CN102912005B - CYP2C8 gene polymorphism detection specific primers and liquid chip - Google Patents
CYP2C8 gene polymorphism detection specific primers and liquid chip Download PDFInfo
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Abstract
The invention discloses CYP2C8 gene detection specific primers and a liquid chip. The liquid chip mainly comprises: various ASPE primers composed of 5' terminal tag sequence and 3' terminal specific primer sequences aiming at target gene mutation site, microspheres coated with anti-tag sequence, and amplification primers. The specific primer sequences comprise: SEQ ID NO.13 and SEQ ID NO.14 aiming at A127T site; SEQ ID NO.15 and SEQ ID NO.16 aiming at A124G site; SEQ ID NO.17 and SEQ ID NO.18 aiming at G145A site; SEQ ID NO.19 and SEQ ID NO.20 aiming at 204delA site; SEQ ID NO.21 and SEQ ID NO.22 aiming at C115G site; and/or SEQ ID NO.23 and SEQ ID NO.24 aiming at G315C site. The matching rate of the detection result obtained by using the liquid chip provided by the invention and a result obtained by using a sequencing method is 100%. Therefore, wild-type and mutant-type parallel detections of a plurality of mutation sites are realized.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, particularly relate to a kind of CYP2C8 genetic polymorphism detection Auele Specific Primer and liquid-phase chip.
Background technology
Cytochrome P 450 Enzyme is a superfamily, and the zymoprotein of being encoded by homologous genes forms, and containing 57 kinds of CYP genes in human genome, corresponding CYP enzyme of encoding, wherein CYP1, CYP2 and CYP3 family is the most important.CYP2C8 is a kind of important liver CYP enzyme, account for 7% of liver CYP total amount, participate in the medicine catalytic process of in the metabolism of I phase nearly 15%, anti-cancer medicine paclitaxel is the specific metabolic substrate of CYP2C8, the 6a-hydroxylating of taxol is the most frequently used mark reaction of CYP2C8 vitro detection, meanwhile, CYP2C8 is also the main CYP enzyme in liver kidney, arachidonic acid being converted into the activated eicosatrienoic acid of tool.
CYP2C8 encoding gene is 30kb, its evolution reading frame codes 490 amino acid, and in its evolution framework regions, found a lot of single nucleotide polymorphism (SNPs) site, this pleomorphism site is relevant to the metabolism of medicine.
The CYP2C8 gene mutation site (pleomorphism site) of target detect of the present invention, it is as shown in the table:
| Sequence number | The content of CYP2C8 site mutation | Write a Chinese character in simplified form |
| 1 | , there is A → T sudden change in the 127th Nucleotide of SEQ ID NO.55 | A127T |
| 2 | , there is A → G sudden change in the 124th Nucleotide of SEQ ID NO.56 | A124G |
| 3 | , there is G → A sudden change in the 145th Nucleotide of SEQ ID NO.57 | G145A |
| 4 | 204th Nucleotide of SEQ ID NO.57, disappearance base A | 204delA |
| 5 | , there is C → G sudden change in the 115th Nucleotide of SEQ ID NO.58 | C115G |
| 6 | , there is G → C sudden change in the 315th Nucleotide of SEQ ID NO.59 | G315C |
At present, the method detect CYP2C8 gene pleiomorphism, analyzed mainly contains: direct sequencing and PCR-RFLP analytical method etc., wherein the most frequently used method has PCR-RFLP analytical method.PCR-RFLP method is the change of the restriction enzyme enzyme recognition site caused based on transgenation, lose as site or produce novel site, by a certain specific fragment of pcr amplification, use digestion with restriction enzyme amplified production again, the size of electrophoresis observation fragment, the transgenation that this method changes for detecting restriction enzyme site, directly can judge genotype, but this method can not be used for the detection in Gene Mutation not producing new restriction enzyme site.Again, these methods above all also exist the limitation detecting flux, can only detect a kind of mutation type at every turn, can not meet the needs of practical application.
