CN102181573A - 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 mutant type 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 one and obtains functional sudden change that 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 mainly is 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, 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 segmental size of electrophoresis observation, this method is used to detect the transgenation that restriction enzyme site changes, and can directly judge genotype, 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 takes place in the 74th Nucleotide of SEQ ID NO.73 | C74T |
| 2 | A → G sudden change takes place in the 123rd Nucleotide of SEQ ID NO.74 | A123G |
| 3 | A → G sudden change takes place in the 181st Nucleotide of SEQ ID NO.75 | A181G |
| 4 | A → G sudden change takes place in the 89th Nucleotide of SEQ ID NO.76 | A89G |
| 5 | G → A sudden change takes place in the 96th Nucleotide of SEQ ID NO.77 | G96A |
| 6 | A → G sudden change takes place in the 106th Nucleotide of SEQ ID NO.78 | A106G |
| 7 | T → G sudden change takes place in the 161st Nucleotide of SEQ ID NO.79 | T161G |
| 8 | A → G sudden change takes place in the 151st Nucleotide of SEQ ID NO.80 | A151G |
| 9 | G → T sudden change takes place in the 122nd Nucleotide of SEQ ID NO.81 | G122T |
Summary of the invention
One of purpose of the present invention provides 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 at every kind of pleomorphism site and the ASPE primer of mutant: every kind of ASPE primer is made up of at the Auele Specific Primer in goal gene mutational site the tag sequence and the 3 ' end of 5 ' end, and described specific primer sequence is selected from: at SEQ ID NO.19 and the SEQ ID NO.20 of C74T, SEQ ID NO.21 and SEQ ID NO.22 at A123G, SEQ ID NO.23 and SEQ ID NO.24 at A181G, SEQ ID NO.25 and SEQ ID NO.26 at A89G, SEQ ID NO.27 and SEQ ID NO.28 at G96A, SEQ ID NO.29 and SEQ ID NO.30 at A106G, SEQ ID NO.31 and SEQ ID NO.32 at T161G, SEQ ID NO.33 and SEQ ID NO.34 at A151G, at more than a pair of among the SEQ ID NO.35 of G122T and the SEQ ID NO.36; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.18;
(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.37～SEQ ID NO.54, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). be used to amplify the primer of needs target sequence that detect, that have corresponding pleomorphism site.
Preferably, described amplimer is selected from: at SEQ ID NO.55 and the SEQ ID NO.56 of C74T, SEQ ID NO.57 and SEQ ID NO.58 at A123G, SEQ ID NO.59 and SEQ ID NO.60 at A181G, SEQ ID NO.61 and SEQ ID NO.62 at A89G, SEQ ID NO.63 and SEQ ID NO.64 at G96A, SEQ ID NO.65 and SEQ ID NO.66 at A106G, SEQ ID NO.67 and SEQ ID NO.68 at T161G, SEQ ID NO.69 and SEQ ID NO.70 at A151G, at more than a pair of among the SEQ ID NO.71 of G122T and the SEQ ID NO.72.
Preferably, described ASPE primer is: the sequence of forming at the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.19 of C74T and SEQ ID NO.2 and SEQ ID NO.20, the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.21 at A123G reaches the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.22, the sequence of being made up of SEQ ID NO.5 and SEQ IDNO.23 at A181G reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.24, the sequence of being made up of SEQ ID NO.7 and SEQ ID NO.25 at A89G reaches the sequence of being made up of SEQ ID NO.8 and SEQ ID NO.26, the sequence of being made up of SEQ ID NO.9 and SEQ ID NO.27 at G96A reaches the sequence of being made up of SEQ ID NO.10 and SEQ ID NO.28, the sequence of being made up of SEQ ID NO.11 and SEQ ID NO.29 at A106G reaches the sequence of being made up of SEQ ID NO.12 and SEQ ID NO.30, the sequence of being made up of SEQ ID NO.13 and SEQ ID NO.31 at T161G reaches the sequence of being made up of SEQ ID NO.14 and SEQ ID NO.32, the sequence of being made up of SEQ ID NO.15 and SEQ ID NO.33 at A151G reaches the sequence of being made up of SEQ ID NO.16 and SEQ ID NO.34, and/or at the sequence of forming by SEQ ID NO.17 and SEQ IDNO.35 of G122T and the sequence of forming 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 need.。Prepared KITLG gene pleiomorphism detects liquid-phase chip and has extraordinary signal-noise ratio, and there is not cross reaction between designed probe and the anti-tag sequence basically, getting of tag sequence label, anti-tag sequence label and combining of tag sequence label and concrete ASPE primer, can avoid cross reaction, realize the parallel detection in a plurality of SNP site.
