CN102559850B - ApoA5 genic mutation detection specific primer and liquid phase chip - Google Patents
ApoA5 genic mutation detection specific primer and liquid phase chip Download PDFInfo
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Abstract
The invention discloses an apoA5 genic mutation detection specific primer and a liquid phase chip, wherein the liquid phase chip mainly includes an ASPE primer formed by 5'end tag sequence and 3'end specific primers, and the specific primers are SEQ ID NO.13 and SEQ ID NO.14 for the mutation of A134G, SEQ ID NO.15 and SEQ ID NO.16 for the mutation of T63C, SEQ ID NO.17 and SEQ ID NO.18 for the mutation of C41G, SEQ ID NO.19 and SEQ ID NO.20 for the mutation of G54T, SEQ ID NO.21 and SEQ ID NO.22 for the mutation of A81G, and/or SEQ ID NO.23 and SEQ ID NO.24 for the mutation of G194A; a microsphere enveloped by anti-tag sequence; and an amplimer. The matching degree of the detection result of the apoA5 genic mutation detection liquid phase chip and the sequencing method is 100%, and the parallel detection of multiple polymorphic sites can be achieved.
Description
Technical field
The present invention bends in biology field, relates to medical science and biotechnology, concrete a kind of apoA5 gene test Auele Specific Primer and the liquid-phase chip of relating to.
Background technology
ApoA5 (apolipoprotein A5 gene, apoA5), lipophorin family member, this assignment of genes gene mapping is in No. 11 long-armed q23 of karyomit(e) districts (11q23), with apoA1/C3/A4 gene cluster at a distance of about 30kb, full length gene 1889bp, this gene has 4 exons, 2 introns and 4 silencers, the protein that a kind of 363 amino acid of encoding forms, has important impact to plasma triglyceride level (triglyceride, TG) level.The relation of plasma TG metabolism and cardiovascular disorder is very close, and NCBI has included more than 20 SNP site at present, the apoA5 gene mutation site of target detect of the present invention, and it is as shown in the table:
| Sequence number | The content of apoA5 site mutation | Write a Chinese character in simplified form |
| 1 | , there is A → G sudden change in the 134th Nucleotide of SEQ ID NO.49 | A134G |
| 2 | , there is T → C sudden change in the 63rd Nucleotide of SEQ ID NO.50 | T63C |
| 3 | , there is C → G sudden change in the 41st Nucleotide of SEQ ID NO.51 | C41G |
| 4 | , there is G → T sudden change in the 54th Nucleotide of SEQ ID NO.52 | G54T |
| 5 | , there is A → G sudden change in the 81st Nucleotide of SEQ ID NO.53 | A81G |
| 6 | , there is G → A sudden change in the 194th Nucleotide of SEQ ID NO.54 | G194A |
At present, the method that apoA5 gene pleiomorphism is detected, analyzed is a lot, as: direct sequencing, PCR-RFLP analytical method etc., wherein the most frequently used is PCR-RFLP analytical method.PCR-RFLP method is the change of the restriction enzyme enzyme recognition site that causes based on transgenation, as lost or generation novel site in site, by a certain specific fragment of pcr amplification, use again digestion with restriction enzyme amplified production, the size of electrophoresis observation fragment, the transgenation that this method changes for detection of restriction enzyme site, can directly judge genotype, but this method can not be for producing the detection in Gene Mutation of new restriction enzyme site.Again, these methods all exist the limitation that detects flux, can only detect a kind of mutation type at every turn, can not meet the needs of practical application.
Summary of the invention
One of object of the present invention is to provide apoA5 gene mutation detection liquid-phase chip.This liquid-phase chip can be used for detecting wild-type and the saltant type of apoA5 gene six kinds of common genotype A134G, T63C, C41G, G54T, A81G and G194A.
An apoA5 gene mutation detection liquid-phase chip, mainly includes:
(A). the ASPE primer of the wild-type designing respectively for every kind of sudden change and saltant type: every kind of ASPE primer holds the Auele Specific Primer for goal gene sudden change to form by the tag sequence and 3 ' of 5 ' end, the Auele Specific Primer of described wild-type and saltant type is respectively: for SEQ ID NO.13 and the SEQ ID NO.14 of A134G sudden change, SEQ ID NO.15 and SEQ ID NO.16 for T63C sudden change, SEQ ID NO.17 and SEQ ID NO.18 for C41G sudden change, SEQID NO.19 and SEQ ID NO.20 for G54T sudden change, SEQ ID NO.21 and SEQ ID NO.22 for A81G sudden change, and/or the SEQ ID NO.23 and the SEQ ID NO.24 that for G194A, suddenly change, described tag sequence is selected from SEQ ID NO.1-12,
(B). there is anti-tag sequence microballoon coated, 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 the sequence in SEQ ID NO.25～SEQ ID NO.36, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). for amplifying the primer that needs target sequence detection, that there is mutational site.