Summary of the invention
An object of the present invention is to provide CYP2C8 genetic polymorphism detection liquid-phase chip, and this liquid-phase chip can be used for detecting CYP2C8 gene six kinds of Common genes type A127T, the wild-type of A124G, G145A, 204delA, C115G and G315C and saltant type.
The technical scheme realizing above-mentioned purpose is as follows:
A kind of CYP2C8 genetic polymorphism detection liquid-phase chip, includes:
(A). the wild-type designed respectively for the different pleomorphism site of CYP2C8 gene and the ASPE primer pair of saltant type: every bar ASPE primer holds the specific primer sequence for goal gene pleomorphism site to form by 5 ' the tag sequence of holding and 3 ', and described specific primer sequence is: for SEQ ID NO.13 and the SEQ ID NO.14 in A127T site; For SEQ ID NO.15 and the SEQ ID NO.16 in A124G site; For SEQ ID NO.17 and the SEQ ID NO.18 in G145A site; For SEQ ID NO.19 and the SEQ ID NO.20 in 204delA site; For SEQ ID NO.21 and the SEQ IDNO.22 in C115G site; And/or for the SEQ ID NO.23 in G315C site and SEQ ID NO.24; Described tag sequence is selected from SEQ IDNO.1 ~ SEQ ID NO.12:
(B). microballoon that have anti-tag sequence bag quilt, that have different colours coding, is also provided with spacer sequence in the middle of described anti-tag sequence is connected with microballoon; Described anti-tag sequence is selected from SEQ ID NO.25 ~ SEQ ID NO.36, and described anti-tag sequence can correspondingly be matched with selected tag complementary in (A);
(C). for amplifying the primer needing that detect, that there is corresponding pleomorphism site target sequence.
Preferably, described amplimer is: for SEQ ID NO.37 and the SEQ ID NO.38 in A127T site; For SEQ ID NO.39 and the SEQ ID NO.40 in A124G site; For SEQ ID NO.41 and the SEQ IDNO.42 in G145A, 204delA site; For SEQ ID NO.43 and the SEQ ID NO.44 in C115G site; And/or for the SEQ IDNO.45 in G315C site and SEQ ID NO.46.
Preferably, for the sequence be made up of SEQ ID NO.1 and SEQ ID NO.13 in A127T site and the sequence that is made up of SEQ ID NO.2 and SEQ ID NO.14; For the sequence be made up of SEQ ID NO.3 and SEQ ID NO.15 in A124G site and the sequence that is made up of SEQ ID NO.4 and SEQ ID NO.16; For the sequence be made up of SEQ ID NO.5 and SEQ IDNO.17 in G145A site and the sequence that is made up of SEQ ID NO.6 and SEQ ID NO.18; For the sequence be made up of SEQID NO.7 and SEQ ID NO.19 in 204delA site and the sequence that is made up of SEQ ID NO.8 and SEQ ID NO.20; For the sequence be made up of SEQ ID NO.9 and SEQ ID NO.21 in C115G site and the sequence that is made up of SEQ ID NO.10 and SEQ ID NO.22; And/or for the sequence be made up of SEQ ID NO.11 and SEQ ID NO.23 in G315C site and the sequence that is made up of SEQID NO.12 and SEQ ID NO.24.
Another object of the present invention is to provide a kind of Auele Specific Primer for CYP2C8 genetic polymorphism detection.
The technical scheme realizing this object is as follows:
For an Auele Specific Primer for CYP2C8 genetic polymorphism detection, described Auele Specific Primer is: for SEQ ID NO.13 and the SEQ ID NO.14 in A127T site; For SEQ ID NO.15 and the SEQ ID NO.16 in A124G site; For SEQ ID NO.17 and the SEQ ID NO.18 in G145A site; For SEQ ID NO.19 and the SEQ IDNO.20 in 204delA site; For SEQ ID NO.21 and the SEQ ID NO.22 in C115G site; And/or for the SEQ IDNO.23 in G315C site and SEQ ID NO.24.
