CN102912008A - 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, and is widely distributed in healthy tissues, mainly is distributed in the tissue such as placenta plamoditrophoblast, small intestine and colonic epithelium, the little periosteum of liver, timid periosteum, lobule of mammary gland and vascular endothelial cell with secretion and excretory function.Human ABCG 2 genes are positioned at 4q22～23,655 amino-acid residues of encoding.ABCG 2 total length 66kb are comprised of 16 exons and 15 introns, and exon is that 60～332bp does not wait.Found that at present the 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 | C → T sudden change occurs in the 184th Nucleotide of SEQ ID NO.27 | C184T |
| 2 | C → A sudden change occurs in the 229th Nucleotide of SEQ ID NO.27 | C229A |
| 3 | C → T sudden change occurs in the 145th Nucleotide of SEQ ID NO.28 | C145T |
At present, the method that the ABCG2 gene pleiomorphism is detected, analyzes seldom, common detection method only has direct sequencing, direct sequencing is the gold mark method of gene test, but owing to there is 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 satisfied.
Summary of the invention
One of purpose of the present invention provides the ABCG2 gene pleiomorphism and detects liquid-phase chip, and this liquid-phase chip can be used for detecting wild-type and the mutant 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 that designs respectively for the different pleomorphism sites of ABCG2 gene and the ASPE primer of mutant: every kind of ASPE primer is comprised of the tag sequence of 5 ' end and 3 ' the end specific primer sequence for the goal gene pleomorphism site, and described specific primer sequence is: for SEQ ID NO.7 and the SEQ ID NO.8 in C184T site; SEQ ID NO.9 and SEQ ID NO.10 for the 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). anti-tag sequence microballoon coated, that have the different colours coding is arranged, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon; 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). being used for amplifying need to primer that detect, that have the target sequence in polymorphism mutational site.
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: the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.7 for the C184T site reaches the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.8; The sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.9 for the C229A site reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.10; And/or for the sequence that is formed by SEQ ID NO.5 and SEQ ID NO.11 in C145T site and the sequence that is formed by SEQ ID NO.6 and SEQ ID NO.12.
Another object of the present invention provides a kind of Auele Specific Primer for the detection of ABCG2 gene pleiomorphism.
Realize that the above-mentioned purpose technical scheme is as follows:
A kind of Auele Specific Primer for the detection of ABCG2 gene pleiomorphism, it is: described specific primer sequence is: for SEQ ID NO.7 and the SEQ ID NO.8 in C184T site; SEQ ID NO.9 and SEQ ID NO.10 for the 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 sequencing technologies commonly used, and realistic especially application needs.Prepared ABCG2 gene pleiomorphism detects liquid-phase chip and has extraordinary signal-noise ratio, and basically there is not cross reaction between designed probe and the anti-tag sequence, choosing and tag sequence label and the specifically combination of ASPE primer of tag sequence label, anti-tag sequence label, can avoid cross reaction, realize the parallel detection in a plurality of SNP site.
2. the present invention has chosen optimum combination by the design experiences of contriver's long-term accumulation and a large amount of experimental implementation from numerous Auele Specific Primers.Designed ASPE primer specificity primer can sensitive be identified the pleomorphism site of target detect specifically, accurately distinguishes the genotype of various types; In same reaction system, between the different Auele Specific Primers, basically do not have cross reaction 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 locus sudden change situation, the also polymorphism situation in a plurality of mutational sites of simultaneously parallel detection, it is consistent to detect effect.
3. detection method step of the present invention is simple, three kinds of pleomorphism sites detect and can finish two amplifications that contain the target sequence in SNP site by a step multiplex PCR, many uncertain factors of existing in the complex operations processes such as repeated multiple times PCR have been avoided, thereby can greatly improve Detection accuracy, embodied accurate while qualitative and quantitative analysis feature.
4. not only to have overcome traditional solid phase chip susceptibility not high in the present invention, and the defective that the repeatability of detected result is poor is improved existing liquid-phase chip technology simultaneously, so that prepared microballoon can be applicable to different test items, has very strong expansion.The fluorescent signal value that detects improves greatly, thereby so that the sensitivity that detects is further enhanced, signal to noise ratio strengthens, and detected result more accurately and reliably.
