CN102304569B - Specific primers and liquid phase chip for polymorphic detection of age-related maculopathy susceptibility 2 (ARMS2) gene and high temperature factor A-1 (HTRA1) gene - Google Patents
Specific primers and liquid phase chip for polymorphic detection of age-related maculopathy susceptibility 2 (ARMS2) gene and high temperature factor A-1 (HTRA1) gene Download PDFInfo
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Abstract
The invention relates to a liquid phase chip for the polymorphic detection of an age-related maculopathy susceptibility 2 (ARMS2) gene and a high temperature factor A-1 (HTRA1) gene. The liquid phase chip comprises allele specific primer extension (ASPE) primers consisting of tag sequences at the 5' end and specific primers aiming at a mutant target gene at the 3' end, microspheres which are wrapped with anti-tag sequences and an amplification primer, wherein the specific primers are SEQ ID No. 10 and SEQ ID No. 11 for a G72T mutant locus of the ARMS2 gene, SEQ ID No. 12 to SEQ ID No. 14 for a C95G/A mutant locus of the HTRA1 gene, SEQ ID No. 15 and SEQ ID No. 16 for a G60A mutant locus of the HTRA1 gene and/or SEQ ID No. 17 and SEQ ID No. 18 for the insertion/deletion (I/D) mutation of the HTRA1 gene. The coincidence rate of a detection result of the liquid phase chip and a sequencing method is up to 100 percent, cross reaction can be prevented, and the parallel detection of multiple polymorphic loci is realized.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete a kind of ARMS2 and HTRA1 gene pleiomorphism detection specificity primer and the liquid-phase chip of relating to.
Background technology
(Age-related macular degeneration is the eye diseases of aging that a kind of common degenerative change owing to the macula lutea position causes AMD), and this disease is destruction center's eyesight and finally cause losing one's sight little by little in age-related macular degeneration.Usually will there be the AMD of soft glassy membrane wart, pigment anomaly and ground pattern atrophy to be called dryness AMD, and will has choroidal neovascularization, retinal pigment epithelium (RPE) disengaging or the Fibrotic AMD of plate-like to be called moist or neovascular AMD.Recently decades, AMD has become western developed country blinding one of the main reasons, and along with China's aging population, retina sufferer numbers such as AMD are also in continuous increase, and its pathogenesis and methods for the treatment of just more and more come into one's own.
Studies show that, be positioned at macular degeneration tumor susceptibility gene 2 (the age-related maculaopathy susceptibility 2gene of euchromosome 10 (10q26), ARMS2 or LOC387715) and essential factors A-1 (the high temperature factor A-1 of high temperature, HTRA1) relevant with the generation development of AMD, it is the main pathogenesis of AMD, wherein, ARMS2 negative gene responsible editor sign indicating number is arranged in the expression of retina plastosome albumen, the site mutation of this gene, to cause the change of its codon encoding amino acid composition, thereby influence the active and expression of albumen.
Studies show that recently, 443 base sequences disappearance/54 base sequences in part 3 ' the UTR zone of the ARMS2 gene that HTRA1 upstream region of gene 4340bp place comprises insert variation (insertion/deletion, I/D) directly related with the generation of AMD, this I/D transgenation will be raised the HTRA1 expression of gene, the change of HTRA1 albumen and downstream signal bioactive molecule thereof has influenced intraocular extracellular matrix metabolic degradation process and vasculogenesis, participates in the AMD pathogenic course then.
The ARMS2 of target detect of the present invention and HTRA1 gene mutation site, it is as shown in the table:
At present, the method that ARMS2 and HTRA1 gene pleiomorphism are detected, analyze is a lot, as: direct sequencing, TaqMan technology, PCR-RFLP analytical method, isothermal duplication, nucleic acid beacons method etc., wherein the most frequently used method has the PCR-RFLP analytical method.The PCR-RFLP method is based on the change of the restriction enzyme enzyme recognition site that transgenation causes, as losing or produce novel site in the site, by a certain specific fragment of pcr amplification, use the digestion with restriction enzyme amplified production again, the size of electrophoresis observation fragment, this method can directly be judged genotype for detection of the transgenation that restriction enzyme site changes, but this method can not be used for not producing the detection in Gene Mutation of new restriction enzyme site.Again, more than these methods all exist the limitation that detects flux, can only detect a kind of mutation type at every turn, can not satisfy the needs of practical application.
