | Wild-type | Wild-type |
| 18 | Wild-type | Wild-type |
| 19 | Wild-type | Wild-type |
| 20 | Wild-type | Wild-type |
The liquid-phase chip of the ASPE primer that embodiment 3 is different is to the detection of karyomit(e) 15q25 section pleomorphism site
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
C77T and A202C site mutation detection liquid-phase chip with karyomit(e) 15q25 section are example; Respectively to the wild-type of C77TA202C 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.10; Accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing that encapsulates on microballoon is selected from SEQ ID NO.21-SEQ ID NO.30.Specifically design shown in following table (table 7).It is said like embodiment 1 and embodiment 2 that synthetic, the anti-tag sequence of ASPE primer encapsulates microballoon, amplimer, detection method.
The design of table 7 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned designing and preparing, by embodiment 2 said testing processes and method sample 21-40 is detected, detected result is following:
Table 8 pattern detection result and Polymorphism Analysis
Table 9 pattern detection result and Polymorphism Analysis
Other is to the liquid-phase chip in different mutational sites, and the ASPE primer uses different Tag sequences, and its result is still reliable and stable, and concrete data are omitted.And the ASPE primer is when selecting among the embodiment 1 collocation of tag sequence and specific primer sequence for use, and effect better (signal to noise ratio is better) is referring to present embodiment test group 2 and test group 5.Other different tag sequences and specific primer sequence collocation, with coming to the same thing of embodiment 2 and present embodiment, concrete data are omitted.
The selection of embodiment 4 karyomit(e) 15q25 section SNP detection specificity primer sequences
One, the design (selection of wild-type and mutant specific primer sequence) of liquid-phase chip preparation
Detecting liquid-phase chip with karyomit(e) 15q25 section A102G and G139C site mutation is example; Respectively to the wild-type of A102G and G139C and the specific primer sequence of mutant design ASPE primer 3 ' end; Comprise preferred specific primer sequence and two alternative specific primer sequences in the embodiment of the invention 1, as shown in table 10.Where,
inner base of SNP loci.
Table 10 specific primer sequence
Detecting liquid-phase chip with karyomit(e) 15q25 section A102G and G139C site mutation is example; Respectively to the wild-type of A102G and G139C and the specific primer sequence of mutant design ASPE primer 3 ' end; The Tag sequence of ASPE primer 5 ' end then is fixed as the best effect sequence among the embodiment 1; And select corresponding with it anti-tag sequence for use, specifically design shown in following table (table 11).It is said like embodiment 1 and embodiment 2 that synthetic, the anti-tag sequence of ASPE primer encapsulates microballoon, amplimer, detection method.
Two of the design of table 11 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned designing and preparing, by embodiment 2 said testing processes and method sample 41-60 is detected, detected result is following:
Table 12 pattern detection result and Polymorphism Analysis
Table 13 pattern detection result and Polymorphism Analysis
Other is to the liquid-phase chip in different mutational sites, and the ASPE primer uses different specific primer sequences, and its result is still reliable and stable, and concrete data are omitted.And the ASPE primer is when selecting among the embodiment 1 collocation of specific primer sequence and tag sequence for use, and effect better (signal to noise ratio is better) is referring to present embodiment test group 7 and test group 10.Other different specific primer sequences and the collocation of tag sequence, with coming to the same thing of embodiment 2 and present embodiment, concrete data are omitted.
More than be to the specifying of possible embodiments of the present invention, but this embodiment is not in order to limiting claim of the present invention, does not allly 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.
Claims (6)
1. the liquid-phase chip that karyomit(e) 15q25 section SNP detects is characterized in that, mainly includes:
(A) the ASPE primer of wild-type that designs respectively to the SNP site of every kind of gene and mutant is right, and every ASPE primer is made up of the Auele Specific Primer that the tag sequence and the 3 ' end of 5 ' end is directed against goal gene SNP site; Said Auele Specific Primer is: to SEQ ID NO.15 and SEQ ID NO.16, the SEQ ID NO.17 of A202C SNP and SEQ ID NO.19 and the SEQ ID NO.20 of SEQ ID NO.18 and/or G139C SNP of the SEQ ID NO.13 of the SEQ ID NO.11 of karyomit(e) 15q25 section A102G SNP and SEQ ID NO.12, C77T SNP and SEQ ID NO.14, C63T SNP; Said tag sequence is selected from the sequence among SEQ ID NO.1～SEQ ID NO.10;
(B) be coated with microballoon special anti-tag sequence, that have the different colours coding respectively; Said anti-tag sequence is selected from the sequence among SEQ ID NO21～SEQ ID NO.30; And said anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing, and said anti-tag sequence also is provided with the spacerarm sequence in the middle of being connected with microballoon;
(C) be used to the increase amplimer of target sequence in A102G, C77T, C63T, A202C and/or G139C SNP site with karyomit(e) 15q25 section.
2. the liquid-phase chip that karyomit(e) 15q25 section SNP according to claim 1 detects; It is characterized in that said amplimer is: to SEQ ID NO.35 and SEQ ID NO.36, the SEQ ID NO.37 of A202C SNP and SEQ ID NO.39 and the SEQ ID NO.40 of SEQ ID NO.38 and/or G139C SNP of the SEQ ID NO.33 of the SEQ ID NO.31 of karyomit(e) 15q25 section A102G SNP and SEQ ID NO.32, C77T SNP and SEQ ID NO.34, C63T SNP.
