CN102912008B - ABCG2 gene polymorphism detection specific primer and liquid phase chip - Google Patents
ABCG2 gene polymorphism detection specific primer and liquid phase chip Download PDFInfo
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Abstract
The invention discloses an ABCG2 gene detection specific primer and a liquid phase chip. The liquid phase chip main comprises each ASPE primer, a microsphere coated by anti-tag sequence and an amplification primer, wherein the ASPE primer comprises 5' end tap sequence and 3' end specific primer sequence aiming at gene mutation site; and the specific primer sequence comprises SEQ ID NO.7 and SEQ ID NO.8 aiming at the C184T site, SEQ ID NO.9 and SEQ ID NO.10 aiming at the C229A site, and/or SEQ ID NO.11 and SEQ ID NO.12 aiming at the C145T site. The consistency between the detection result of the liquid phase chip and that of a sequencing method reaches 100 percent, so that multi-mutational-site wild type and mutant type parallel detection is realized.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete a kind of ABCG2 gene pleiomorphism detection specificity primer and the liquid-phase chip of relating to.
Background technology
ABCG2 is abc transport superfamily protein (ATP-binding cassette transporter superfamily, ABC transporter superfamily) G subfamily the 2nd member.ABCG2 is positioned cytolemma, widely distributed in healthy tissues, is mainly distributed in the tissue with secretion and excretory function as in placenta plamoditrophoblast, small intestine and colonic epithelium, the little periosteum of liver, timid periosteum, lobule of mammary gland and vascular endothelial cell.Mankind ABCG 2 genes are positioned at 4q22～23,655 amino-acid residues of encoding.ABCG 2 total length 66kb, are made up of 16 exons and 15 introns, exon be 60～332bp not etc.Found that at present ABCG2 gene has more than 40 SNPs, wherein 3 of target detect of the present invention SNPs sites are the most common.
The ABCG2 gene mutation site (pleomorphism site) of target detect of the present invention, it is as shown in the table:
| Sequence number | The content of ABCG2 site mutation | Write a Chinese character in simplified form |
| 1 | , there is C → T sudden change in the 184th Nucleotide of SEQ ID NO.27 | C184T |
| 2 | , there is C → A sudden change in the 229th Nucleotide of SEQ ID NO.27 | C229A |
| 3 | , there is C → T sudden change in the 145th Nucleotide of SEQ ID NO.28 | C145T |
At present, the method that ABCG2 gene pleiomorphism is detected, analyzed is little, common detection method only has direct sequencing, direct sequencing is the gold mark method of gene test, but owing to there being the limitation that detects flux, a kind of mutation type can only be detected at every turn, the needs of practical application can not be met.
Summary of the invention
One of object of the present invention is to provide ABCG2 gene pleiomorphism and detects liquid-phase chip, and this liquid-phase chip can be used for detecting wild-type and the saltant type of ABCG2 gene three kinds of common genotype C184T, C229A and C145T.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind of ABCG2 gene pleiomorphism detects liquid-phase chip, includes:
(A). the wild-type designing respectively for the different pleomorphism sites of ABCG2 gene and the ASPE primer of saltant type: every kind of ASPE primer is made up of for the specific primer sequence of goal gene pleomorphism site tag sequence and the 3 ' end of 5 ' end, and described specific primer sequence is: for SEQ ID NO.7 and the SEQ ID NO.8 in C184T site; For SEQ ID NO.9 and the SEQ ID NO.10 in C229A site; And/or for SEQ ID NO.11 and the SEQ ID NO.12 in C145T site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.6;
(B). there is microballoon that anti-tag sequence is coated with, that there is different colours coding, in the middle of described anti-tag sequence is connected with microballoon, be also provided with spacerarm sequence; Described anti-tag sequence is selected from SEQ ID NO.13～SEQ ID NO.18, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). need primer detection, that there is the target sequence in polymorphism mutational site for amplifying.
Preferably, described amplimer is: for SEQ ID NO.22 and the SEQ ID NO.23 in C184T, C229A site; And/or for SEQ ID NO.24 and the SEQ ID NO.25 in C145T site.
