CN102191337A - Specific primers and liquid phase chip for detecting polymorphism of cyckin-dependent kinase 5 regulatorysubunit-associated protein 1-like 1(CDKAL1) gene - Google Patents
Specific primers and liquid phase chip for detecting polymorphism of cyckin-dependent kinase 5 regulatorysubunit-associated protein 1-like 1(CDKAL1) gene Download PDFInfo
- Publication number
- CN102191337A CN102191337A CN2011101538766A CN201110153876A CN102191337A CN 102191337 A CN102191337 A CN 102191337A CN 2011101538766 A CN2011101538766 A CN 2011101538766A CN 201110153876 A CN201110153876 A CN 201110153876A CN 102191337 A CN102191337 A CN 102191337A
- Authority
- CN
- China
- Prior art keywords
- seq
- sequence
- phase chip
- tag
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention discloses specific primers and a liquid phase chip for detecting the polymorphism of a CDKAL1 gene. The liquid phase chip mainly comprises specific primers consisting of a tag sequence at a 5' end and specific primer sequences of polymorphic sites of a target gene, microspheres and amplification primers, wherein the specific primer sequences are one or more pairs from SEQ ID No.9 and SEQ ID No.10 for A107C, SEQ ID No.11 and SEQ ID No.12 for G323C, SEQ ID No.13 and SEQ ID No.14 for A210G and SEQ ID No.15 and SEQ ID No.16 for A75G; the tag sequence may be a sequence from SEQ ID No.1 to SEQ ID No.8. The prepared liquid phase chip for detecting the polymorphism of the CDKAL1 gene has a very high signal-to-noise ratio, and the cross reaction of a designed probe and an anti-tae sequence is prevented basically.
Description
Technical field
The present invention bends in biology field, relates to medical science and biotechnology, concrete a kind of CDKAL1 gene pleiomorphism detection specificity primer and the liquid-phase chip of relating to.
Background technology
CDK5 regulator subunit associated protein 1 analogue 1 (cyckin-dependent kinase 5 regulatorysubunit-associated protein 1-like 1, CDKAL1) gene is the encoding gene of CDK5 (Cyclin Dependent Kinase 5) regulator subunit associated protein 1, be positioned at the short arm of a chromosome 22.3 districts No. 6, its signaling zone is positioned on the 5th intron, expresses at human pancreas and Skeletal Muscle Cell camber.CDK5 is playing an important role aspect protection β cell function, the lowering blood glucose, and CDK5 can cause the β cytopathy under the sugared toxic effect of height, and the protein of CDKAL1 genes encoding can suppress the activation of CDK5.If the CDKAL1 gene locus morphs, then the restraining effect to CDK5 weakens, and causes the β cell function impaired, and insulin secretion reduces.The CDKAL1 gene passes through to influence the secreting function of beta Cell of islet, thereby influences secretion of insulin.
The CDKAL1 gene mutation site of target detect of the present invention, it is as shown in the table:
| Sequence number | The content of CDKAL1 site mutation | Write a Chinese character in simplified form |
| 1 | A → C sudden change takes place in the 107th Nucleotide of SEQ ID NO 31 | ?A107C |
| 2 | G → C sudden change takes place in the 323rd Nucleotide of SEQ ID NO 31 | ?G323C |
| 3 | A → G sudden change takes place in the 210th Nucleotide of SEQ ID NO 32 | ?A210G |
| 4 | A → G sudden change takes place in the 75th Nucleotide of SEQ ID NO 33 | ?A75G |
At present, the method that the CDKAL1 gene pleiomorphism is detected, analyzes is a lot, as: direct sequencing, quantitative fluorescent PCR, PCR-RFLP analytical method, TaqMan technology or the like, wherein the most frequently used method have quantitative fluorescent PCR and PCR-RFLP analytical method.Easy and simple to handle, advantages such as the result quick, quantification that quantitative fluorescent PCR has, still, this technology exists sample easily to pollute, and cross reaction easily takes place, the shortcoming that false positive rate is high; And 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.Once more, 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 the CDKAL1 gene pleiomorphism and detects liquid-phase chip, and this liquid-phase chip can be used for detecting wild-type and the mutant of CDKAL1 gene four kinds of common genotype A107C, G323C, A210G and A75G.