Major advantage of the present invention is:
1. the detected result of detection liquid-phase chip provided by the present invention and the identical rate of sequencing are up to 100%, and the time required for detecting is well below conventional sequencing technologies, and realistic especially application needs.Prepared CYP2C8 genetic polymorphism detection liquid-phase chip has extraordinary signal-noise ratio, and substantially there is not cross reaction between designed probe and anti-tag sequence, choosing and the combination of tag sequence label and concrete ASPE primer of tag sequence label, anti-tag sequence label, can cross reaction be avoided, realize the parallel detection of multiple pleomorphism site.
2. the present invention passes through the design experiences of contriver's long term accumulation and a large amount of experimental implementation, have chosen optimum combination from numerous Auele Specific Primers.The ASPE primer specificity primer of the present invention's design can the sensitive mutational site identifying target detect specifically, accurately distinguishes the genotype of various type; In same reaction system, substantially there is not cross reaction between different Auele Specific Primers, between the pcr amplification product that detects of Auele Specific Primer and non-targeted, detection specificity is good, and cross reacting rate is lower than 3%; Except Single locus catastrophe can be detected, also can the polymorphism situation in the simultaneously multiple mutational site of parallel detection, Detection results is consistent.
3. detection method step of the present invention is simple, six kinds of pleomorphism sites detect the amplification that can complete five target sequences containing pleomorphism site by a step multiplex PCR, avoid the many uncertain factors existed in the complex operations processes such as repeated multiple times PCR, thus greatly can improve Detection accuracy, embody accurate qualitative and quantitative analysis feature simultaneously.
4. not only to overcome conventional solid chip susceptibility not high in the present invention, and the defect of the repeatability difference of detected result, improves existing liquid-phase chip technology simultaneously, makes prepared microballoon can be applicable to different test items, has very strong expansion.The fluorescent signal value detected improves greatly, thus the sensitivity detected is further enhanced, and signal to noise ratio strengthens, and detected result more accurately and reliably.
5. the CYP2C8 gene mutation detection liquid-phase chip in the present invention is the technical scheme that the parallel detection of multiple sites Multi-genotype provides a kind of difference design, and creates significant technique effect.Meanwhile, due to technical characteristics such as high-throughput high specifics, the demand of more realistic application.Liquid-phase chip technology of the present invention will represent the technology trends of biological target detection.
Embodiment
Embodiment 1CYP2C8 genetic polymorphism detection liquid-phase chip, mainly includes:
One, ASPE primer
For wild-type and the saltant type of CYP2C8 gene six kinds of Common genes type A127T, A124G, G145A, 204delA, C115G and G315C, design specific primer sequence respectively.ASPE primer is made up 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 1CYP2C8 gene
Every bar ASPE primer comprises two parts, and 5 ' end is for for the specificity tag sequence of anti-tag sequence on corresponding microballoon, and 3 ' end is saltant type or the special primer segments (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 bar primer after synthesis is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Two, the microballoon of anti-tag sequence bag quilt
According to designed ASPE Auele Specific Primer fragment, select tag sequence, between the anti-tag sequence reducing each microballoon to greatest extent and tag and the ASPE Auele Specific Primer fragment secondary structure that may be formed, the anti-tag sequence that 12 kinds of microballoons numberings of selection are corresponding on microballoon is as shown in table 2:
The anti-tag sequence that table 2 microballoon numbering is corresponding on microballoon
The 12 kinds of microballoon purchased from American Luminex companies selected, are coated in anti-tag sequence on microballoon.Be connected with the spacer sequence of 5-10 T between anti-tag sequence and microballoon, before each anti-tag sequence, namely add the spacer sequence of the preceding paragraph 5-10 T, anti-tag sequence is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.By the anti-tag sequence sterilizing ddH of synthesis
