CN102181573B - Specific primers and liquid-phase chip for detection of KITLG gene - Google Patents
Specific primers and liquid-phase chip for detection of KITLG gene Download PDFInfo
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Abstract
The invention discloses specific primers and a liquid-phase chip for the single nucleotide polymorphism (SNP) detection of the kit ligand gene (KITLG). The liquid-phase chip mainly comprises allele specific primers (ASPE), each of which consists of a tag sequence at a 5' end and specific primers aiming at mutational sites of target genes at a 3' end, microspheres wrapped by an anti-tag sequence and amplification primers, wherein sequences of the specific primers are one or more from the following pairs of sequences: SEQ ID No. 19 and SEQ ID No. 20, SEQ ID No. 21 and SEQ ID No. 22, SEQ ID No. 23 and SEQ ID No. 24, SEQ ID No. 25 and SEQ ID No. 26, SEQ ID No. 27 and SEQ ID No. 28, SEQ ID No. 29 and SEQ ID No. 30, SEQ ID No. 31 and SEQ ID No. 32, SEQ ID No. 33 and SEQ ID No. 34 and SEQ ID No.35 and SEQ ID No. 36. In the detection liquid-phase chip, the coincidence rate of detection results and a sequencing method is up to 100 percent, so the parallel detection of the wild type and mutanttype of polymorphic loci is realized.
Description
Technical field
The present invention bends in biology field, relates to medical science and biotechnology, concrete a kind of KITLG gene test Auele Specific Primer and the liquid-phase chip of relating to.
Background technology
KITLG (the KIT ligand gene) is the KIT ligand gene that is positioned at 12p22.The KITLG sudden change is a functional sudden change of acquisition, can increase the melanic quantity of pigment cell, i.e. KITLG transgenation causes familial progressive hyperpigmentation.
At present, few to the report that the KITLG gene pleiomorphism detects, analyzes, the method for its research is mainly direct sequencing and PCR-RFLP analytical method.The PCR-RFLP method is based on the change of the restriction enzyme enzyme recognition site that transgenation causes, lose or the generation novel site as the site, by a certain specific fragment of pcr amplification, use again the digestion with restriction enzyme amplified production, the size of electrophoresis observation fragment, this method can directly judge 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.In addition, above method all exists the limitation that detects flux, can only detect a kind of mutation type at every turn, can not satisfy the needs of practical application.
The KITLG gene mutation site of target detect of the present invention, it is as shown in the table:
| Sequence number | The content of KITLG site mutation | Write a Chinese character in simplified form |
| 1 | C → T sudden change occurs in the 74th Nucleotide of SEQ ID NO.73 | C74T |
| 2 | A → G sudden change occurs in the 123rd Nucleotide of SEQ ID NO.74 | A123G |
| 3 | A → G sudden change occurs in the 181st Nucleotide of SEQ ID NO.75 | A181G |
| 4 | A → G sudden change occurs in the 89th Nucleotide of SEQ ID NO.76 | A89G |
| 5 | G → A sudden change occurs in the 96th Nucleotide of SEQ ID NO.77 | G96A |
| 6 | A → G sudden change occurs in the 106th Nucleotide of SEQ ID NO.78 | A106G |
| 7 | T → G sudden change occurs in the 161st Nucleotide of SEQ ID NO.79 | T161G |
| 8 | A → G sudden change occurs in the 151st Nucleotide of SEQ ID NO.80 | A151G |
| 9 | G → T sudden change occurs in the 122nd Nucleotide of SEQ ID NO.81 | G122T |
Summary of the invention
One of purpose of the present invention is to provide the KITLG gene pleiomorphism and detects liquid-phase chip, and this liquid-phase chip can be used for detecting wild-type and the mutant of KITLG gene nine kinds of common genotype C74T, A123G, A181G, A89G, G96A, A106G, T161G, A151G and G122T.