A kind of CDKAL1 gene pleiomorphism detects liquid-phase chip, mainly includes:
A. wild-type and the mutant specificity ASPE primer that designs respectively at 4 kinds of common pleomorphism sites of CDKAL1 gene, every kind of ASPE primer is made up of the tag sequence of 5 ' end and the specific primer sequence at the goal gene pleomorphism site of 3 ' end, and described specific primer sequence is selected from respectively: at the SEQ ID NO.9 of A107C and SEQ ID NO.10, at the SEQ ID NO.11 of G323C and SEQ ID NO.12, at the SEQ ID NO.13 of A210G and SEQ ID NO.14, at more than a pair of among the SEQ ID NO.15 of A75G and the SEQ ID NO.16; Described tag sequence is selected from the sequence among SEQ ID NO.1～SEQ IDNO.8;
B. be coated with the microballoon of special anti-tag sequence respectively, described anti-tag sequence can be correspondingly with A in selected tag sequence complementary pairing, described anti-tag sequence is selected from the sequence among SEQ ID NO.17～SEQ ID NO.24, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon, above-mentioned every kind of microballoon has the different colours coding;
C. be used for increasing have A107C, the primer of the above pleomorphism site target sequence of G323C, A210G and A75G.
Preferably, described amplimer is: at the SEQ ID NO.25 of A107C and/or G323C and SEQ ID NO.26, at the SEQ ID NO.27 of A210G and SEQ ID NO.28, at more than a pair of among the SEQ ID NO.29 of A75G and the SEQ ID NO.30.
Preferably, described ASPE primer is: the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.9 at A107C reaches the sequence of being made up of SEQ ID NO.2 and SEQ ID NO.10, the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.11 at G323C reaches the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.12, the sequence of being made up of SEQ ID NO.5 and SEQ IDNO.13 at A210G reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.14, more than a pair of in the sequence of forming at the sequence of forming by SEQ ID NO.7 and SEQ ID NO.15 of A75G and by SEQ ID NO.8 and SEQ ID NO.16.
Another object of the present invention provides the specific primer sequence that a kind of CDKAL1 of being used for gene pleiomorphism detects.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind ofly be used for the specific primer sequence that the CDKAL1 gene pleiomorphism detects, it is selected from: at the SEQ IDNO.9 of A107C and SEQ ID NO.10, at the SEQ ID NO.11 of G323C and SEQ ID NO.12, at the SEQ IDNO.13 of A210G and SEQ ID NO.14, at more than a pair of among the SEQ ID NO.15 of A75G and the SEQ ID NO.16.
Major advantage of the present invention is:
1. the identical rate of the detected result of liquid-phase chip provided by the present invention and sequencing is up to 100%, and detects the needed time well below sequencing technologies commonly used, realistic especially application need.In very a large amount of Auele Specific Primers, through test in a large number, reaction is verified, obtains the liquid-phase chip system of optimum combination.Prepared CDKAL1 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, choosing 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. the present invention has chosen optimum combination by the design experiences of contriver's long-term accumulation and a large amount of experimental implementation from numerous Auele Specific Primers.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, 4 kinds of pleomorphism sites detect and can finish 4 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 CDKAL1 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 1CDKAL1 gene pleiomorphism detects liquid-phase chip, mainly includes:
One, ASPE primer
Wild-type and mutant at CDKAL1 gene four kinds of common genotype A107C, G323C, A210G and A75G 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 1CDKAL1 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 8 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
8 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 CDKAL1 gene four kinds of common genotype A107C, G323C, A210G and A75G, design of amplification primers amplifies three target sequences that contain pleomorphism site respectively to (seeing Table 3), and wherein A107C and G323C are positioned at same amplified production.
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 1CDKAL1 gene test liquid-phase chips are to the detection of sample
The prescription of described various solution is as follows:
MES damping fluid (pH5.0) prescription (250m1) of 50mM:
2 * Tm hybridization buffer
| Reagent | The source | Final concentration | The consumption of every 250ml |
| 1MTris-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, obtain DNA to be detected about DNA extraction.
Two, the pcr amplification of testing sample
Design three pairs of primers, one step of multiplex PCR amplifies three target sequences that contain CDKAL1 gene four kinds of common genotype A107C, G323C, A210G and A75G respectively, wherein A107C and G323C are positioned at same amplified production, the product size is respectively 605bp, 589bp and 282bp, and primer sequence (SEQ ID NO.25-30) 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.25-30 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 the corresponding 16 kinds of microballoons of every group selection
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 CDKAL1 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 CDKAL1 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.As seen CDKAL1 gene pleiomorphism provided by the present invention detects liquid-phase chip can detect CDKAL1 gene polymorphism sites type exactly, and the result is reliable and stable.