2o is made into the stock solution of 100nmol/ml.Described spacerarm is for by anti-tag and microsphere surface is spaced apart or anti-tag is placed in the sequence of hydrophilic environments.By arranging the spacer sequence of suitable length between anti-tag sequence and microballoon, can reduce sterically hindered, improving the efficiency of hybridization and the specificity of hybridization.Common spacer sequence comprises poly dT, i.e. poly (dT), and oligomerization four polyoxyethylene glycol and (CH2) n spacerarm (n >=3), as (CH2) 12, (CH2) 18 etc.In addition, if there is poly (dA) interference, poly (TTG) can also be used as spacerarm.Spacerarm of the present invention is preferably 5-10 T, and the process of microballoon bag quilt is as follows:
Get 5 × 10 respectively
6the carboxylated microballoon (purchased from Luminex company) of individual above-mentioned numbering is suspended in (pH4.5) in the MES solution of 50ul 0.1mol/L, adds the anti-tag molecule (100nmol/ml) of 10ul synthesis.EDC (N-(3-Dimethylaminopropyl-N-ethylcarbodiimide) (purchased from the Pierce Chemical company) working fluid of preparation 10ng/ml.The EDC working fluid of 2.5ul is added, constant-temperature incubation 30 minutes, then the EDC working fluid adding 2.5ul in microsphere suspensions, then constant-temperature incubation 30 minutes.After reaction terminates, the Tween-20 washing with 0.02% once, then is washed once with the SDS liquid of 0.1%.The microballoon being coated with anti-tag sequence after washing is resuspended in the Tris-EDTA solution [10mmol/L Tris (pH8.0)] of 100ul, in 1mmol/LEDTA, 2-8 DEG C keeps in Dark Place.
Three, the primer of the target sequence containing mutational site is amplified
For CYP2C8 gene six kinds of Common genes type A127T, A124G, G145A, 204delA, C115G and G315C, design of amplification primers, to (see table 3), amplifies five target sequences containing pleomorphism site respectively.
Table 3 amplifies the primer of the target sequence with pleomorphism site
All primers are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every bar primer after synthesis is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Embodiment 2 uses CYP2C8 gene test liquid-phase chip described in embodiment 1 to the detection of sample
The formula of described various solution is as follows:
The MES damping fluid (pH5.0) formula (250ml) of 50mM:
2 × Tm hybridization buffer
| Reagent | Source | Final concentration | The consumption of every 250ml |
| 1MTris-HCl，pH8.0 | SigmaT3038 | 0.2M | 50ml |
| 5MNaCl | Sigma S5150 | 0.4M | 20ml |
| Triton X-100 | Sigma T8787 | 0.16％ | 0.4ml |
4 DEG C are stored in after filtration.
ExoSAP-IT test kit purchased from American USB company.
Biotin labeled dCTP is purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
With reference to " molecular cloning " about the methods involving of DNA extraction, obtain DNA to be detected.
Two, the pcr amplification of testing sample
Design five pairs of primers, multiplex PCR one step amplifies the five objective sequences respectively containing CYP2C8 gene six kinds of Common genes type A127T, A124G, G145A, 204delA, C115G and G315C, wherein, G145A and 204delA is positioned at same amplified production, product size is respectively 340bp, 384bp, 433bp, 343bp and 420bp, and primer sequence (SEQ IDNO.37-46) is shown in shown in above-mentioned table 3.
First multiple PCR primer working fluid is prepared: the primer stock solution 100ul respectively getting SEQ ID NO.37-46 respectively, in 1.5ml Eppendorf tube, mixes and is multiple PCR primer working fluid.Multi-PRC reaction system is as follows:
Pcr amplification program is: 95 DEG C of 3min; 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 40s, 30 circulations; 72 DEG C of 10min; 4 DEG C save backup.
Three, the enzyme of PCR primer cuts process
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 DEG C, hatch 15min for 80 DEG C, the enzyme that deactivation is unnecessary.Product after enzyme cuts process 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, thus make biotin labelings multiple on reacted product band.
First the ASPE primer working fluid of mixing is prepared: get the corresponding wild-type of gene to be detected and saltant type ASPE primer stock solution 10ul respectively 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:
Response procedures is: 96 DEG C of 2min; 94 DEG C of 30s, 54 DEG C of 1min, 72 DEG C of 2min, 30 circulations; 4 DEG C save backup.