2. pass through design experiences and a large amount of operations of testing of the present inventor's long-term accumulation, from numerous Auele Specific Primers, chosen optimum combination.The genotype of various types is accurately distinguished in the mutational site that the ASPE primer specificity primer of the present invention design can sensitive recognition objective specifically detects; 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 single site mutation situation, also the polymorphism situation in a plurality of mutational sites of parallel detection simultaneously detects the effect unanimity.
3. detection method step of the present invention is simple, nine kinds of pleomorphism sites detect and can finish 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 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.
5. the KITLG gene mutation detection liquid-phase chip among the present invention provides a kind of technical scheme of different designs for the parallel detection of a plurality of sites several genes type, and has produced significant technique effect.Simultaneously, since technical characterictics such as high-throughput high specific, more realistic demands of applications.The technology trends that on behalf of biological target, liquid-phase chip technology of the present invention will detect.
Embodiment
Embodiment 1KITLG gene pleiomorphism detects liquid-phase chip, mainly includes:
One, ASPE primer
Wild-type and mutant at KITLG gene nine kinds of common genotype C74T, A123G, A181G, A89G, G96A, A106G, T161G, A151G and G122T design specific primer sequence 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 1KITLG 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 18 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
18 kinds of microballoons selecting are available from U.S. Luminex company, with anti-tag sequence bag by with microballoon on.Be connected with the spacerarm sequence of 5-10 T between anti-tag sequence and the microballoon, promptly 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 to be 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 10ul synthetic anti-tag molecule (100nmol/ml).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 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 the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Embodiment 2 utilization embodiment 1 described KITLG gene test liquid-phase chip is to the detection of sample
The prescription of described various solution is as follows:
MES damping fluid (pH5.0) prescription (250ml) of 50mM:
2 * Tm hybridization buffer
| Reagent | The source | Final concentration | The consumption of every 250ml |
| 1MTris-HCl，pH8.0 | 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 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, obtain DNA to be detected about DNA extraction.
Two, the pcr amplification of testing sample
Design nine pairs of primers, one step of multiplex PCR amplifies nine target sequences that contain KITLG gene nine kinds of common genotype C74T, A123G, A181G, A89G, G96A, A106G, T161G, A151G and G122T respectively, 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 the above-mentioned table 3.
At first prepare the multiple PCR primer working fluid: the primer stock solution 100ul that respectively gets SEQ ID NO.55-72 respectively mixes and is the multiple PCR primer working fluid in the 1.5ml Eppendorf tube.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 blended ASPE primer working fluid: get gene corresponding wild type to be detected and mutant ASPE primer stock solution 10ul respectively in the 1.5ml Eppendorf tube, add 10mmol/L Tris Buffer and mend, mix and be ASPE mix primer working fluid to 200ul.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 18 kinds of bags of every group selection by the microballoon of anti-tag
5Individual/ml);
2. the microballoon of getting every kind of numbering of 1ul respectively is 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. the ASPE reaction solution of getting 5-25ul is used ddH in corresponding hole
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 is by Luminex serial analysis instrument detecting.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 KITLG gene polymorphism sites originally, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Being provided with 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 can detect KITLG gene polymorphism sites type exactly, and the 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 liquid-phase chip of the ASPE primer that embodiment 3 is different is to the detection of KITLG gene polymorphism sites
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
Detecting liquid-phase chip with KITLG gene A 123G and A106G site mutation is example, respectively at 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 then is selected from SEQ ID NO.1-SEQ ID NO.18, accordingly, bag is selected from SEQ ID NO.37-SEQ ID NO.54 by the anti-tag sequence with corresponding tag sequence complementary pairing on microballoon.Specific design is shown in following table (table 8).Synthetic, the anti-tag sequence bag of ASPE primer is described like embodiment 1 and embodiment 2 by microballoon, amplimer, detection method.