5. the ABCG2 gene mutation detection liquid-phase chip among the present invention provides a kind of technical scheme of different designs for the parallel detection of a plurality of sites Multi-genotype, and has produced significant technique effect.Simultaneously, since the technical characterictics such as high-throughput high specific, the demand of more realistic application.Liquid-phase chip technology of the present invention will represent the technology trends that biological target detects.
Embodiment
Embodiment 1 ABCG2 gene pleiomorphism detects liquid-phase chip, mainly includes:
One, ASPE primer
Wild-type and mutant for ABCG2 gene three kinds of common genotype C184T, C229A and C145T design respectively specific primer sequence.The ASPE primer is comprised of " Tag sequence+specific primer sequence ".The ASPE primer sequence is as shown in the table:
The ASPE primer sequence of table 1 ABCG2 gene (Tag sequence+specific primer sequence)
Every the ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence for anti-tag sequence on the corresponding microballoon, and 3 ' end is mutant or the special primer fragment (as shown in Table 1 above) of wild-type.All ASPE primers are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with respectively the stock solution of 100pmol/mL with 10mmol/L Tris Buffer.
Two, the coated microballoon of anti-tag sequence
According to designed ASPE Auele Specific Primer fragment, select the tag sequence, reduce to greatest extent between the anti-tag sequence of each microballoon and secondary structure that tag and ASPE Auele Specific Primer fragment may form, corresponding anti-tag sequence is as shown in table 2 on 6 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
6 kinds of microballoons selecting are available from U.S. Luminex company, with the anti-tag sequence coated with microballoon on.Be connected with the spacerarm sequence of 5-10 T between anti-tag sequence and the microballoon, namely add the spacerarm sequence of the preceding paragraph 5-10 T before each anti-tag sequence, the anti-tag sequence is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.With synthetic anti-tag sequence sterilization ddH
2O is made into the stock solution of 100nmol/ml.Described spacerarm is for being used for anti-tag and microsphere surface is spaced apart or anti-tag is placed the sequence of hydrophilic environments.By the spacerarm sequence of suitable length is set between anti-tag sequence and microballoon, can reduce sterically hinderedly, improve the efficient of hybridization and the specificity of hybridization.Common spacerarm sequence comprises poly dT, i.e. poly (dT), and oligomerization four polyoxyethylene glycol and (CH2) n spacerarm (n 〉=3) are such as (CH2) 12, (CH2) 18 etc.In addition, if exist poly (dA) to disturb, can also use poly (TTG) as spacerarm.Spacerarm of the present invention is preferably 5-10 T, and the process that microballoon is coated with is as follows:
Get respectively 5 * 10
6The carboxylated microballoon of individual above-mentioned numbering (available from Luminex company) is suspended in the MES solution of 50ul 0.1mol/L (pH4.5), adds the synthetic anti-tag molecule (100nmol/ml) of 10ul.EDC (N-(3-Dimethylaminopropyl-N-ethylcarbodiimide) (available from the Pierce Chemical company) working fluid of preparation 10ng/ml.The EDC working fluid that adds 2.5ul in the microballoon suspension, constant-temperature incubation 30 minutes adds the EDC working fluid of 2.5ul again, and constant-temperature incubation is 30 minutes again.After reaction finished, the Tween-20 washing with 0.02% was once washed once with 0.1% SDS liquid again.The microballoon that is coated with the anti-tag sequence after the washing is resuspended in the Tris-EDTA solution [10mmol/L Tris (pH8.0)] of 100ul, and among the 1mmol/LEDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains the mutational site
For ABCG2 gene three kinds of common genotype C184T, C229A and C145T, design of amplification primers amplifies respectively two target sequences that contain pleomorphism site 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 respectively the stock solution of 100pmol/mL with 10mmol/L Tris Buffer.
Embodiment 2 uses embodiment 1 described ABCG2 gene pleiomorphism to detect liquid-phase chip to the detection of sample
The prescription of described various solution is as follows:
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 | Sigma T3038 | 0.2M | 50ml |
| 5MNaCl | Sigma S5150 | 0.4M | 20ml |
| Triton X-100 | Sigma T8787 | 0.16％ | 0.4ml |
Be stored in 4 ℃ after the filtration.
The ExoSAP-IT test kit is available from U.S. USB company.
Biotin labeled dCTP is available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
With reference to " molecular cloning " methods involving about DNA extraction, obtain DNA to be detected.