Preferably, described ASPE primer is: for the sequence being made up of SEQ ID NO.1 and SEQ ID NO.7 in C184T site and the sequence that is made up of SEQ ID NO.2 and SEQ ID NO.8; For the sequence being formed by SEQ ID NO.3 and SEQ ID NO.9 in C229A site and the sequence that formed by SEQ ID NO.4 and SEQ ID NO.10; And/or for the sequence being formed by SEQ ID NO.5 and SEQ ID NO.11 in C145T site and the sequence that formed by SEQ ID NO.6 and SEQ ID NO.12.
Another object of the present invention is to provide a kind of Auele Specific Primer detecting for ABCG2 gene pleiomorphism.
Realize above-mentioned purpose technical scheme as follows:
The Auele Specific Primer detecting for ABCG2 gene pleiomorphism, it is: described specific primer sequence is: for SEQ ID NO.7 and the SEQ ID NO.8 in C184T site; For SEQ ID NO.9 and the SEQ ID NO.10 in C229A site; And/or for SEQ ID NO.11 and the SEQ ID NO.12 in C145T site.
Major advantage of the present invention is:
1. the identical rate of the detected result of detection liquid-phase chip provided by the present invention and sequencing is up to 100%, and detects the needed time well below conventional sequencing technologies, and realistic especially application needs.Prepared ABCG2 gene pleiomorphism detects liquid-phase chip and has extraordinary signal-noise ratio, and between designed probe and anti-tag sequence, substantially there is not cross reaction, 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 multiple SNP site.
2. the present invention, by the design experiences of the long-term accumulation of contriver and a large amount of experimental implementation, has chosen optimum combination from numerous Auele Specific Primers.Designed ASPE primer specificity primer can the sensitive pleomorphism site of identifying specifically target detect, accurately distinguishes the genotype of various types; In same reaction system, between different Auele Specific Primers, substantially there is not cross reaction between Auele Specific Primer and the pcr amplification product of non-target detect, detection specificity is good, and cross reacting rate is lower than 3%; Except detecting Single locus sudden change situation, the also polymorphism situation in the multiple mutational sites of parallel detection simultaneously, detects effect consistent.
3. detection method step of the present invention is simple, three kinds of pleomorphism sites detect the amplification that can complete by a step multiplex PCR two target sequences that contain SNP site, the many uncertain factors that exist in the complex operations processes such as repeated multiple times PCR are avoided, thereby can greatly improve Detection accuracy, embody accurate while qualitative and quantitative analysis feature.
4. not only to have overcome traditional solid phase chip susceptibility not high in the present invention, and the poor defect of repeatability of detected result is improved existing liquid-phase chip technology simultaneously, makes prepared microballoon can be applicable to different test items, has very strong expansion.The fluorescent signal value detecting improves greatly, thereby the sensitivity detecting is further enhanced, and signal to noise ratio strengthens, and detected result more accurately and reliably.
5. the technical scheme that the ABCG2 gene mutation detection liquid-phase chip in the present invention provides a kind of difference to conceive for the parallel detection of multiple sites Multi-genotype, and produced significant technique effect.Meanwhile, due to the technical characterictics such as high-throughput high specific, the demand of more realistic application.Liquid-phase chip technology of the present invention is by the technology trends that is representing that biological target detects.
Embodiment
Embodiment 1 ABCG2 gene pleiomorphism detects liquid-phase chip, mainly includes:
One, ASPE primer
For wild-type and the saltant type of ABCG2 gene three kinds of common genotype C184T, C229A and C145T, design respectively specific primer sequence.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 1 ABCG2 gene
Every ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence for anti-tag sequence on corresponding microballoon, and 3 ' end is saltant type or the special primer fragment (as shown in Table 1 above) of wild-type.All ASPE primers are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with respectively the stock solution of 100pmol/mL with 10mmol/L Tris Buffer.