Summary of the invention
One of purpose of the present invention provides a kind of ARMS2 and the HTRA1 gene pleiomorphism detects liquid-phase chip, this liquid-phase chip can be used for detecting wild-type and the mutant of ARMS2 gene common mutations site G72T, and wild-type and the mutant of HTRA1 gene common mutations C95G/A, G60A and I/D.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind of ARMS2 and HTRA1 gene pleiomorphism detect liquid-phase chip, mainly include:
(A). the wild-type that designs respectively at every kind of sudden change and the ASPE primer of mutant: every kind of ASPE primer is made up of at the Auele Specific Primer of goal gene sudden change tag sequence and the 3 ' end of 5 ' end, and described Auele Specific Primer is respectively: at SEQ ID NO.10 and the SEQ ID NO.11 in ARMS2 gene G72T mutational site, SEQ ID NO.12-14 at HTRA1 gene C 95G/A mutational site, SEQ ID NO.15 and SEQ ID NO.16 at HTRA1 gene G60A mutational site, and/or at SEQ ID NO.17 and the SEQ ID NO.18 of HTRA1 gene I/D sudden change; Described tag sequence is selected from SEQ ID NO.1-9;
(B). microballoon anti-tag sequence bag quilt, 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 the sequence among SEQ ID NO.19～SEQ ID NO.27, 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 sudden change.
Preferably, described amplimer is: at the SEQ ID NO.28 in ARMS2 gene G72T mutational site and SEQ IDNO.29, at the SEQ ID NO.30 in HTRA1 gene C 95G/A mutational site and SEQ ID NO.31, at the SEQ ID NO.32 in HTRA1 gene G60A mutational site and SEQ ID NO.33 and/or at SEQ IDNO.34 and the SEQ ID NO.35 of HTRA1 gene I/D sudden change..
Preferably, described ASPE primer is: the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.10 reaches the sequence of being made up of SEQ IDNO.2 and SEQ ID NO.11, the sequence of being formed by SEQ ID NO.3 and SEQ ID NO.12, the sequence of being made up of SEQ IDNO.4 and SEQ ID NO.13 reaches the sequence of being made up of SEQ ID NO.5 and SEQ ID NO.14, the sequence of being made up of SEQ IDNO.6 and SEQ ID NO.15 reaches the sequence of being made up of SEQ ID NO.7 and SEQ ID NO.16, and/or the sequence of being formed by SEQID NO.8 and SEQ ID NO.17 and the sequence formed by SEQ ID NO.9 and SEQ ID NO.18.
Another object of the present invention provides a kind of Auele Specific Primer for ARMS2 and the detection of HTRA1 gene pleiomorphism.
Concrete technical scheme is as follows:
A kind of Auele Specific Primer that detects for ARMS2 and HTRA1 gene pleiomorphism mainly includes: at the SEQ ID NO.10 in ARMS2 gene G72T mutational site and SEQ ID NO.11, at the SEQ ID NO.12-14 in HTRA1 gene C 95G/A mutational site, at the SEQ ID NO.15 in HTRA1 gene G60A mutational site and SEQ ID NO.16 and/or at SEQ ID NO.17 and the SEQ ID NO.18 of HTRA1 gene I/D sudden change.
Major advantage of the present invention is:
1. the identical rate of detection method 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.Because in very a large amount of Auele Specific Primers, through a large amount of tests, the reaction checking, obtain the liquid-phase chip system of optimum combination, prepared ARMS2 and HTRA1 gene pleiomorphism detect liquid-phase chip and have extraordinary signal-noise ratio, and there is not cross reaction between designed probe and the anti-tag sequence basically, 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 of a plurality of pleomorphism sites.
2. the present invention has chosen optimum combination by the design experiences of contriver's long-term accumulation and the operation of a large amount of experiments from numerous Auele Specific Primers.The ASPE primer specificity primer of the present invention's design can sensitive be identified the mutational site of target detect specifically, accurately distinguishes the genotype of various types; In same reaction system, between the different Auele Specific Primers, do not have cross reaction basically between the pcr amplification product of Auele Specific Primer and non-target detect, detection specificity is good, and cross reacting rate is lower than 3%; Except detecting the single polymorphism situation of ARMS2 and HTRA1 gene separately, the also polymorphism situation of parallel detection ARMS2 and a plurality of sudden changes of HTRA1 gene simultaneously, this polymorphism comprises single nucleotide polymorphism (SNP) and insertion/deletion mutantion (I/D) etc., detects the effect unanimity.