A kind of KITLG gene pleiomorphism detects liquid-phase chip, includes:
(A). the wild-type that designs respectively for every kind of pleomorphism site and the ASPE primer of mutant: every kind of ASPE primer is comprised of the tag sequence of 5 ' end and 3 ' the end Auele Specific Primer for the goal gene mutational site, described specific primer sequence is selected from: for SEQ ID NO.19 and the SEQ ID NO.20 of C74T, SEQ ID NO.21 and SEQ ID NO.22 for A123G, SEQ ID NO.23 and SEQ ID NO.24 for A181G, SEQ ID NO.25 and SEQ ID NO.26 for A89G, SEQ ID NO.27 and SEQ ID NO.28 for G96A, SEQ ID NO.29 and SEQ ID NO.30 for A106G, SEQ ID NO.31 and SEQ ID NO.32 for T161G, SEQ ID NO.33 and SEQ ID NO.34 for A151G, for more than a pair of in the SEQ ID NO.35 of G122T and SEQ ID NO.36, described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.18,
(B). that the anti-tag sequence is coated with, as to have different colours coding microballoon 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 in SEQ ID NO.37～SEQ ID NO.54, 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.
preferably, described amplimer is selected from: for SEQ ID NO.55 and the SEQ ID NO.56 of C74T, SEQ ID NO.57 and SEQ ID NO.58 for A123G, SEQ ID NO.59 and SEQ ID NO.60 for A181G, SEQ ID NO.61 and SEQ ID NO.62 for A89G, SEQ ID NO.63 and SEQ ID NO.64 for G96A, SEQ ID NO.65 and SEQ ID NO.66 for A106G, SEQ ID NO.67 and SEQ ID NO.68 for T161G, SEQ ID NO.69 and SEQ ID NO.70 for A151G, for more than a pair of in the SEQ ID NO.71 of G122T and SEQ ID NO.72.
preferably, described ASPE primer is: the sequence that forms for the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.19 of C74T and SEQ ID NO.2 and SEQ ID NO.20, the sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.21 for A123G reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.22, the sequence that is comprised of SEQ ID NO.5 and SEQ IDNO.23 for A181G reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.24, the sequence that is comprised of SEQ ID NO.7 and SEQ ID NO.25 for A89G reaches the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.26, the sequence that is comprised of SEQ ID NO.9 and SEQ ID NO.27 for G96A reaches the sequence that is comprised of SEQ ID NO.10 and SEQ ID NO.28, the sequence that is comprised of SEQ ID NO.11 and SEQ ID NO.29 for A106G reaches the sequence that is comprised of SEQ ID NO.12 and SEQ ID NO.30, the sequence that is comprised of SEQ ID NO.13 and SEQ ID NO.31 for T161G reaches the sequence that is comprised of SEQ ID NO.14 and SEQ ID NO.32, the sequence that is comprised of SEQ ID NO.15 and SEQ ID NO.33 for A151G reaches the sequence that is comprised of SEQ ID NO.16 and SEQ ID NO.34, and/or reach for the sequence that is formed by SEQ ID NO.17 and SEQ IDNO.35 of G122T the sequence that is formed by SEQ ID NO.18 and SEQ ID NO.36.
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 detect the needed time well below sequencing technologies commonly used, realistic especially application needs.。Prepared KITLG gene pleiomorphism detects liquid-phase chip and has extraordinary signal-noise ratio, and basically there is not cross reaction between designed probe and anti-tag sequence, getting 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 design experiences of the long-term accumulation by the present inventor and the operation of great many of experiments, chosen optimum combination 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 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, also the polymorphism situation in a plurality of mutational sites of parallel detection simultaneously, detect effect consistent.
3. detection method step of the present invention is simple, nine kinds of pleomorphism sites detect and can complete nine 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, 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 of detection be further enhanced, and signal to noise ratio strengthens, and detected result more accurately and reliably.
5. the KITLG gene mutation detection liquid-phase chip in 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, 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 will represent the technology trends that biological target detects.