Preferably, described amplimer is: the SEQ ID NO.37 suddenling change for A134G and SEQ ID NO.38, the SEQ ID NO.39 suddenling change for T63C and SEQ ID NO.40, the SEQ ID NO.41 suddenling change for C41G and SEQ ID NO.42, the SEQ ID NO.43 suddenling change for G54T and SEQ ID NO.44, the SEQ ID NO.45 suddenling change for A81G and SEQ IDNO.46 and/or the SEQ ID NO.47 and the SEQ ID NO.48 that for G194A, suddenly change.
Preferably, described ASPE primer is: the sequence being comprised of SEQ ID NO.1 and SEQ ID NO.13 and the sequence being comprised of SEQ IDNO.2 and SEQ ID NO.14, the sequence being formed by SEQ ID NO.3 and SEQ ID NO.15 and the sequence being formed by SEQ IDNO.4 and SEQ ID NO.16, the sequence being formed by SEQ ID NO.5 and SEQ ID NO.17 and the sequence being formed by SEQ IDNO.6 and SEQ ID NO.18, the sequence being formed by SEQ ID NO.7 and SEQ ID NO.19 and the sequence being formed by SEQ IDNO.8 and SEQ ID NO.20, the sequence being formed by SEQ ID NO.9 and SEQ ID NO.21 and the sequence being formed by SEQ IDNO.10 and SEQ ID NO.22, and/or the sequence being formed by SEQ ID NO.11 and SEQ ID NO.23 and the sequence that formed by SEQ ID NO.12 and SEQ ID NO.24.
Another object of the present invention is to provide a kind of Auele Specific Primer for apoA5 detection in Gene Mutation.
Concrete technical scheme is as follows:
An Auele Specific Primer for apoA5 detection in Gene Mutation, includes: the SEQ ID NO.13 suddenling change for A134G and SEQ ID NO.14, the SEQ ID NO.15 suddenling change for T63C and SEQ ID NO.16, the SEQID NO.17 suddenling change for C41G and SEQ ID NO.18, the SEQ ID NO.19 suddenling change for G54T and SEQ ID NO.20, the SEQ ID NO.21 suddenling change for A81G and SEQ ID NO.22 and/or the SEQ ID NO.23 and the SEQ ID NO.24 that for G194A, suddenly change.
Major advantage of the present invention is:
1. the identical rate of the detected result of apoA5 gene mutation 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.In the Auele Specific Primer very a large amount of, through lot of experiments, reaction checking, obtain the liquid-phase chip system of optimum combination, prepared liquid-phase chip has extraordinary signal-noise ratio, and does not substantially have cross reaction between designed probe and anti-tag sequence, the choosing and the combination of tag sequence label and concrete ASPE primer of tag sequence label, anti-tag sequence label, can avoid cross reaction, realize the parallel detection in a plurality of mutational sites.
2. the ASPE primer specificity primer of the present invention design can the sensitive mutational 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, also the polymorphism situation in a plurality of mutational sites of parallel detection simultaneously, detects effect consistent.
3. detection method step of the present invention is simple, six kinds of pleomorphism sites detect and can complete the amplification of two target sequences that contain SNP site by a step multiplex PCR, the many uncertain factors that exist 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 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.
Embodiment
ApoA5 gene mutation detection liquid-phase chip described in embodiment 1, mainly includes:
One, ASPE primer
Wild-type and saltant type for six kinds of common genotype A134G, T63C, C41G, G54T, A81G and G194A of apoA5 gene, design respectively specific primer sequence.ASPE primer is comprised 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 1apoA5 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 12 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
12 kinds of microballoons selecting are purchased from U.S. Luminex company, by anti-tag sequence coated with microballoon on.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 for spaced apart by anti-tag and microballoon table picture 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 of individual above-mentioned numbering (purchased 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) (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 six kinds of common genotype A134G, T63C, C41G, G54T, A81G and G194A of apoA5 gene, design of amplification primers, to (in Table 3), amplifies respectively six 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.
The detection of apoA5 gene test liquid-phase chip to sample described in embodiment 2 utilization embodiment 1
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 |
| 5M NaCl | 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 U.S. USB company.