3. detection method step of the present invention is simple, can can finish four amplifications that contain the target sequence of pleomorphism 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 improve the detection accuracy rate greatly, embodied qualitative, quantitative analysis feature of accurate while.
4. not only to have overcome traditional solid phase chip susceptibility not high in the present invention, and the defective of the repeatability difference of detected result is improved existing liquid-phase chip technology simultaneously, makes prepared microballoon can be applicable to different test items, has very strong expansion.The fluorescent signal value that detects improves greatly, thereby makes the sensitivity that detects be further enhanced, and signal to noise ratio strengthens, and detected result more accurately and reliably.
Embodiment
Embodiment 1ARMS2 and HTRA1 gene pleiomorphism detect liquid-phase chip, mainly include:
One, ASPE primer
At wild-type and the mutant of ARMS2 gene common mutations site G72T, and wild-type and the mutant in the common I/D sudden change of HTRA1 gene and C95G/A, G60A mutational site, specific primer sequence designed respectively.The ASPE primer is made up of " Tag sequence+specific primer sequence ".The ASPE primer sequence is as shown in the table:
The ASPE primer sequence (Tag sequence+specific primer sequence) of table 1ARMS2 and HTRA1 gene
Every the ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence at anti-tag sequence on the corresponding microballoon, and 3 ' end is mutant or the special primer fragment (shown in above-mentioned table 1) of wild-type.All ASPE primers are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Two, the microballoon of anti-tag sequence bag quilt
According to designed ASPE Auele Specific Primer fragment, select the tag sequence, reduce between the anti-tag sequence of each microballoon to greatest extent and secondary structure that tag and ASPE Auele Specific Primer fragment may form, corresponding anti-tag sequence is as shown in table 2 on 9 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
Nine kinds of microballoons selecting are available from U.S. Luminex company, with anti-tag sequence bag by on microballoon.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 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 of microballoon bag quilt is as follows:
Get 5 * 10 respectively
6The carboxylated microballoon of individual above-mentioned numbering (available from Luminex company) is suspended in the MES solution of 50ul 0.1mol/L (pH4.5), adds 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.Add the EDC working fluid of 2.5ul in the microballoon suspension, constant temperature was hatched 30 minutes, added the EDC working fluid of 2.5ul again, and constant temperature was hatched 30 minutes again.After reaction finished, the Tween-20 washing with 0.02% was once washed once with 0.1% SDS liquid again.The microballoon that is coated with the anti-tag sequence after the washing is resuspended in the Tris-EDTA solution [10mmol/LTris (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
At wild-type and the mutant of ARMS2 gene common mutations site G72T, and wild-type and the mutant in the common I/D sudden change of HTRA1 gene and C95G/A, G60A mutational site, design of amplification primers is to (seeing Table 3).
Design 3 pairs of amplimers respectively at ARMS2 mutational site G72T and HTRA1 mutational site C95G/A, G60A, amplify three target sequences that contain the mutational site; Insertion/deletion mutantion at the HTRA1 gene, design 1 pair of amplimer, when target sequence is the insert type homozygote, PCR product length is 679bp, when the target detect sequence is the absence type homozygote, PCR product length is 290bp, and when to detect sequence be insert type/deletion heterozygote, there were two kinds of 679bp and 290bp simultaneously in the PCR product.
Table 3 amplifies the primer of the target sequence with pleomorphism site
All primers are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with the stock solution of 100pmol/mL respectively with 10mmol/LTris Buffer.
It is as follows to the prescription of the described various solution of detection of sample that embodiment 2 uses embodiment 1 described ARMS2 and HTRA1 gene pleiomorphism to detect liquid-phase chip:
MES damping fluid (pH5.0) prescription (250ml) of 50mM:
2 * Tm hybridization buffer
| Reagent | The source | Final concentration | The consumption of every 250ml |
| MTris-HCl，pH8.0 | SigmaT3038 | 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 four pairs of primers, multiplex PCR one step amplifies respectively at three target sequences that contain the mutational site of ARMS2 mutational site G72T and HTRA1 mutational site C95G/A, G60A, and the product size is respectively 207bp, 220bp and 237bp; Insertion/deletion mutantion at the HTRA1 gene, design 1 pair of amplimer, when target sequence is the insert type homozygote, PCR product length is 679bp, when the target detect sequence is the absence type homozygote, PCR product length is 290bp, and when to detect sequence be insert type/deletion heterozygote, there were two kinds of 679bp and 290bp simultaneously in the PCR product.Primer sequence (SEQ ID NO.28-35) is seen shown in the above-mentioned table 3.