Embodiment
Embodiment 1KITLG gene pleiomorphism detects liquid-phase chip, mainly includes:
One, ASPE primer
Wild-type and mutant for KITLG gene nine kinds of common genotype C74T, A123G, A181G, A89G, G96A, A106G, T161G, A151G and G122T 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 (Tag sequence+specific primer sequence) of table 1KITLG gene
Every the ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence for anti-tag sequence on 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, on 18 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
18 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 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 in 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 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, then add the EDC working fluid of 2.5ul, then constant-temperature incubation 30 minutes.After reaction finished, the Tween-20 with 0.02% washed once, then washed once with 0.1% SDS liquid.The microballoon that is coated with the anti-tag sequence after washing is resuspended in the Tris-EDTA solution [10mmol/LTris (pH8.0)] of 100ul, in 1mmol/LEDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains the mutational site
For KITLG gene nine kinds of common genotype C74T, A123G, A181G, A89G, G96A, A106G, T161G, A151G and G122T, design of amplification primers is to (seeing Table 3), and the coldest days of the year end bar of increasing respectively contains the target sequence of pleomorphism site.
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 the described KITLG gene test of embodiment 1 liquid-phase chip to the detection of sample
The formula of described various solution is as follows:
MES damping fluid (pH5.0) formula (250ml) of 50mM:
2 * Tm hybridization buffer
| Reagent | The source | Final concentration | The consumption of every 250ml |
| 1MTris-HCl，pH8.0 | SigmaT3038 | 0.2M | 50ml |
| 5M NaCl | Sigma S5150 | 0.4M | 20ml |
| Triton X-100 | Sigma T8787 | 0.16％ | 0.4ml |
Be stored in 4 ℃ after 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 nine pairs of primers, one step of multiplex PCR amplifies the nine objective sequences that contain respectively KITLG gene nine kinds of common genotype C74T, A123G, A181G, A89G, G96A, A106G, T161G, A151G and G122T, the product size is respectively 299bp, 267bp, 372bp, 309bp, 341bp, 294bp, 396bp, 428bp and 278bp, and primer sequence (SEQ ID NO.55-72) is seen shown in 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.55-72 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;
2.37 ℃ hatch 15min, 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, mix biotin labeled dCTP in reaction process, thereby make a plurality of biotin labeling on 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 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 is 2.5 * 10 to the microballoon of 18 kinds of coated anti-tag 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, the vortex mixing;
5. get the above-mentioned microballoon suspension of 25ul in 96 hole filter plate corresponding holes, 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. 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, detect on the Luminex instrument.
Six, result detects and data analysis
The 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 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 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 KITLG 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 KITLG genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.As seen KITLG gene pleiomorphism provided by the present invention detects liquid-phase chip and can detect exactly KITLG gene polymorphism sites type, and result is reliable and stable.
One of table 4 pattern detection result (MFI)
Table 5 pattern detection result (MFI)
Table 6 sample KITLG transgenation ratio (%)
Table 7 sample KITLG 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 | 89GG | 89GG |
| 5 | Wild-type | Wild-type |
| 6 | 122GT | 122GT |
| 7 | 74TT | 74TT |
| 8 | Wild-type | Wild-type |
| 9 | 181AG | 181AG |
| 10 | 161TG | 161TG |
| 11 | Wild-type | Wild-type |
| 12 | 96GA | 96GA |
| 13 | 74TT | 74TT |
| 14 | Wild-type | Wild-type |
| 15 | Wild-type | Wild-type |
| 16 | Wild-type | Wild-type |
| 17 | Wild-type | Wild-type |
| 18 | Wild-type | Wild-type |
| 19 | 106GG | 106GG |
| 20 | Wild-type | Wild-type |
The detection of the liquid-phase chip of the ASPE primer that embodiment 3 is different to the KITLG gene polymorphism sites
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
Detect liquid-phase chip as example take KITLG Gene A 123G and A106G site mutation, respectively for the wild-type of A123G and A106G and the specific primer sequence of mutant design ASPE primer 3 ' end, the Tag sequence of ASPE primer 5 ' end is selected from SEQ ID NO.1-SEQ ID NO.18, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing that is coated on microballoon is selected from SEQ ID NO.37-SEQ ID NO.54.Specific design is as shown in following table (table 8).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 8 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned design preparation, by the described testing process of embodiment 2 and method, sample 21-40 is detected, detected result is as follows:
Table 9 pattern detection result and Polymorphism Analysis
Table 10 pattern detection result and Polymorphism 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 in embodiment 1 collocation of tag sequence and specific primer sequence, and effect better (signal to noise ratio is better) is referring to the present embodiment test group 2.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.