Two, the coated microballoon of anti-tag sequence
According to designed ASPE Auele Specific Primer fragment, select tag sequence, reduce to greatest extent between the anti-tag sequence of each microballoon and secondary structure that tag and ASPE Auele Specific Primer fragment may form, on 6 kinds of microballoons numberings of selection and microballoon, corresponding anti-tag sequence is as shown in table 2:
Corresponding anti-tag sequence on table 2 microballoon numbering and microballoon
6 kinds of microballoons selecting are purchased from Luminex company of the U.S., by coated anti-tag sequence and microballoon.Between anti-tag sequence and microballoon, be connected with the spacerarm sequence of 5-10 T, before each anti-tag sequence, add the spacerarm sequence of the preceding paragraph 5-10 T, anti-tag sequence is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.By synthetic anti-tag sequence sterilizing ddH
2o is made into the stock solution of 100nmol/ml.Described spacerarm is for by anti-tag and microsphere surface is spaced apart or anti-tag is placed in to the sequence of hydrophilic environments.By the spacerarm sequence of suitable length is set between anti-tag sequence and microballoon, can reduce sterically hinderedly, improve the efficiency of hybridization and the specificity of hybridization.Common spacerarm sequence comprises poly dT, i.e. poly (dT), and oligomerization four polyoxyethylene glycol and (CH2) n spacerarm (n >=3), 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 coated process of microballoon is as follows:
Get respectively 5 × 10
6the carboxylated microballoon (purchased from Luminex company) of individual above-mentioned numbering 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) (purchased from the Pierce Chemical company) working fluid of preparation 10ng/ml.Toward the EDC working fluid that adds 2.5ul in microballoon suspension, constant-temperature incubation 30 minutes, then add the EDC working fluid of 2.5ul, then constant-temperature incubation 30 minutes.After reaction finishes, the Tween-20 with 0.02% washs once, then washs once with 0.1% SDS liquid.The Tris-EDTA solution [10mmol/L Tris (pH8.0)] that the microballoon that is coated with anti-tag sequence after washing is resuspended in to 100ul, in 1mmol/LEDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains mutational site
For ABCG2 gene three kinds of common genotype C184T, C229A and C145T, design of amplification primers, to (in table 3), amplifies respectively two target sequences that contain pleomorphism site.
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 primer after synthetic is mixed with respectively the stock solution of 100pmol/mL with 10mmol/L Tris Buffer.
Embodiment 2 uses ABCG2 gene pleiomorphism described in embodiment 1 to detect the detection of liquid-phase chip to sample
The formula of described various solution is as follows:
MES damping fluid (pH5.0) formula (250ml) of 50mM:
2 × Tm hybridization buffer
| Reagent | Source | Final concentration | The consumption of every 250ml |
| 1MTris-HCl，pH8.0 | Sigma T3038 | 0.2M | 50ml |
| 5MNaCl | Sigma S5150 | 0.4M | 20ml |
| Triton X-100 | Sigma T8787 | 0.16％ | 0.4ml |
After filtration, be stored in 4 ℃.
ExoSAP-IT test kit is purchased from USB company of the U.S..
Biotin labeled dCTP is purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
Methods involving with reference to " molecular cloning " about DNA extraction, obtains DNA to be detected.
Two, the pcr amplification of testing sample
Design two pairs of primers, multiplex PCR one step amplifies the two objective sequences that contain respectively ABCG2 gene three kinds of common genotype C184T, C229A and C145T, wherein, C184T and C229A are positioned at same amplified production, product size is respectively 456bp and 313bp, and primer sequence (SEQ ID NO.22-25) is shown in shown in above-mentioned table 3.
First prepare multiple PCR primer working fluid: respectively get respectively the primer stock solution 100ul of SEQ ID NO.22-25 in 1.5ml Eppendorf tube, mix and be multiple PCR primer working fluid.Multi-PRC reaction system is as follows:
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. hatch 15min for 37 ℃, hatch 15min for 80 ℃, the enzyme that deactivation is unnecessary.Enzyme is cut product after treatment and 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 multiple biotin labeling on reacted product band.