Five, hybridization
1., according to the ASPE primer of design, the corresponding 12 kinds of microballoons (as described in Example 1) of every group selection, often kind of microballoon concentration is 2.5 × 10
5individual/ml.Often kind of microballoon is encoded with different colours respectively, simultaneously often kind of microsphere surface is connected to the specific oligonucleotide sequence (anti-tag) of one section of 24bp, and the tag sequence specific that these anti-tag sequences can be held with corresponding ASPE primer 5 ' respectively combines;
2. get 1ul respectively and often plant the microballoon of numbering 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 microsphere suspensions of 25ul in the 96 corresponding holes of hole filter plate, control wells adds the ddH of 25ul
2o;
6. the ASPE reaction solution getting 5-25ul, in corresponding hole, uses ddH
2o complements to 50ul;
7. encase 96 orifice plates with lucifuge with masking foil, 95 DEG C of 60s, 37 DEG C of 15min hatch 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;
Microballoon is resuspended in 1 × Tm hybridization buffer of 75ul by 11., adds the SA-PE (SA-PE) that 15ul concentration is 10ug/ml;
Hatch 15min for 12.37 DEG C, detect on Luminex instrument.
Six, result detects and data analysis
Reaction after product is detected by Luminex serial analysis instrument.Detected result is as shown in table 4, table 5 and table 6.
Following requirement is had to fluorescent value (MFI) and data processing:
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 represent with 0);
3. meet the data of above two conditions, by following formulae discovery sudden change ratio:
Sudden change ratio=saltant type NET MFI ÷ (saltant type NETMFI+ 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 saltant type homozygote.
Use present method to detect 20 increments CYP2C8 gene polymorphism sites 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.Detect with sequencing and compare with liquid-phase chip result, calculate the identical rate of classifying method detected result provided by the present invention.Present method detect 20 increments this CYP2C8 genotype call results and the sequencing result rate of coincideing reach 100%.Visible CYP2C8 genetic polymorphism detection liquid-phase chip provided by the present invention can detect CYP2C8 gene polymorphism sites type exactly, and result is reliable and stable.
One of table 4 pattern detection result (MFI)
Two of table 5 pattern detection result (MFI)
Table 6 sample CYP2C8 transgenation ratio (%)
Table 7 sample CYP2C8 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 | 145GA | 145GA |
| 4 | Wild-type | Wild-type |
| 5 | Wild-type | Wild-type |
| 6 | 127TT | 127TT |
| 7 | Wild-type | Wild-type |
| 8 | 145AA、315CC | 145AA、315CC |
| 9 | Wild-type | Wild-type |
| 10 | Wild-type | Wild-type |
| 11 | Wild-type | Wild-type |
| 12 | 124GG | 124GG |
| 13 | Wild-type | Wild-type |
| 14 | Wild-type | Wild-type |
| 15 | Wild-type | Wild-type |
| 16 | Wild-type | Wild-type |
| 17 | 204delA | 204delA |
| 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 CYP2C8 gene polymorphism sites
One, the design (selection of Tag sequence and Anti-Tag sequence) prepared of liquid-phase chip
Liquid-phase chip is detected for CYP2C8 Gene A 124G and C115G site mutation, respectively for the specific primer sequence that wild-type and the saltant type design ASPE primer 3 ' of A124G and C115G are held, the Tag sequence that ASPE primer 5 ' is held then is selected from SEQ ID NO.1-SEQ ID NO.12, accordingly, be coated in and microballoon is selected from SEQ ID NO.25-SEQ ID NO.36 with the anti-tag sequence that corresponding tag complementary matches.Specific design is as shown in following table (table 8).The synthesis of ASPE primer, anti-tag sequence bag by microballoon, amplimer, detection method etc. as described in embodiment 1 and embodiment 2.
Design prepared by table 8 liquid-phase chip
Two, sample detection
Adopt liquid-phase chip prepared by above-mentioned design, detect sample 21-40 by testing process described in embodiment 2 and method, detected result is as follows:
Table 8 pattern detection result and Polymorphism Analysis
Table 8 pattern detection result and Polymorphism Analysis
Other is for the liquid-phase chip in different mutational site, and ASPE primer uses different Tag sequences, and its result is still reliable and stable, and concrete data are omitted.And ASPE primer is when selecting that in embodiment 1, tag sequence and specific primer sequence are arranged in pairs or groups, effect better (signal to noise ratio is better), see the present embodiment test group 2 and test group 5.Other different tag sequence and specific primer sequence are arranged in pairs or groups, and with coming to the same thing of embodiment 2 and the present embodiment, concrete data are omitted.