Embodiment 4 is with the selection of KITLG gene pleiomorphism detection specificity primer sequence
One, the design (selection of wild-type and mutant specific primer sequence) of liquid-phase chip preparation
Detect liquid-phase chip as example take KITLG Gene A 181G and T161G site mutation, take the complementary sequence forward or backwards of this place, mutational site target sequence as template, respectively for the wild-type of A181G and T161G 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 present invention 1, as shown in table 11.Wherein,
Interior base is pleomorphism site.
Table 11 specific primer sequence
Detect liquid-phase chip as example take KITLG Gene A 181G and T161G site mutation, select different specific primer sequences for A181G and T161G, the Tag sequence of ASPE primer 5 ' end is fixed as the best effect sequence in embodiment 1, and select with it corresponding anti-tag sequence, specific design is as shown in following table (table 12).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 12 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned design preparation, by the described testing process of embodiment 2 and method, sample 41-60 is detected, detected result is as follows:
Table 13 pattern detection result and Polymorphism Analysis
Table 14 pattern detection result and Polymorphism Analysis
And the ASPE primer is when selecting in embodiment 1 collocation of specific primer sequence and tag sequence, and effect better (signal to noise ratio is better) is referring to the present embodiment test group 7 and test group 10.The different specific primer sequences of complementary sequence forward or backwards and the collocation of tag sequence that other derives from place, target detect site sequence, be still in embodiment 1 specific primer sequence and tag sequence arranging effect better.Other is for specific primer sequence and the collocation of tag sequence in different mutational sites, and with coming to the same thing of embodiment 2 and the present embodiment, namely the selected Auele Specific Primer of embodiment 1, have better signal to noise ratio, detects effect also better, and concrete data are omitted.
Be more than for the illustrating of possible embodiments of the present invention, but this embodiment limits the scope of the claims 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 scope of the claims of the present invention.
Claims (5)
1. a KITLG gene pleiomorphism detects liquid-phase chip, it is characterized in that, includes:
(A). the wild-type that designs respectively for every kind of pleomorphism site 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 mutational site, described specific primer sequence is: for SEQ ID NO.19 and the SEQ ID NO.20 of C74T, and be selected from SEQ ID NO.21 and SEQ ID NO.22 for A123G, SEQ ID NO.23 and SEQ ID NO.24 for A181G, SEQ ID NO.25 and SEQ ID NO.26 for A89G, SEQ ID NO.27 and SEQ ID NO.28 for G96A, SEQ ID NO.29 and SEQ ID NO.30 for A106G, SEQ ID NO.31 and SEQ ID NO.32 for T161G, SEQ ID NO.33 and SEQ ID NO.34 for A151G, for more than a pair of in the SEQ ID NO.35 of G122T and SEQ ID NO.36, described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.18,
(B). that the anti-tag sequence is coated with, as to have different colours coding microballoon 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 in SEQ ID NO.37～SEQ ID NO.54, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). be used for amplifying and need to detect, primer with target sequence of corresponding pleomorphism site, described amplimer is: for SEQ ID NO.55 and the SEQ ID NO.56 of C74T, and be selected from SEQ ID NO.57 and SEQ ID NO.58 for A123G, SEQ ID NO.59 and SEQ ID NO.60 for A181G, SEQ ID NO.61 and SEQ ID NO.62 for A89G, SEQ ID NO.63 and SEQ ID NO.64 for G96A, SEQ ID NO.65 and SEQ ID NO.66 for A106G, SEQ ID NO.67 and SEQ ID NO.68 for T161G, SEQ ID NO.69 and SEQ ID NO.70 for A151G, for more than a pair of in the SEQ ID NO.71 of G122T and SEQ ID NO.72.