3. the liquid-phase chip that karyomit(e) 15q25 section SNP according to claim 1 detects; It is characterized in that said ASPE primer is to being: the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.11 to karyomit(e) 15q25 section A102G SNP reaches the sequence of being made up of SEQ ID NO.2 and SEQ ID NO.12; The sequence of being made up of SEQ ID NO.3 and SEQ ID NO.13 to C77T SNP reaches the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.14; The sequence of being made up of SEQ ID NO.5 and SEQ ID NO.15 to C63T SNP reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.16; The sequence of being made up of SEQ ID NO.7 and SEQ ID NO.17 to A202C SNP reaches the sequence of being made up of SEQ ID NO.8 and SEQ ID NO.18; And/or the sequence of being made up of SEQ ID NO.9 and SEQ ID NO.19 that is directed against G139C SNP reaches the sequence of being made up of SEQ ID NO.10 and SEQ ID NO.20.
4. the liquid-phase chip that karyomit(e) 15q25 section SNP according to claim 3 detects is characterized in that, mainly includes:
(A) the ASPE primer is to being: the sequence of being made up of SEQ ID NO.1 and SEQ IDNO.11 to karyomit(e) 15q25 section A102G SNP reaches the sequence of being made up of SEQ ID NO.2 and SEQ ID NO.12; The sequence of being made up of SEQ ID NO.3 and SEQ ID NO.13 to C77T SNP reaches the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.14; The sequence of being made up of SEQ ID NO.5 and SEQ ID NO.15 to C63T SNP reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.16; The sequence of being made up of SEQ ID NO.7 and SEQ ID NO.17 to A202C SNP reaches the sequence of being made up of SEQ ID NO.8 and SEQ ID NO.18; Reach the sequence of forming by SEQ ID NO.10 and SEQ ID NO.20 with the sequence of forming by SEQ ID NO.9 and SEQ ID NO.19 to G139C SNP;
(B) be coated with microballoon special anti-tag sequence, that have the different colours coding respectively; Said anti-tag sequence is selected from the sequence among SEQ ID NO.21～SEQ ID NO.30; And said anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing, and said anti-tag sequence also is provided with the spacerarm sequence in the middle of being connected with microballoon;
(C) to SEQ ID NO.35 and SEQ ID NO.36, the SEQ ID NO.37 of A202C SNP and SEQ ID NO.39 and the SEQ ID NO.40 of SEQ ID NO.38 and G139C SNP of the SEQ ID NO.33 of the SEQ ID NO.31 of karyomit(e) 15q25 section A102G SNP and SEQ ID NO.32, C77T SNP and SEQ ID NO.34, C63T SNP.
5. the liquid-phase chip that detects according to each described karyomit(e) 15q25 section SNP of claim 1-4 is characterized in that said spacerarm sequence is 5-10 T.
6. the Auele Specific Primer that detects of a karyomit(e) 15q25 section SNP; It is characterized in that, include to the SEQ ID NO.11 of karyomit(e) 15q25 section A102G SNP and SEQ ID NO.12, to the SEQ ID NO.13 of C77T SNP and SEQ ID NO.14, to the SEQ ID NO.15 of C63T SNP and SEQ ID NO.16, to the SEQ ID NO.17 of A202C SNP and SEQ ID NO.18 and/or to SEQ ID NO.19 and the SEQ ID NO.20 of G139C SNP.
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| WO2004005886A2 (en) * | 2002-07-02 | 2004-01-15 | Genelink, Inc. | Kits and methods for assessing cardiovascular health |
| US20070037177A1 (en) * | 2005-08-12 | 2007-02-15 | Weinshilboum Richard M | CYP19A1 polymorphisms |
| US20080299094A1 (en) * | 2004-07-01 | 2008-12-04 | Medical Research Fund Of Tel Aviv | Methods and Kits for Predicting Liver Fibrosis Progression Rate in Chronic Hepatitis C Patients |
| CN101565749A (en) * | 2009-04-15 | 2009-10-28 | 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 |
| CN101580875A (en) * | 2009-06-09 | 2009-11-18 | 广州益善生物技术有限公司 | Specific sequence, liquid-phase chip and method for SNP detection of genes related to therapeutic effectiveness of platinum medicaments |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004005886A2 (en) * | 2002-07-02 | 2004-01-15 | Genelink, Inc. | Kits and methods for assessing cardiovascular health |
| US20080299094A1 (en) * | 2004-07-01 | 2008-12-04 | Medical Research Fund Of Tel Aviv | Methods and Kits for Predicting Liver Fibrosis Progression Rate in Chronic Hepatitis C Patients |
| US20070037177A1 (en) * | 2005-08-12 | 2007-02-15 | Weinshilboum Richard M | CYP19A1 polymorphisms |
| CN101565749A (en) * | 2009-04-15 | 2009-10-28 | 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 |
| CN101580875A (en) * | 2009-06-09 | 2009-11-18 | 广州益善生物技术有限公司 | Specific sequence, liquid-phase chip and method for SNP detection of genes related to therapeutic effectiveness of platinum medicaments |
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