Two, the pcr amplification of testing sample
Design two pairs of primers, one step of multiplex PCR 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 the same amplified production, the product size is respectively 456bp and 313bp, and primer sequence (SEQ ID NO.22-25) is seen shown in the above-mentioned table 3.
At first prepare the multiple PCR primer working fluid: respectively get respectively the primer stock solution 100ul of SEQ ID NO.22-25 in the 1.5ml Eppendorf tube, mix and be the multiple PCR primer working fluid.The multi-PRC reaction system is as follows:
The pcr amplification program is: 95 ℃ of 3min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 40s, 30 circulations; 72 ℃ of 10min; 4 ℃ save backup.
Three, the enzyme of PCR product is cut processing
1. get the reacted product of 7.5ul PCR, add 1ul 10 * SAP damping fluid, 1ul SAP enzyme and 0.5ul Exo-I enzyme;
2. hatch 15min for 37 ℃, hatch 15min for 80 ℃, the enzyme that deactivation is unnecessary.The product that enzyme is cut after the processing is directly used in follow-up ASPE primer extension reaction.
Four, site-specific primer extension reaction (ASPE)
Utilize the ASPE primer of above-mentioned design to carry out primer extension reaction, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on the reacted product band.
At first prepare the ASPE primer working fluid that mixes: get respectively the corresponding wild-type of gene to be detected and mutant ASPE primer stock solution 10ul in the 1.5ml Eppendorf tube, add 10mmol/L Tris Buffer and 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, simultaneously every kind of microsphere surface is connected with respectively the specific oligonucleotide sequence (anti-tag) of one section 24bp, and these anti-tag sequences can be respectively and the tag sequence specific combination of corresponding ASPE primer 5 ' end;
2. get respectively the microballoon of every kind of numbering of 1ul in the Eppendorf tube of 1.5ml;
3. microballoon is in 〉=centrifugal the 1-2min of 10000g;
4. supernatant discarded, microballoon is resuspended in 2 * Tm hybridization buffer of 100ul, the vortex mixing;
5. get the above-mentioned microballoon suspension of 25ul in the corresponding hole of 96 hole filter plates, control wells adds the ddH of 25ul
2O;
6. get the ASPE reaction solution of 5-25ul in corresponding hole, use ddH
2O complements to 50ul;
7. encase 96 orifice plates with lucifuge with masking foil, 95 ℃ of 60s, 37 ℃ of 15min are hatched hybridization;
8. the microballoon after the hybridization is in 〉=centrifugal the 2-5min of 3000g;
9. remove supernatant, microballoon is resuspended in 1 * Tm hybridization buffer of 75ul;
10. microballoon is in 〉=centrifugal the 2-5min of 3000g;
11. microballoon is resuspended in 1 * Tm hybridization buffer of 75ul, adding 15ul concentration is the SA-PE (SA-PE) of 10ug/ml;
12.37 ℃ hatch 15min, on the Luminex instrument, detect.
Six, the result detects and data analysis
The reaction after product detects by Luminex serial analysis instrument.Detected result is shown in table 4, table 5 and table 6.
Fluorescent value (MFI) and data processing there are following requirement:
1. each site need have an allelotrope MFI at least greater than 300 and greater than 10 * PCR negative control MFI;
2.NET MFI=sample MFI-PCR negative control MFI (NET MFI less than 0 with 0 the expression);
3. satisfy the data of above two conditions, calculate sudden change ratio by following formula:
Sudden change ratio=mutant NET MFI ÷ (mutant 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 mutant 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: 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 ABCG2 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.As seen ABCG2 gene pleiomorphism provided by the present invention detects liquid-phase chip and can detect exactly ABCG2 gene polymorphism sites type, and the 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.) | The 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 liquid-phase chip of the ASPE primer that embodiment 3 is different is to the detection of ABCG2 gene SNP site
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
Detect liquid-phase chip as example take ABCG2 gene C 184T site mutation, respectively for the wild-type of C184T 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.6, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing that is coated on the microballoon is selected from SEQ ID NO.13-SEQ ID NO.18.Specific design is shown in following table (table 7).Synthetic, the coated microballoon of anti-tag sequence of ASPE primer, amplimer, detection method are described like embodiment 1 and embodiment 2.