2. KITLG gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that, described ASPE primer is: the sequence that the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.19 and SEQ ID NO.2 and SEQ ID NO.20 form, and the sequence of selecting free SEQ ID NO.3 and SEQ ID NO.21 to form reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.22, the sequence that is comprised of SEQ ID NO.5 and SEQ ID NO.23 reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.24, the sequence that is comprised of SEQ ID NO.7 and SEQ ID NO.25 reaches the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.26, the sequence that is comprised of SEQ ID NO.9 and SEQ ID NO.27 reaches the sequence that is comprised of SEQ ID NO.10 and SEQ ID NO.28, the sequence that is comprised of SEQ ID NO.11 and SEQ ID NO.29 reaches the sequence that is comprised of SEQ ID NO.12 and SEQ ID NO.30, the sequence that is comprised of SEQ ID NO.13 and SEQ ID NO.31 reaches the sequence that is comprised of SEQ ID NO.14 and SEQ ID NO.32, the sequence that is comprised of SEQ ID NO.15 and SEQ ID NO.33 reaches the sequence that is comprised of SEQ ID NO.16 and SEQ ID NO.34, more than a pair of in the sequence that forms with the sequence that is formed by SEQ ID NO.17 and SEQ ID NO.35 and by SEQ ID NO.18 and SEQ ID NO.36.
3. KITLG gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that,
(A). described ASPE primer is: the sequence that the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.19 and SEQ ID NO.2 and SEQ ID NO.20 form, the sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.21 reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.22, the sequence that is comprised of SEQ ID NO.5 and SEQ ID NO.23 reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.24, the sequence that is comprised of SEQ ID NO.7 and SEQ ID NO.25 reaches the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.26, the sequence that is comprised of SEQ ID NO.9 and SEQ ID NO.27 reaches the sequence that is comprised of SEQ ID NO.10 and SEQ ID NO.28, the sequence that is comprised of SEQ ID NO.11 and SEQ ID NO.29 reaches the sequence that is comprised of SEQ ID NO.12 and SEQ ID NO.30, the sequence that is comprised of SEQ ID NO.13 and SEQ ID NO.31 reaches the sequence that is comprised of SEQ ID NO.14 and SEQ ID NO.32, the sequence that is comprised of SEQ ID NO.15 and SEQ ID NO.33 reaches the sequence that is comprised of SEQ ID NO.16 and SEQ ID NO.34, reach with the sequence that is formed by SEQ ID NO.17 and SEQ ID NO.35 the sequence that is formed by SEQ ID NO.18 and SEQ ID NO.36,
(B). that the anti-tag sequence is coated with, as to have different colours coding microballoon 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 in SEQ ID NO.37～SEQ ID NO.54, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). described amplimer is: for SEQ ID NO.55 and the SEQ ID NO.56 of C74T, SEQ ID NO.57 and SEQ ID NO.58 for A123G, SEQ ID NO.59 and SEQ ID NO.60 for A181G, SEQ ID NO.61 and SEQ ID NO.62 for A89G, SEQ ID NO.63 and SEQ ID NO.64 for G96A, SEQ ID NO.65 and SEQ ID NO.66 for A106G, SEQ ID NO.67 and SEQ ID NO.68 for T161G, for the SEQ ID NO.69 of A151G and SEQ ID NO.70 with for SEQ ID NO.71 and the SEQ ID NO.72 of G122T.
4. according to claim 1-3 described KITLG gene pleiomorphisms of any one detect liquid-phase chip, it is characterized in that, described spacerarm sequence is 5-10 T.
5. one kind is used for the Auele Specific Primer that the KITLG gene pleiomorphism detects, it is characterized in that, described Auele Specific Primer is selected from: for SEQ ID NO.19 and the SEQ ID NO.20 of C74T, and be selected from SEQ ID NO.21 and SEQ ID NO.22 for A123G, SEQ ID NO.23 and SEQ ID NO.24 for A181G, SEQ ID NO.25 and SEQ ID NO.26 for A89G, SEQ ID NO.27 and SEQ ID NO.28 for G96A, SEQ ID NO.29 and SEQ ID NO.30 for A106G, SEQ ID NO.31 and SEQ ID NO.32 for T161G, SEQ ID NO.33 and SEQ ID NO.34 for A151G, for more than a pair of in the SEQ ID NO.35 of G122T and SEQ ID NO.36.
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| CN101812511B (en) * | 2009-12-29 | 2012-08-22 | 广州益善生物技术有限公司 | CYP3A4 gene SNP detection specific primer, liquid-phase chip and method |
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