The selection of embodiment 4CYP2C8 genetic polymorphism detection specific primer sequence
One, the design (selection of wild-type and saltant type specific primer sequence) prepared of liquid-phase chip
Liquid-phase chip is detected for the pleomorphism site of CYP2C8 Gene A 127T and G145A, with the complementary sequence forward or backwards of this place, mutational site target sequence for template, respectively for the specific primer sequence that wild-type and the saltant type design ASPE primer 3 ' of A127T and G145A are held, comprise preferred specific primer sequence in the embodiment of the present invention 1 and 2 alternative specific primer sequences, as shown in table 9.Wherein,
interior base is pleomorphism site.
Table 9 specific primer sequence
Liquid-phase chip is detected for the pleomorphism site of CYP2C8 Gene A 127T and G145A, different specific primer sequences is selected for A127T and G145A, the Tag sequence that ASPE primer 5 ' is held then is fixed as the best effect sequence in embodiment 1, and select the anti-tag sequence corresponded, specific design is as shown in following table (table 10).The synthesis of ASPE primer, anti-tag sequence bag by microballoon, amplimer, detection method etc. as described in embodiment 1 and embodiment 2.
Design two prepared by table 10 liquid-phase chip
Two, sample detection
Adopt liquid-phase chip prepared by above-mentioned design, detect sample 41-60 by testing process described in embodiment 2 and method, detected result is as follows:
Table 11 pattern detection result and Polymorphism Analysis
Table 11 pattern detection result and Polymorphism Analysis
From the present embodiment, when ASPE primer selects that in embodiment 1, specific primer sequence and tag sequence are arranged in pairs or groups, effect better (signal to noise ratio is better), see the present embodiment test group 7 and test group 10.Other derives from the different specific primer sequence of the complementary sequence forward or backwards of place, target detect site sequence and tag sequence is arranged in pairs or groups, with coming to the same thing of embodiment 2 and the present embodiment, namely be still that the specific primer sequence described in embodiment 2 is better from different tag sequence arranging effects, concrete data are omitted.Other multiple specific primer sequence for different mutational sites and tag sequence are arranged in pairs or groups, with coming to the same thing of embodiment 2 and the present embodiment, the Auele Specific Primer namely selected by embodiment 1, has better signal to noise ratio, Detection results is also better, and concrete data are omitted.
Be more than illustrating for possible embodiments of the present invention, but this embodiment be not used to limit the scope of the claims of the present invention, allly do not depart from the equivalence that skill of the present invention spirit does and implement or change, all should be contained in the scope of the claims of the present invention.
Claims (5)
1. a CYP2C8 genetic polymorphism detection liquid-phase chip, is characterized in that, includes:
(A). the wild-type designed respectively for the different pleomorphism site of CYP2C8 gene and the ASPE primer pair of saltant type: every bar ASPE primer holds the specific primer sequence for goal gene pleomorphism site to form by 5 ' the tag sequence of holding and 3 ', and described specific primer sequence is: for SEQ ID NO.13 and the SEQ ID NO.14 in A127T site; And the SEQ ID NO.15 be selected from for A124G site and SEQ ID NO.16; For SEQ ID NO.17 and the SEQ ID NO.18 in G145A site; For SEQ ID NO.19 and the SEQ ID NO.20 in 204delA site; With at least one pair of in the SEQ ID NO.21 in C115G site and SEQID NO.22; Described tag sequence is selected from SEQ ID NO.1 ~ SEQ ID NO.12;
(B). microballoon that have anti-tag sequence bag quilt, that have different colours coding, is also provided with spacer sequence in the middle of described anti-tag sequence is connected with microballoon; Described anti-tag sequence is selected from SEQ ID NO.25 ~ SEQ ID NO.36, and described anti-tag sequence can correspondingly be matched with selected tag complementary in (A);
(C). for amplifying the primer needing that detect, that there is corresponding pleomorphism site target sequence;
Described amplimer is: for SEQ ID NO.37 and the SEQ ID NO.38 in A127T site; And the SEQ ID NO.39 be selected from for A124G site and SEQ ID NO.40; For SEQ ID NO.41 and the SEQ IDNO.42 in G145A, 204delA site; With at least one pair of in the SEQ ID NO.43 in C115G site and SEQ ID NO.44.