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 six pairs of primers, multiplex PCR one step amplifies five kinds of common genotype A134G, T63C, C41G, G54T, A81G and G194A target sequence of totally six that contains respectively apoA5 gene, product size is respectively 281bp, 325bp, 185bp, 154bp, 376bp and 483bp, and primer sequence (SEQ ID NO.37-48) 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.37-48 in 1.5ml Eppendorf tube, mix and be multiple PCR primer working fluid.Multi-PRC reaction system is as follows:
2 * damping fluid is (containing Mg
2+) 25ul
DNTP (each 2.5mmol/L) 4ul
Taq enzyme (5U/ul) 0.2ul
Multiple PCR primer working fluid (each 8.3pmol/mL) 6ul
Template DNA (10ng/ul) 2ul
ddH
2O 12.8ul
Be total to 50ul
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;
Hatch 15min for 2.37 ℃, hatch 15min for 80 ℃, the enzyme that deactivation is unnecessary.The product that enzyme is cut after 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 reacted product band.
First the ASPE primer working fluid that preparation mixes: get respectively the corresponding wild-type of apoA5 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:
10 * damping fluid 2ul
MgCl
2(50mmol/L) 0.5ul
Biotin-dCTP(400umol/L) 0.25ul
DATP, dGTP, dTTP mixed solution (each 100umol/L) 1ul
Tsp enzyme (5U/ul) 0.25ul
ASPE primer working fluid (each 500nmol/L) 1ul mixing
Enzyme is cut the pcr amplification product 5ul of processing
ddH
2O 10ul
Be total to 20ul
Response procedures is: 96 ℃ of 2min; 94 ℃ of 30s, 54 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ save backup.
Five, hybridization
1. according to the ASPE primer of design, (microballoon concentration is 2.5 * 10 to the corresponding 12 kinds of microballoons of every group selection
5individual/ml); ;
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. with masking foil, encase 96 orifice plates with lucifuge, 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 with 0 expression);
3. meet the data of above two conditions, by following formula, calculate sudden change ratio:
Sudden change ratio=saltant type NET MFI ÷ (saltant type NET MFI+ 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 apoA5 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.With sequencing, detect with liquid-phase chip result and compare, calculate the identical rate of classifying method detected result provided by the present invention.Present method detects 20 increments apoA5 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.Visible apoA5 gene pleiomorphism provided by the present invention detects liquid-phase chip can detect apoA5 gene polymorphism sites type exactly, and result is reliable and stable.
One of table 4 pattern detection result (MFI)
Two (MFI) of table 5 pattern detection result
Table 6 sample apoA5 transgenation ratio (%)
Table 7 sample apoA5 gene mutation type analytical results
| Catalogue number(Cat.No.) | Liquid-phase chip detected result | Sequencing result |
| 1 | 54TT | 54TT |
| 2 | Wild-type | Wild-type |
| 3 | 134AG | 134AG |
| 4 | Wild-type | Wild-type |
| 5 | 63GG | 63GG |
| 6 | 81GG | 81GG |
| 7 | Wild-type | Wild-type |
| 8 | 134GG | 134GG |
| 9 | Wild-type | Wild-type |
| 10 | 41GG | 41GG |
| 11 | 54TT、194GA | 54TT、194GA |
| 12 | Wild-type | Wild-type |
| 13 | Wild-type | Wild-type |
| 14 | 41CG | 41CG |
| 15 | 134GG | 134GG |
| 16 | Wild-type | Wild-type |
| 17 | Wild-type | Wild-type |
| 18 | Wild-type | Wild-type |
| 19 | 81GG | 81GG |
| 20 | Wild-type | Wild-type |
The detection of the liquid-phase chip of the ASPE primer that embodiment 3 is different to apoA5 gene polymorphism sites
One, the design that prepared by liquid-phase chip (selection of Tag sequence and Anti-Tag sequence)
Take apoA5 Gene A 134G and C41G site mutation, to detect liquid-phase chip be example, respectively for the wild-type of A134G and C41G 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.12, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing being coated on microballoon is selected from SEQ ID NO.25-SEQ ID NO.36.Specific design is as shown in following table (table 8).Synthetic, the anti-tag sequence of ASPE primer are coated with microballoon, amplimer, detection method like described in embodiment 1 and embodiment 2.
Design prepared by table 8 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 9 sample A134G detected result and Polymorphism Analysis
Table 10 sample C41G detected result and Polymorphism 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 and test group 5.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.