One of table 4 pattern detection result (MFI)
The table 5 sample CDKAL1 ratio (%) that suddenlys change
Table 6 sample CDKAL1 gene mutation type analytical results
| Catalogue number(Cat.No.) | The liquid-phase chip detected result | Sequencing result |
| 1 | Wild-type | Wild-type |
| 2 | Wild-type | Wild-type |
| 3 | Wild-type | Wild-type |
| 4 | Wild-type | Wild-type |
| 5 | Wild-type | Wild-type |
| 6 | Wild-type | Wild-type |
| 7 | Wild-type | Wild-type |
| 8 | 107CC、210GG | 107CC、210GG |
| 9 | Wild-type | Wild-type |
| 10 | Wild-type | Wild-type |
| 11 | Wild-type | Wild-type |
| 12 | Wild-type | Wild-type |
| 13 | 323GC | 323GC |
| 14 | Wild-type | Wild-type |
| 15 | Wild-type | Wild-type |
| 16 | Wild-type | Wild-type |
| 17 | 210GG | 210GG |
| 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 CDKAL1 gene polymorphism sites
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
Detecting liquid-phase chip with CDKAL1 gene A 107C and G323C site mutation is example, respectively at the wild-type of A107C and G323C 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.8, accordingly, bag is selected from SEQ ID NO.17-SEQ ID NO.24 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.
The design of table 7 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned design preparation, by embodiment 2 described testing processes and method sample 21-40 is detected, detected result is as follows:
Table 8 pattern detection result and 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 4CDKAL1 gene polymorphism sites 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 CDKAL1 gene polymorphism sites A210G and A75G site mutation is example, complementary sequence forward or backwards with place, mutational site target sequence is a template, respectively at the wild-type of A210G and A75G 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 a pleomorphism site.
Table 10 specific primer sequence
Detecting liquid-phase chip with CDKAL1 gene polymorphism sites A210G and A75G site mutation respectively is example, select different specific primer sequences for use at A210G and A75G, 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 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
From present embodiment as can be seen, when the ASPE primer was selected among the embodiment 1 collocation of specific primer sequence and tag sequence for use, effect better (signal to noise ratio is better) was 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.The specific primer sequence in other different mutational sites and the collocation of tag sequence, 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 CDKAL1 gene pleiomorphism detects liquid-phase chip, it is characterized in that, mainly includes:
A. wild-type and the mutant specificity ASPE primer that designs respectively at the different pleomorphism sites of CDKAL1 gene, every kind of ASPE primer is made up of the tag sequence of 5 ' end and the specific primer sequence at the goal gene pleomorphism site of 3 ' end, and described specific primer sequence is selected from respectively: at the SEQ ID NO.9 of A107C and SEQ ID NO.10, at the SEQID NO.11 of G323C and SEQ ID NO.12, at the SEQ ID NO.13 of A210G and SEQ ID NO.14, at more than a pair of among the SEQ IDNO.15 of A75G and the SEQ ID NO.16; Described tag sequence is selected from the sequence among SEQ ID NO.1～SEQ ID NO.8;
B. be coated with the microballoon of special anti-tag sequence respectively, described anti-tag sequence can be correspondingly with A in selected tag sequence complementary pairing, described anti-tag sequence is selected from the sequence among SEQ ID NO.17～SEQ ID NO.24, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon, above-mentioned every kind of microballoon has the different colours coding;
C. be used for increasing have A107C, the primer of the above pleomorphism site target sequence of G323C, A210G and A75G.
2. CDKAL1 gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that described amplimer is selected from: at the SEQ ID NO.25 of A107C and/or G323C and SEQ ID NO.26, at the SEQ ID NO.27 of A210G and SEQ ID NO.28, at more than a pair of among the SEQ ID NO.29 of A75G and the SEQ ID NO.30.
3. CDKAL1 gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that described ASPE primer is selected from: the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.9 at A107C reaches the sequence of being made up of SEQ ID NO.2 and SEQ IDNO.10, the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.11 at G323C reaches the sequence of being made up of SEQ IDNO.4 and SEQ ID NO.12, the sequence of being made up of SEQ ID NO.5 and SEQ ID NO.13 at A210G reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.14, more than a pair of in the sequence of forming at the sequence of forming by SEQ ID NO.7 and SEQ ID NO.15 of A75G and by SEQ ID NO.8 and SEQ ID NO.16.