2. CYP2C8 genetic polymorphism detection liquid-phase chip according to claim 1, it is characterized in that, described ASPE primer is: for the sequence be made up of SEQ ID NO.1 and SEQ ID NO.13 and the sequence be made up of SEQ ID NO.2 and SEQ IDNO.14 in A127T site; And be selected from for the sequence be made up of SEQ ID NO.3 and SEQ ID NO.15 in A124G site and the sequence that is made up of SEQ ID NO.4 and SEQ ID NO.16; For the sequence be made up of SEQ ID NO.5 and SEQ IDNO.17 in G145A site and the sequence that is made up of SEQ ID NO.6 and SEQ ID NO.18; For the sequence be made up of SEQID NO.7 and SEQ ID NO.19 in 204delA site and the sequence that is made up of SEQ ID NO.8 and SEQ ID NO.20; With at least one pair of in the sequence be made up of SEQ ID NO.9 and SEQ ID NO.21 for C115G site and the sequence that is made up of SEQ ID NO.10 and SEQ ID NO.22.
3. CYP2C8 genetic polymorphism detection liquid-phase chip according to claim 1, is characterized in that,
(A). described ASPE primer is: for the sequence be made up of SEQ ID NO.1 and SEQ ID NO.13 in A127T site and the sequence that is made up of SEQ ID NO.2 and SEQ ID NO.14; And be selected from for the sequence be made up of SEQ ID NO.3 and SEQ ID NO.15 in A124G site and the sequence that is made up of SEQ ID NO.4 and SEQ ID NO.16; For the sequence be made up of SEQ ID NO.5 and SEQ ID NO.17 in G145A site and the sequence that is made up of SEQ ID NO.6 and SEQ ID NO.18; For the sequence be made up of SEQ ID NO.7 and SEQ ID NO.19 and the sequence be made up of SEQ ID NO.8 and SEQ IDNO.20 in 204delA site; For at least one pair of in the sequence be made up of SEQ ID NO.9 and SEQ ID NO.21 in C115G site and the sequence that is made up of SEQ IDNO.10 and SEQ ID NO.22;
(B). microballoon that have anti-tag sequence bag quilt, that have different colours coding, is also provided with spacer sequence in the middle of described anti-tag sequence is connected with microballoon; Described anti-tag sequence is selected from SEQ ID NO.25 ~ SEQ ID NO.36, and described anti-tag sequence can correspondingly be matched with selected tag complementary in (A);
(C). described amplimer is: for SEQ ID NO.37 and the SEQ ID NO.38 in A127T site; And the SEQ ID NO.39 be selected from for A124G site and SEQ ID NO.40; For SEQ ID NO.41 and the SEQ ID NO.42 in G145A, 204delA site; For at least a pair in the SEQ ID NO.43 in C115G site and SEQ ID NO.44.
4. the CYP2C8 genetic polymorphism detection liquid-phase chip according to any one of claim 1-3, is characterized in that, described spacerarm is 5-10 T.
5. for an Auele Specific Primer for CYP2C8 genetic polymorphism detection, it is characterized in that, described Auele Specific Primer is: for SEQ ID NO.13 and the SEQ ID NO.14 in A127T site; And the SEQ ID NO.15 be selected from for A124G site and SEQ IDNO.16; For SEQ ID NO.17 and the SEQ ID NO.18 in G145A site; For SEQ ID NO.19 and the SEQ ID NO.20 in 204delA site; With at least one pair of in the SEQ ID NO.21 in C115G site and SEQ ID NO.22.
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