At first prepare the multiple PCR primer working fluid: respectively get the primer stock solution 100ul of SEQ ID NO.28-35 respectively 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 ℃ of preservations are standby.
Three, the enzyme of PCR product is cut processing
1. get the reacted product of 7.5ul PCR, add 1ul 10 * SAP damping fluid, 1ul SAP enzyme and 0.5ul Exo-I enzyme;
2.37 ℃ hatch 15min, hatch 15min for 80 ℃, the enzyme that deactivation is unnecessary.The product that enzyme is cut after the processing 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 the corresponding wild-type of gene to be detected and mutant ASPE primer stock solution 10ul respectively 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 ℃ of preservations are standby.
Five, hybridization
1. according to the ASPE primer of design, (every kind of microballoon concentration is 2.5 * 10 to the corresponding 9 kinds of microballoons of every group selection
5Individual/ml);
2. get the microballoon of every kind of numbering of 1ul respectively 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 Streptavidin-phycoerythrin (SA-PE) of 10ug/ml;
12.37 ℃ hatch 15min, on the Luminex instrument, detect.
Six, the result detects and data analysis
The reaction after product 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 NET MFI+ wild-type NET MFI)
4. rule of thumb the sudden change ratio of each detection site is determined threshold value (cut-off value), to divide wild-type homozygote, heterozygote and mutant homozygote.
Use present method to detect 20 increments ARMS2 and HTRA1 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 this ARMS2 and the identical rate of HTRA1 genotype detection result and sequencing result of 20 increments and reaches 100%.As seen ARMS2 provided by the present invention and HTRA1 gene pleiomorphism detect liquid-phase chip and can detect ARMS2 and HTRA1 gene type exactly, and the result is reliable and stable.
Table 4 pattern detection result (MFI)
Table 5 sample ARMS2 and HTRA1 transgenation ratio (%)
Table 6 sample ARMS2 and HTRA1 gene mutation type analytical results
| Catalogue number(Cat.No.) | The liquid-phase chip detected result | Sequencing result |
| 1 | Wild-type | Wild-type |
| 2 | 95GG | 95GG |
| 3 | 60AA | 60AA |
| 4 | deletion | deletion |
| 5 | Wild-type | Wild-type |
| 6 | 72TT | 72TT |
| 7 | Wild-type | Wild-type |
| 8 | Wild-type | Wild-type |
| 9 | Wild-type | Wild-type |
| 10 | Wild-type | Wild-type |
| 11 | Wild-type | Wild-type |
| 12 | Wild-type | Wild-type |
| 13 | 95AA | 95AA |
| 14 | 60GA | 60GA |
| 15 | Wild-type | Wild-type |
| 16 | 95CG | 95CG |
| 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 ARMS2 and HTRA1 gene polymorphism sites
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
G60A sudden change detection liquid-phase chip with ARMS2 gene G72T site mutation and HTRA1 gene is example, respectively at the wild-type of G72T and G60A 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.9, accordingly, bag is selected from SEQ ID NO.19-SEQ ID NO.27 by the anti-tag sequence with corresponding tag sequence complementary pairing on microballoon.Specific design is shown in following table (table 7).Synthetic, the anti-tag sequence bag of ASPE primer is described like embodiment 1 and embodiment 2 by microballoon, amplimer, detection method.
One of 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 Polymorphism Analysis
Table 9 pattern detection result and Polymorphism Analysis
Other is at the liquid-phase chip in different mutational sites, and the ASPE primer uses different Tag sequences, and its result is still reliable and stable, and concrete data are omitted.And the ASPE primer is when selecting among the embodiment 1 collocation of tag sequence and specific primer sequence for use, and effect better (signal to noise ratio is better) is referring to present embodiment test group 1 and test group 5.Other different tag sequences and specific primer sequence collocation, with coming to the same thing of embodiment 2 and present embodiment, concrete data are omitted.