4. CDKAL1 gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that, mainly includes:
A. specificity ASPE primer: at the sequence of forming by SEQ ID NO.1 and SEQ ID NO.9 of A107C and the sequence of forming by SEQID NO.2 and SEQ ID NO.10, the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.11 at G323C reaches the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.12, the sequence of being made up of SEQ ID NO.5 and SEQ IDNO.13 at A210G reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.14, reach the sequence of forming by SEQ ID NO.8 and SEQ ID NO.16 with the sequence of forming by SEQ IDNO.7 and SEQ ID NO.15 at A75G;
B. be coated with the microballoon of special anti-tag sequence respectively, described anti-tag sequence can be correspondingly with A in selected tag sequence complementary pairing, described anti-tag sequence is selected from the sequence among SEQ ID NO.17～SEQ ID NO.24, also is provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon;
C. described amplimer is: at the SEQ ID NO.25 of A107C and G323C and SEQ ID NO.26, at the SEQ ID NO.27 of A210G and SEQ ID NO.28 with at SEQ ID NO.29 and the SEQ ID NO.30 of A75G.
5. detect liquid-phase chip according to each described CDKAL1 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 CDKAL1 gene pleiomorphism detects, it is characterized in that described specific primer sequence is: at the SEQ ID NO.9 of A107C and SEQ ID NO.10, at the SEQ ID NO.11 of G323C and SEQ IDNO.12, at the SEQ ID NO.13 of A210G and SEQ ID NO.14, at more than a pair of among the SEQ ID NO.15 of A75G and the SEQ IDNO.16.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110153876 CN102191337B (en) | 2011-06-09 | 2011-06-09 | Specific primers and liquid phase chip for detecting polymorphism of cyckin-dependent kinase 5 regulatorysubunit-associated protein 1-like 1(CDKAL1) gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110153876 CN102191337B (en) | 2011-06-09 | 2011-06-09 | Specific primers and liquid phase chip for detecting polymorphism of cyckin-dependent kinase 5 regulatorysubunit-associated protein 1-like 1(CDKAL1) gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102191337A true CN102191337A (en) | 2011-09-21 |
| CN102191337B CN102191337B (en) | 2013-06-19 |
Family
ID=44600202
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110153876 Expired - Fee Related CN102191337B (en) | 2011-06-09 | 2011-06-09 | Specific primers and liquid phase chip for detecting polymorphism of cyckin-dependent kinase 5 regulatorysubunit-associated protein 1-like 1(CDKAL1) gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102191337B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108359728A (en) * | 2018-04-04 | 2018-08-03 | 良培基因生物科技（武汉）有限公司 | Nucleic acid sequence, kit and its detection method for detecting diabetes risk site |
| CN115651984A (en) * | 2022-10-20 | 2023-01-31 | 山东大学 | Application of biomarker in evaluating drug resistance of tumor taxus chemotherapy |
-
2011
- 2011-06-09 CN CN 201110153876 patent/CN102191337B/en not_active Expired - Fee Related
Non-Patent Citations (5)
| Title |
|---|
| 《DIABETES》 20071231 Pascoe L等 Common variants of the novel type 2 diabetes genes CDKAL1 and HHEX/IDE are associated with decreased pancreatic beta-cell function 第56卷, * |
| PASCOE L等: "Common variants of the novel type 2 diabetes genes CDKAL1 and HHEX/IDE are associated with decreased pancreatic β-cell function", 《DIABETES》 * |
| RONG R等: "Association analysis of variation in/near FTO,CDKAL1,SLC30A8,HHEX,EXT2,IGF2BP2,LOC387761,and CDKN2B with type 2 diabetes and related quantitative traits in Pima Indians", 《DIABETES》 * |
| STEINTHORSDOTTIR V等: "A variant in CDKAL1 influences insulin response and risk of type 2 diabetes", 《NATURE GENETICS》 * |
| 杨雨晗等: "GWAS在2型糖尿病相关基因研究中的应用", 《中国生物化学与分子生物学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108359728A (en) * | 2018-04-04 | 2018-08-03 | 良培基因生物科技（武汉）有限公司 | Nucleic acid sequence, kit and its detection method for detecting diabetes risk site |