The design of table 8 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 9 pattern detection result and Polymorphism Analysis
Table 10 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 2.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.
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
Detecting liquid-phase chip with KITLG gene A 181G and T161G site mutation is example, complementary sequence forward or backwards with this place, mutational site target sequence is a template, respectively at 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 invention 1, as shown in table 11.Wherein,
Interior base is a pleomorphism site.
Table 11 specific primer sequence
Detecting liquid-phase chip with KITLG gene A 181G and T161G site mutation is example, select different specific primer sequences for use at A181G and T161G, the Tag sequence of ASPE primer 5 ' end then is fixed as the best effect sequence among the embodiment 1, and select corresponding with it anti-tag sequence for use, specific design is shown in following table (table 12).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 12 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 13 pattern detection result and Polymorphism Analysis
Table 14 pattern detection result and Polymorphism Analysis
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 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 still are that specific primer sequence and tag sequence arranging effect are better among the embodiment 1.Other is at the specific primer sequence and the collocation of tag sequence in different mutational sites, and with coming to the same thing of embodiment 2 and present embodiment, promptly embodiment 1 selected Auele Specific Primer has better signal to noise ratio, and it is also better to detect effect, and 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 (6)
1. a KITLG gene pleiomorphism detects liquid-phase chip, it is characterized in that, includes:
(A). the wild-type that designs respectively at every kind of pleomorphism site and the ASPE primer of mutant: every kind of ASPE primer is made up of at the specific primer sequence in goal gene mutational site the tag sequence and the 3 ' end of 5 ' end, and described specific primer sequence is selected from: at SEQ ID NO.19 and the SEQ ID NO.20 of C74T, SEQ ID NO.21 and SEQ IDNO.22 at A123G, SEQ ID NO.23 and SEQ ID NO.24 at A181G, SEQ ID NO.25 and SEQ IDNO.26 at A89G, SEQ ID NO.27 and SEQ ID NO.28 at G96A, SEQ ID NO.29 and SEQ IDNO.30 at A106G, SEQ ID NO.31 and SEQ ID NO.32 at T161G, SEQ ID NO.33 and SEQ IDNO.34 at A151G, at more than a pair of among the SEQ ID NO.35 of G122T and the SEQ ID NO.36; Described tag sequence is selected from SEQ IDNO.1～SEQ ID NO.18;
(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.37～SEQ ID NO.54, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). be used to amplify the primer of needs target sequence that detect, that have corresponding pleomorphism site.
2. KITLG gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that described amplimer is selected from: at SEQ ID NO.55 and the SEQ ID NO.56 of C74T, SEQ ID NO.57 and SEQ ID NO.58 at A123G, SEQ ID NO.59 and SEQ ID NO.60 at A181G, SEQ ID NO.61 and SEQ ID NO.62 at A89G, SEQ ID NO.63 and SEQ ID NO.64 at G96A, SEQ ID NO.65 and SEQ ID NO.66 at A106G, SEQ ID NO.67 and SEQ ID NO.68 at T161G, SEQ ID NO.69 and SEQ ID NO.70 at A151G, at more than a pair of among the SEQ ID NO.71 of G122T and the SEQ ID NO.72.
3. KITLG gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that described ASPE primer is: the sequence that sequence of being made up of SEQ ID NO.1 and SEQ ID NO.19 and SEQ ID NO.2 and SEQ ID NO.20 form, the sequence of being made up of SEQID NO.3 and SEQ ID NO.21 reaches the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.22, the sequence of being made up of SEQ IDNO.5 and SEQ ID NO.23 reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.24, the sequence of being made up of SEQ IDNO.7 and SEQ ID NO.25 reaches the sequence of being made up of SEQ ID NO.8 and SEQ ID NO.26, the sequence of being made up of SEQ IDNO.9 and SEQ ID NO.27 reaches the sequence of being made up of SEQ ID NO.10 and SEQ ID NO.28, the sequence of being made up of SEQ IDNO.11 and SEQ ID NO.29 reaches the sequence of being made up of SEQ ID NO.12 and SEQ ID NO.30, the sequence of being made up of SEQ IDNO.13 and SEQ ID NO.31 reaches the sequence of being made up of SEQ ID NO.14 and SEQ ID NO.32, the sequence of being made up of SEQ IDNO.15 and SEQ ID NO.33 reaches the sequence of being made up of SEQ ID NO.16 and SEQ ID NO.34, and/or sequence of forming by SEQ ID NO.17 and SEQ ID NO.35 and the sequence formed by SEQ ID NO.18 and SEQ ID NO.36.