First the ASPE primer working fluid that preparation mixes: get respectively the corresponding wild-type of gene to be detected and saltant type ASPE primer stock solution 10ul in 1.5ml Eppendorf tube, add 10mmol/L Tris Buffer to mend to 200ul, mix and be ASPE mix primer working fluid.The system of ASPE reaction is as follows:
Response procedures is: 96 ℃ of 2min; 94 ℃ of 30s, 54 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ save backup.
Five, hybridization
1. according to the ASPE primer of design, every kind of microballoon concentration of the corresponding 4 kinds of microballoons of every group selection (as described in Example 1) is 2.5 × 10
5individual/m.Every kind of microballoon is encoded with different colours respectively, while, every kind of microsphere surface was connected with respectively the specific oligonucleotide sequence (anti-tag) of one section of 24bp, the tag sequence specific combination that these anti-tag sequences can be held with corresponding ASPE primer 5 ' respectively;
2. get respectively the microballoon of every kind of numbering of 1ul in the Eppendorf tube of 1.5ml;
3. microballoon is in >=centrifugal the 1-2min of 10000g;
4. supernatant discarded, microballoon is resuspended in 2 × Tm hybridization buffer of 100ul, and vortex mixes;
5. get the above-mentioned microballoon suspension of 25ul in the 96 corresponding holes of hole filter plate, control wells adds the ddH of 25ul
2o;
6. get the ASPE reaction solution of 5-25ul in corresponding hole, use ddH
2o complements to 50ul;
7. encase 96 orifice plates with lucifuge with masking foil, 95 ℃ of 60s, 37 ℃ of 15min are hatched hybridization;
8. the microballoon after hybridization is in >=centrifugal the 2-5min of 3000g;
9. remove supernatant, microballoon is resuspended in 1 × Tm hybridization buffer of 75ul;
10. microballoon is in >=centrifugal the 2-5min of 3000g;
11. are resuspended in microballoon in 1 × Tm hybridization buffer of 75ul, and adding 15ul concentration is the SA-PE (SA-PE) of 10ug/ml;
Hatch 15min for 12.37 ℃, on Luminex instrument, detect.
Six, result detects and data analysis
Reaction after product detects by Luminex serial analysis instrument.Detected result is as shown in table 4, table 5 and table 6.
Fluorescent value (MFI) and data processing are had to following requirement:
1. each site need have at least an allelotrope MFI to be greater than 300 and be greater than 10 × PCR negative control MFI;
2.NET MFI=sample MFI-PCR negative control MFI (NET MFI be less than 0 represent with 0);
3. meet the data of above two conditions, calculate sudden change ratio by following formula:
Sudden change ratio=saltant type NET MFI ÷ (saltant type NETMFI+ wild-type NET MFI)
4. the sudden change ratio definite threshold (cut-off value) to each detection site rule of thumb, to divide wild-type homozygote, heterozygote and saltant type homozygote.
Use present method to detect 20 increments ABCG2 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 liquid-phase chip result and compare with sequencing, calculate the identical rate of classifying method detected result provided by the present invention.Present method detects 20 increments ABCG2 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.Visible ABCG2 gene pleiomorphism provided by the present invention detects liquid-phase chip can detect ABCG2 gene polymorphism sites type exactly, and result is reliable and stable.