| CN115651984A (en) * | 2022-10-20 | 2023-01-31 | 山东大学 | Application of biomarker in evaluating drug resistance of tumor taxus chemotherapy |
| CN115651984B (en) * | 2022-10-20 | 2023-04-14 | 山东大学 | Application of biomarker in evaluating tumor taxus chemotherapy drug resistance |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102191337B (en) | 2013-06-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102134605A (en) | Single-nucleotide polymorphism (SNP) detection specific primer of CYP2C19 gene and liquid phase chip | |
| CN103374609B (en) | ABO gene mutation detection specific primers and liquid chip | |
| CN102191337B (en) | Specific primers and liquid phase chip for detecting polymorphism of cyckin-dependent kinase 5 regulatorysubunit-associated protein 1-like 1(CDKAL1) gene | |
| CN102994621A (en) | PTPN11 gene mutation detection specificity primer and liquid chip thereof | |
| CN102912002B (en) | ABCB2 gene polymorphism detection specific primers and liquid chip | |
| CN102181573B (en) | Specific primers and liquid-phase chip for detection of KITLG gene | |
| CN102952866B (en) | Specific primer and liquid chip for detecting polymorphism of DPYD (dihydropyrimidine dehydrogenase) gene | |
| CN102191336B (en) | MYC gene single nucleotide polymorphism (SNP) detection specific primer and liquid-phase chip | |
| CN102304567B (en) | Polymorphic detection specific primers and liquid phase chip in 8 q 24 section of chromosome | |
| CN102191335B (en) | Specific primer and liquid-phase chip for SNP (single nucleotide polymorphism) detection of PIGU (phosphatidylinositol glycan anchor biosynthesis, class U) genes | |
| CN102888444B (en) | Factor II and Factor V genetic polymorphism detection specific primer and liquid-phase chip | |
| CN102952872B (en) | Specific primer and liquid chip for detecting polymorphism of SLCO1B3 gene | |
| CN102952868B (en) | Specific primer and liquid chip for detecting polymorphism of SLC15A2 gene | |
| CN102952867B (en) | Specific primer and liquid chip for detecting polymorphism of SLC22A6 gene | |
| CN102952869B (en) | Specific primer and liquid chip for detecting polymorphism of SLCO1B1 gene | |
| CN102952870B (en) | Specific primer and liquid chip for detecting polymorphism of SLC22A2 gene | |
| CN102191334A (en) | Signal transducer and activator of transcription (STAT)4 gene polymorphism detection specific primer and liquid-phase chip | |
| CN102304564B (en) | Specific primers and liquid phase chip for single nucleotide polymorphism (SNP) detection in 15 q 25 section of chromosome | |
| CN102912005A (en) | CYP2C8 gene polymorphism detection specific primers and liquid chip | |
| CN103374607A (en) | ABCC (ATP (adenosine triphosphate)-binding cassette, sub-family C)1 gene mutation detection specific primers and liquid chip | |
| CN103849675B (en) | RTEL1 detection in Gene Mutation Auele Specific Primer and liquid-phase chip | |
| CN102304566B (en) | Specific primers and liquid phase chip for polymorphic detection of human hedgehog interacting protein (HHIP) gene | |
| CN103374606A (en) | CHEK1 (checkpoint kinase 1) gene mutation detection specific primers and liquid chip | |
| CN103571918A (en) | Specific detection primers and detection liquid phase chip for FYCO1 gene mutation | |
| CN102912003A (en) | ABCB1 gene polymorphism detection specific primers and liquid chip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee |
Owner name: SUREXAM BIOTECHNOLOGY CO., LTD. Free format text: FORMER NAME: GUANGZHOU YISHAN BIOTECHNOLOGY CO., LTD. |
|
| CP01 | Change in the name or title of a patent holder |
Address after: Five 510663 Guangdong city of Guangzhou province Guangzhou Science City Moon Road No. 80, Guangzhou technology innovation base B, C Patentee after: Surexam Biological Technology Co., Ltd. Address before: Five 510663 Guangdong city of Guangzhou province Guangzhou Science City Moon Road No. 80, Guangzhou technology innovation base B, C Patentee before: Guangzhou Yishan Biotechnology Co., Ltd. |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130619 Termination date: 20140609 |
|
| EXPY | Termination of patent right or utility model |