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. an apoA5 gene mutation detection liquid-phase chip, is characterized in that, mainly includes:
(A). the ASPE primer of the wild-type designing respectively for every kind of sudden change and saltant type: every kind of ASPE primer holds the Auele Specific Primer for goal gene sudden change to form by the tag sequence and 3 ' of 5 ' end, the Auele Specific Primer of described wild-type and saltant type is: for SEQ ID NO.13 and the SEQ ID NO.14 of A134G sudden change, and be selected from SEQ ID NO.15 and the SEQ ID NO.16 for T63C sudden change, SEQ ID NO.17 and SEQ ID NO.18 for C41G sudden change, SEQ ID NO.19 and SEQ ID NO.20 for G54T sudden change, SEQ ID NO.21 and SEQ ID NO.22 for A81G sudden change, with the SEQ ID NO.23 suddenling change for G194A and at least one pair of in SEQ ID NO.24, described tag sequence is selected from SEQ ID NO.1-12,
(B). there is anti-tag sequence microballoon coated, 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 the sequence in SEQ ID NO.25～SEQ ID NO.36, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). for amplifying the primer that needs target sequence detection, that there is mutational site, amplimer is: the SEQ ID NO.37 and the SEQ ID NO.38 that for A134G, suddenly change, and be selected from SEQ ID NO.39 and SEQ ID NO.40, the SEQ ID NO.41 suddenling change for C41G and SEQ ID NO.42, the SEQ ID NO.43 suddenling change for G54T and SEQ ID NO.44, the SEQ ID NO.45 suddenling change for A81G and SEQ ID NO.46 and the SEQ ID NO.47 suddenling change for G194A and at least one pair of in SEQ ID NO.48 for T63C sudden change.
2. apoA5 gene mutation detection liquid-phase chip according to claim 1, it is characterized in that, described ASPE primer is: the sequence being comprised of SEQ ID NO.1 and SEQ ID NO.13 and the sequence being comprised of SEQ ID NO.2 and SEQ ID NO.14, and select the sequence that free SEQ ID NO.3 and SEQ ID NO.15 form and the sequence being formed by SEQ ID NO.4 and SEQ ID NO.16, the sequence being formed by SEQ ID NO.5 and SEQ ID NO.17 and the sequence being formed by SEQ ID NO.6 and SEQ ID NO.18, the sequence being formed by SEQ ID NO.7 and SEQ ID NO.19 and the sequence being formed by SEQ ID NO.8 and SEQ ID NO.20, the sequence being formed by SEQ ID NO.9 and SEQ ID NO.21 and the sequence being formed by SEQ ID NO.10 and SEQ ID NO.22, with at least one pair of in the sequence being formed by SEQ ID NO.11 and SEQ ID NO.23 and the sequence that formed by SEQ ID NO.12 and SEQ ID NO.24.
3. apoA5 gene mutation detection liquid-phase chip according to claim 1, is characterized in that,
(A). described ASPE primer is: the sequence being comprised of SEQ ID NO.1 and SEQ ID NO.13 and the sequence being comprised of SEQ ID NO.2 and SEQ ID NO.14, the sequence being formed by SEQ ID NO.3 and SEQ ID NO.15 and the sequence being formed by SEQ ID NO.4 and SEQ ID NO.16, the sequence being formed by SEQ ID NO.5 and SEQ ID NO.17 and the sequence being formed by SEQ ID NO.6 and SEQ ID NO.18, the sequence being formed by SEQ ID NO.7 and SEQ ID NO.19 and the sequence being formed by SEQ ID NO.8 and SEQ ID NO.20, the sequence being formed by SEQ ID NO.9 and SEQ ID NO.21 and the sequence being formed by SEQ ID NO.10 and SEQ ID NO.22, with the sequence being formed by SEQ ID NO.11 and SEQ ID NO.23 and the sequence that formed by SEQ ID NO.12 and SEQ ID NO.24,
(B). there is anti-tag sequence microballoon coated, 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 the sequence in SEQ ID NO.25～SEQ ID NO.36, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). described amplimer is: the SEQ ID NO.37 suddenling change for A134G and SEQ ID NO.38, the SEQ ID NO.39 suddenling change for T63C and SEQ ID NO.40, the SEQ ID NO.41 suddenling change for C41G and SEQ ID NO.42, the SEQ ID NO.43 suddenling change for G54T and SEQ ID NO.44, the SEQ ID NO.45 suddenling change for A81G and SEQ ID NO.46 and the SEQ ID NO.47 and the SEQ ID NO.48 that for G194A, suddenly change.
4. according to the apoA5 gene mutation detection liquid-phase chip described in claim 1-3 any one, it is characterized in that, described spacerarm is 5-10 T.
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| Zhi-Kai Liu，et al.Associations of polymorphisms in the apolipoprotein A1/C3/A4/A5 gene cluster with familial combined hyperlipidaemia in Hong Kong Chinese.《Atherosclerosis》.2009,第208卷第427-432页. * |
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