One of table 4 pattern detection result (MFI)
Table 5 sample ABCG2 transgenation ratio (%)
| Sample number | C184T | C229A | C145T |
| 1 | 2％ | 2％ | 1％ |
| 2 | 1％ | 2％ | 3％ |
| 3 | 1％ | 2％ | 2％ |
| 4 | 1％ | 1％ | 2％ |
| 5 | 2％ | 47％ | 2％ |
| 6 | 2％ | 1％ | 1％ |
| 7 | 1％ | 2％ | 2％ |
| 8 | 2％ | 1％ | 2％ |
| 9 | 2％ | 2％ | 2％ |
| 10 | 2％ | 2％ | 2％ |
| 11 | 98％ | 1％ | 2％ |
| 12 | 1％ | 1％ | 98％ |
| 13 | 1％ | 2％ | 2％ |
| 14 | 2％ | 1％ | 1％ |
| 15 | 1％ | 98％ | 1％ |
| 16 | 2％ | 1％ | 2％ |
| 17 | 2％ | 2％ | 1％ |
| 18 | 2％ | 2％ | 1％ |
| 19 | 2％ | 2％ | 2％ |
| 20 | 3％ | 1％ | 1％ |
Table 6 sample ABCG2 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 | Wild-type | Wild-type |
| 4 | Wild-type | Wild-type |
| 5 | 229CA | 229CA |
| 6 | Wild-type | Wild-type |
| 7 | Wild-type | Wild-type |
| 8 | Wild-type | Wild-type |
| 9 | Wild-type | Wild-type |
| 10 | Wild-type | Wild-type |
| 11 | 184TT | 184TT |
| 12 | 145TT | 145TT |
| 13 | Wild-type | Wild-type |
| 14 | Wild-type | Wild-type |
| 15 | 229AA | 229AA |
| 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 detection of the liquid-phase chip of the ASPE primer that embodiment 3 is different to ABCG2 gene SNP site
One, the design (selection of Tag sequence and Anti-Tag sequence) that prepared by liquid-phase chip
Detect liquid-phase chip as example take ABCG2 gene C 184T site mutation, respectively for the wild-type of C184T and the specific primer sequence of saltant type design ASPE primer 3 ' end, the Tag sequence of ASPE primer 5 ' end is selected from SEQ ID NO.1-SEQ ID NO.6, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing being coated on microballoon is selected from SEQ ID NO.13-SEQ ID NO.18.Specific design is as shown in following table (table 7).Synthetic, the anti-tag sequence of ASPE primer are coated with microballoon, amplimer, detection method etc. as described in embodiment 1 and embodiment 2.
Design prepared by table 7 liquid-phase chip
Two, sample detection
The liquid-phase chip that adopts above-mentioned design to prepare, detects sample 21-40 by testing process described in embodiment 2 and method, and detected result is as follows:
Table 8 pattern detection result and gene SNP analysis
Other is for the liquid-phase chip in different mutational sites, and ASPE primer uses different Tag sequences, and its result is still reliable and stable, and concrete data are omitted.And ASPE primer is while selecting in embodiment 1 collocation of tag sequence and specific primer sequence, effect better (signal to noise ratio is better), referring to the present embodiment test group 1.Other different tag sequences and specific primer sequence collocation, with coming to the same thing of embodiment 2 and the present embodiment, concrete data are omitted.
The selection of embodiment 4 ABCG2 gene pleiomorphism detection specificity primer sequences
One, the design (selection of wild-type and saltant type specific primer sequence) that prepared by liquid-phase chip
Detect liquid-phase chip as example take the SNP site of ABCG2 Gene C2 29A, take the complementary sequence forward or backwards of this place, mutational site target sequence as template, respectively for the wild-type of C229A and the specific primer sequence of saltant type design ASPE primer 3 ' end, comprise preferred specific primer sequence and 2 alternative specific primer sequences in the embodiment of the present invention 1, as shown in table 9.Wherein,
interior base is pleomorphism site.
Table 9 specific primer sequence
Detect liquid-phase chip as example take the SNP site of ABCG2 Gene C2 29A, select different specific primer sequences for C229A, the Tag sequence of ASPE primer 5 ' end is fixed as the best effect sequence in embodiment 1, and select the anti-tag sequence of answering in contrast, specific design is as shown in following table (table 10).Synthetic, the anti-tag sequence of ASPE primer are coated with microballoon, amplimer, detection method etc. as described in embodiment 1 and embodiment 2.