4. KITLG gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that,
(A). described ASPE primer is: the sequence that sequence of being made up of SEQ ID NO.1 and SEQ ID NO.19 and SEQ ID NO.2 and SEQ ID NO.20 form, the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.21 reaches the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.22, the sequence of being made up of SEQ ID NO.5 and SEQ ID NO.23 reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.24, the sequence of being made up of SEQ ID NO.7 and SEQ ID NO.25 reaches the sequence of being made up of SEQ ID NO.8 and SEQ ID NO.26, the sequence of being made up of SEQ ID NO.9 and SEQ ID NO.27 reaches the sequence of being made up of SEQ ID NO.10 and SEQ ID NO.28, the sequence of being made up of SEQ ID NO.11 and SEQ ID NO.29 reaches the sequence of being made up of SEQ IDNO.12 and SEQ ID NO.30, the sequence of being made up of SEQ ID NO.13 and SEQ ID NO.31 reaches the sequence of being made up of SEQ IDNO.14 and SEQ ID NO.32, the sequence of being made up of SEQ ID NO.15 and SEQ ID NO.33 reaches the sequence of being made up of SEQ IDNO.16 and SEQ ID NO.34, reach the sequence of forming by SEQID NO.18 and SEQ ID NO.36 with the sequence of forming by SEQ ID NO.17 and SEQ ID NO.35;
(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.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: at SEQ ID NO.55 and the SEQ ID NO.56 of C74T, SEQID NO.57 and SEQ ID NO.58 at A123G, SEQ ID NO.59 and SEQ ID NO.60 at A181G, SEQ IDNO.61 and SEQ ID NO.62 at A89G, SEQ ID NO.63 and SEQ ID NO.64 at G96A, SEQ IDNO.65 and SEQ ID NO.66 at A106G, SEQ ID NO.67 and SEQ ID NO.68 at T161G, at the SEQ IDNO.69 of A151G and SEQ ID NO.70 with at SEQ ID NO.71 and the SEQ ID NO.72 of G122T.
5. detect liquid-phase chip according to each described KITLG gene pleiomorphism of claim 1-4, it is characterized in that described spacerarm sequence is 5-10 T.
6. 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: at SEQ ID NO.19 and the SEQ ID NO.20 of C74T, SEQ ID NO.21 and SEQ ID NO.22 at A123G, SEQ ID NO.23 and SEQ ID NO.24 at A181G, SEQ ID NO.25 and SEQ ID NO.26 at A89G, SEQ ID NO.27 and SEQ ID NO.28 at G96A, SEQ ID NO.29 and SEQ ID NO.30 at A106G, SEQ ID NO.31 and SEQ ID NO.32 at T161G, SEQ ID NO.33 and SEQ ID NO.34 at A151G, at more than a pair of among the SEQ ID NO.35 of G122T and the SEQ ID NO.36.
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| CN101812511A (en) * | 2009-12-29 | 2010-08-25 | 广州益善生物技术有限公司 | CYP3A4 gene SNP detection specific primer, liquid-phase chip and method |
| CN101863972A (en) * | 2009-04-14 | 2010-10-20 | 中国科学院上海生命科学研究院 | Pigment synthesis KIT ligand and application thereof |
| WO2010141362A1 (en) * | 2009-05-31 | 2010-12-09 | The Trustees Of The University Of Pennsylvania | Compositions and methods for diagnosing the occurrence or likelihood of occurrence of testicular germ cell cancer |
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| CN101863972A (en) * | 2009-04-14 | 2010-10-20 | 中国科学院上海生命科学研究院 | Pigment synthesis KIT ligand and application thereof |
| WO2010141362A1 (en) * | 2009-05-31 | 2010-12-09 | The Trustees Of The University Of Pennsylvania | Compositions and methods for diagnosing the occurrence or likelihood of occurrence of testicular germ cell cancer |
| CN101812511A (en) * | 2009-12-29 | 2010-08-25 | 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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