The design of table 7 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned design preparation, by embodiment 2 described testing processes and method sample 21-40 is detected, detected result is as follows:
Table 8 pattern detection result and gene SNP analysis
Other is for 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, and effect better (signal to noise ratio is better) is referring to present embodiment test group 1.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 ABCG2 gene pleiomorphism detection specificity primer sequences
One, the design (selection of wild-type and mutant specific primer sequence) of liquid-phase chip preparation
Detect liquid-phase chip as example take the SNP site of ABCG2 Gene C2 29A, take the forward or backwards complementary sequence of this place, mutational site target sequence as template, respectively for the wild-type of C229A and the specific primer sequence of mutant design ASPE primer 3 ' end, comprise preferred specific primer sequence and 2 alternative specific primer sequences in the embodiment of the 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 then is fixed as the best effect sequence among the embodiment 1, and select with it corresponding anti-tag sequence, specific design is shown in following table (table 10).Synthetic, the coated microballoon of anti-tag sequence of ASPE primer, amplimer, detection method are described like embodiment 1 and embodiment 2.
Two of the design of table 10 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned design preparation, by embodiment 2 described testing processes and method sample 41-60 is detected, detected result is as follows:
Table 11 pattern detection result and Polymorphism Analysis
By present embodiment as seen, when the ASPE primer was selected among the embodiment 1 collocation of specific primer sequence and tag sequence, effect better (signal to noise ratio is better) was referring to present embodiment test group 4.Other derives from different specific primer sequences and the collocation of tag sequence of the forward or backwards complementary sequence of place, target detect site sequence, with coming to the same thing of embodiment 2 and present embodiment, namely still be that the specific primer sequence described in the embodiment 1 is better from different tag sequence arranging effects, concrete data are omitted.Other is for multiple specific primer sequence and the collocation of tag sequence in different mutational sites, with coming to the same thing of embodiment 2 and present embodiment, namely embodiment 1 selected Auele Specific Primer has better signal to noise ratio, it is also better to detect effect, and concrete data are omitted.
More than be for the specifying of possible embodiments of the present invention, but this embodiment limits claim of the present invention, allly do not break away from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the claim of the present invention.
Claims (6)
1. an ABCG2 gene pleiomorphism detects liquid-phase chip, it is characterized in that, includes:
(A). the wild-type that designs respectively for the different pleomorphism sites of ABCG2 gene and the ASPE primer of mutant: every kind of ASPE primer is comprised of the tag sequence of 5 ' end and 3 ' the end specific primer sequence for the goal gene pleomorphism site, and described specific primer sequence is: for SEQ ID NO.7 and the SEQ ID NO.8 in C184T site; SEQ ID NO.9 and SEQ ID NO.10 for the 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). anti-tag sequence microballoon coated, that have the different colours coding is arranged, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon; 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). be used for amplifying the primer that needs target sequence detection, that have corresponding pleomorphism site.
2. ABCG2 gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that 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.
3. ABCG2 gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that described ASPE primer is: the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.7 for the C184T site reaches the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.8; The sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.9 for the C229A site reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.10; And/or for the sequence that is formed by SEQ ID NO.5 and SEQ ID NO.11 in C145T site and the sequence that is formed by SEQ ID NO.6 and SEQ ID NO.12.
4. ABCG2 gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that,
(A). described ASPE primer is: the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.7 for the C184T site reaches the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.8; The sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.9 for the C229A site reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.10; Reach the sequence that is formed by SEQ ID NO.6 and SEQ ID NO.12 with the sequence that is formed by SEQ ID NO.5 and SEQ ID NO.11 for the C145T site;
(B). anti-tag sequence microballoon coated, that have the different colours coding is arranged, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon; 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 SEQ ID NO.22 and the SEQ ID NO.23 in C184T, C229A site; With SEQ ID NO.24 and the SEQ ID NO.25 for the C145T site.
5. ABCG2 gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that described spacerarm is 5-10 T.
6. one kind is used for the Auele Specific Primer that the ABCG2 gene pleiomorphism detects, and it is characterized in that described specific primer sequence is: for SEQ ID NO.7 and the SEQ ID NO.8 in C184T site; SEQ ID NO.9 and SEQ ID NO.10 for the C229A site; And/or for SEQ ID NO.11 and the SEQ ID NO.12 in C145T site.
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