Two of design prepared by table 10 liquid-phase chip
Two, sample detection
The liquid-phase chip that adopts above-mentioned design to prepare, detects sample 41-60 by testing process described in embodiment 2 and method, and detected result is as follows:
Table 11 pattern detection result and Polymorphism Analysis
From the present embodiment, when ASPE primer is selected in embodiment 1 collocation of specific primer sequence and tag sequence, effect better (signal to noise ratio is better), referring to the present embodiment test group 4.Other derives from different specific primer sequences and the collocation of tag sequence of the complementary sequence forward or backwards of place, target detect site sequence, with coming to the same thing of embodiment 2 and the present embodiment, be still that the specific primer sequence described in embodiment 1 is better from different tag sequence arranging effects, concrete data are omitted.Other multiple specific primer sequence for different mutational sites and the collocation of tag sequence, with coming to the same thing of embodiment 2 and the present embodiment, the selected Auele Specific Primer of embodiment 1, has better signal to noise ratio, detect effect also better, concrete data are omitted.
Be more than for the illustrating of possible embodiments of the present invention, but this embodiment is not in order to limit the scope of the claims of the present invention, allly do not depart from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the scope of the claims of the present invention.
Claims (4)
1. ABCG2 gene pleiomorphism detects a liquid-phase chip, it is characterized in that, includes:
(A). the wild-type designing respectively for the different pleomorphism sites of ABCG2 gene and the ASPE primer of saltant type: every kind of ASPE primer is made up of for the specific primer sequence of goal gene pleomorphism site tag sequence and the 3 ' end of 5 ' end, and described specific primer sequence is: for SEQ ID NO.9 and the SEQ ID NO.10 in C229A site; And/or for SEQ ID NO.11 and the SEQ ID NO.12 in C145T site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.6;
(B). there is microballoon that anti-tag sequence is coated with, that there is different colours coding, in the middle of described anti-tag sequence is connected with microballoon, be also provided with spacerarm sequence; Described anti-tag sequence is selected from SEQ ID NO.13～SEQ ID NO.18, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). for amplifying the primer that needs the target sequence detecting, there is corresponding pleomorphism site; Described amplimer is: for SEQ ID NO.22 and the SEQ ID NO.23 in C229A site; And/or for the SEQ ID NO.24 in C145T site and
SEQ ID NO.25。
2. ABCG2 gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that, described ASPE primer is: for the sequence being made up of SEQ ID NO.3 and SEQ ID NO.9 in C229A site and the sequence that is made up of SEQ ID NO.4 and SEQ ID NO.10; And/or for the sequence being formed by SEQ ID NO.5 and SEQ ID NO.11 in C145T site and the sequence that formed by SEQ ID NO.6 and SEQ ID NO.12.
3. ABCG2 gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that, comprises
(A). described ASPE primer is: for the sequence being made up of SEQ ID NO.3 and SEQ ID NO.9 in C229A site and the sequence that is made up of SEQ ID NO.4 and SEQ ID NO.10; With the sequence being formed by SEQ ID NO.5 and SEQ ID NO.11 for C145T site and the sequence being formed by SEQ ID NO.6 and SEQ ID NO.12;
(B). there is microballoon that anti-tag sequence is coated with, that there is different colours coding, in the middle of described anti-tag sequence is connected with microballoon, be also provided with spacerarm sequence; Described anti-tag sequence is selected from SEQ ID NO.13～SEQ ID NO.18, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). for amplifying the primer that needs the target sequence detecting, there is corresponding pleomorphism site; Described amplimer is: for SEQ ID NO.22 and the SEQ ID NO.23 in C229A site; With SEQ ID NO.24 and the SEQ ID NO.25 for C145T site.
4. ABCG2 gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that, described spacerarm is 5-10 T.
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| FUNCTIONAL ASSESSMENT OF ABCG2 (BCRP) GENE POLYMORPHISMS TO PROTEIN EXPRESSION IN HUMAN PLACENTA;Daisuke Kobayashi et al.;《DRUG METABOLISM AND DISPOSITION》;20051231;第33卷(第1期);94-101 * |
| 肖宇山, 胡丽莉.ABCG2基因单核苷酸多态性研究进展.《广东药学院学报》.2010,第26卷(第5期),540-545. |
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