The selection of embodiment 4ARMS2 and HTRA1 gene pleiomorphism detection specificity primer sequence
One, the design (selection of wild-type and mutant specific primer sequence) of liquid-phase chip preparation
C95G/A sudden change detection liquid-phase chip with ARMS2 gene G72T site mutation and HTRA1 gene is example, respectively at the wild-type of G72T and C95G/A 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 10.Wherein,
Interior base is pleomorphism site.
Table 10 specific primer sequence
C95G/A sudden change detection liquid-phase chip with ARMS2 gene G72T site mutation and HTRA1 gene is example respectively, select different specific primer sequences for use at G72T and C95G/A, 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 for use, specific design is shown in following table (table 11).Synthetic, the anti-tag sequence bag of ASPE primer is described like embodiment 1 and embodiment 2 by microballoon, amplimer, detection method.
Two of the design of table 11 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned design preparation, by embodiment 2 described testing processes and method sample 41-60 is detected, detected result is as follows:
Table 12 pattern detection result and Polymorphism Analysis
Table 13 pattern detection result and Polymorphism Analysis
| Sequence number | The C95-w detected value | The C95G-m detected value | The C95A-m detected value |
| NO. | Group1 | Group2 | Group3 | Group1 | Group2 | Group3 | Group1 | Group2 | Group3 |
| Negative control | 19 | 16 | 19 | 21 | 24 | 29 | 24 | 26 | 25 |
| 41 | 3510 | 3056 | 2876 | 57 | 148 | 60 | 67 | 102 | 161 |
| 42 | 3319 | 2681 | 2675 | 95 | 149 | 179 | 50 | 113 | 112 |
| 43 | 3004 | 2509 | 2585 | 61 | 155 | 136 | 61 | 64 | 92 |
| 44 | 3684 | 3079 | 2909 | 69 | 71 | 113 | 86 | 157 | 128 |
| 45 | 2929 | 2260 | 2485 | 110 | 112 | 190 | 125 | 170 | 164 |
| 46 | 52 | 110 | 94 | 2623 | 2117 | 1898 | 73 | 79 | 88 |
| 47 | 3671 | 3149 | 3319 | 80 | 83 | 133 | 102 | 123 | 119 |
| 48 | 3627 | 3324 | 2988 | 80 | 102 | 108 | 100 | 188 | 130 |
| 49 | 2868 | 2227 | 2308 | 129 | 176 | 168 | 56 | 61 | 63 |
| 50 | 3663 | 2959 | 2959 | 124 | 217 | 164 | 115 | 179 | 212 |
| 51 | 3398 | 3044 | 2915 | 102 | 116 | 137 | 65 | 70 | 72 |
| 52 | 2519 | 1890 | 1757 | 80 | 138 | 155 | 81 | 169 | 129 |
| 53 | 2843 | 2236 | 2045 | 65 | 139 | 162 | 102 | 169 | 111 |
| 54 | 3151 | 2736 | 2399 | 84 | 107 | 130 | 65 | 119 | 100 |
| 55 | 100 | 105 | 150 | 67 | 164 | 153 | 2917 | 2512 | 2366 |
| 56 | 3557 | 2906 | 3108 | 98 | 164 | 117 | 111 | 177 | 127 |
| 57 | 2931 | 2312 | 2563 | 124 | 186 | 157 | 51 | 136 | 65 |
| 58 | 3392 | 3018 | 2786 | 96 | 140 | 180 | 83 | 122 | 93 |
| 59 | 3057 | 2284 | 2612 | 115 | 175 | 138 | 82 | 166 | 106 |
| 60 | 3123 | 2538 | 2759 | 87 | 146 | 117 | 84 | 160 | 134 |
Table 14 pattern detection result and Polymorphism Analysis
Other is at the liquid-phase chip in different mutational sites, and the ASPE primer uses different specific primer sequences, and its result is still reliable and stable, and concrete data are omitted.And the ASPE primer is when selecting among the embodiment 1 collocation of specific primer sequence and tag sequence for use, and effect better (signal to noise ratio is better) is referring to present embodiment test group 7 and experimental group 10.Other different specific primer sequences and the collocation of tag sequence, with coming to the same thing of embodiment 2 and present embodiment, concrete data are omitted.
More than be at the specifying of possible embodiments of the present invention, but this embodiment is not in order to limiting claim of the present invention, does not allly break away from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the claim of the present invention.
Claims (1)
1. an ARMS2 and HTRA1 gene pleiomorphism detect liquid-phase chip, it is characterized in that, mainly include:
(A). the wild-type that designs respectively at every kind of sudden change and the ASPE primer of mutant: every kind of ASPE primer is made up of at the Auele Specific Primer of goal gene sudden change tag sequence and the 3 ' end of 5 ' end, described Auele Specific Primer is: at SEQ ID NO.10 and the SEQ ID NO.11 in ARMS2 gene G72T mutational site, and be selected from SEQ ID NO.12-14 at HTRA1 gene C 95G/A mutational site, SEQ ID NO.15 and SEQ ID NO.16 at HTRA1 gene G60A mutational site, with at more than a pair of among the SEQ ID NO.17 of HTRA1 gene insert type/absence type sudden change and the SEQ ID NO.18; Described tag sequence is selected from SEQ ID NO.1-9;
(B). microballoon anti-tag sequence bag quilt, 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 the sequence among SEQ ID NO.19～SEQ ID NO.27, 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 sudden change, described amplimer is: at SEQ ID NO.28 and the SEQ ID NO.29 in ARMS2 gene G72T mutational site, and be selected from the SEQ ID NO.30 in HTRA1 gene C 95G/A mutational site and SEQ ID NO.31, at the SEQ ID NO.32 in HTRA1 gene G60A mutational site and SEQ ID NO.33 with at more than a pair of among the SEQ ID NO.34 of HTRA1 gene insert type/absence type sudden change and the SEQ ID NO.35.
2,. ARMS2 according to claim 1 and HTRA1 gene pleiomorphism detect liquid-phase chip, it is characterized in that, described ASPE primer is: the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.10 reaches the sequence of being made up of SEQ ID NO.2 and SEQ ID NO.11, and is selected from the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.12, the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.13 reaches the sequence of being made up of SEQ ID NO.5 and SEQ ID NO.14, the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.15 reaches the sequence of being made up of SEQ ID NO.7 and SEQ ID NO.16, more than a pair of in the sequence of forming with the sequence of being formed by SEQ ID NO.8 and SEQ ID NO.17 and by SEQ ID NO.9 and SEQ ID NO.18.
3, ARMS2 according to claim 1 and HTRA1 gene pleiomorphism detect liquid-phase chip, it is characterized in that,
(A). described ASPE primer is: the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.10 reaches the sequence of being made up of SEQ ID NO.2 and SEQ ID NO.11, the sequence of being formed by SEQ ID NO.3 and SEQ ID NO.12, the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.13 reaches the sequence of being made up of SEQ ID NO.5 and SEQ ID NO.14, the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.15 reaches the sequence of being made up of SEQ ID NO.7 and SEQ ID NO.16, reach the sequence of being formed by SEQ ID NO.9 and SEQ ID NO.18 with the sequence of being formed by SEQ ID NO.8 and SEQ ID NO.17;
(B). microballoon anti-tag sequence bag quilt, 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 the sequence among SEQ ID NO.19～SEQ ID NO.27, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). described amplimer is: at the SEQ ID NO.28 in ARMS2 gene G72T mutational site and SEQ ID NO.29, at the SEQ ID NO.30 in HTRA1 gene C 95G/A mutational site and SEQ ID NO.31, at the SEQ ID NO.32 in HTRA1 gene G60A mutational site and SEQ ID NO.33 with at SEQ ID NO.34 and the SEQ ID NO.35 of HTRA1 gene insert type/absence type sudden change.
4, detect liquid-phase chip according to each described ARMS2 of claim 1-3 and HTRA1 gene pleiomorphism, it is characterized in that described spacerarm is 5-10 T.
5, a kind of Auele Specific Primer for ARMS2 and the detection of HTRA1 gene pleiomorphism, it is characterized in that, mainly include: at SEQ ID NO.10 and the SEQ ID NO.11 in ARMS2 gene G72T mutational site, and be selected from SEQ ID NO.12-14 at HTRA1 gene C 95G/A mutational site, at the SEQ ID NO.15 in HTRA1 gene G60A mutational site and SEQ ID NO.16 with at more than a pair of among the SEQ ID NO.17 of HTRA1 gene insert type/absence type sudden change and the SEQ ID NO.18.
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