CN102191337B - 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
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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 22.3rd district No. 6, its signaling zone is positioned on the 5th intron, expresses at mankind's pancreas and Skeletal Muscle Cell camber.CDK5 is playing an important role aspect protection β cell function, reduction blood sugar, and CDK5 can cause the β cytopathy under the effect of the sugared toxicity of height, and the protein of CDKAL1 genes encoding can suppress the activation of CDK5.If the CDKAL1 gene locus morphs, the restraining effect of CDK5 weakened, cause the β cell function impaired, insulin secretion reduces.The CDKAL1 gene passes through to affect the secreting function of beta Cell of islet, thereby affects the secretion of Regular 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 occurs in the 107th Nucleotide of SEQ ID NO 31 | A107C |
| 2 | G → C sudden change occurs in the 323rd Nucleotide of SEQ ID NO 31 | G323C |
| 3 | A → G sudden change occurs in the 210th Nucleotide of SEQ ID NO 32 | A210G |
| 4 | A → G sudden change occurs 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 etc., wherein the most frequently used method has quantitative fluorescent PCR and PCR-RFLP analytical method.Easy and simple to handle, the advantages such as result quick, quantification that quantitative fluorescent PCR has, still, this technology exists sample easily to pollute, and cross reaction easily occurs, 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, 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.Again, above 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 is to provide 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. the wild-type and the mutant specificity ASPE primer that design respectively for 4 kinds of common polymorphisms sites of CDKAL1 gene, every kind of ASPE primer is comprised of the tag sequence of 5 ' end and the specific primer sequence for the goal gene pleomorphism site of 3 ' end, described specific primer sequence is selected from respectively: for SEQ ID NO.9 and the SEQ ID NO.10 of A107C, SEQ ID NO.11 and SEQ ID NO.12 for G323C, SEQ ID NO.13 and SEQ ID NO.14 for A210G, for more than a pair of in the SEQ ID NO.15 of A75G and SEQ ID NO.16, described tag sequence is selected from the sequence in SEQ ID NO.1～SEQ IDNO.8,
B. be coated with respectively the microballoon of special anti-tag sequence, described anti-tag sequence can be correspondingly with A in selected tag sequence complementary pairing, described anti-tag sequence is selected from the sequence in 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 the primer that amplification has the above pleomorphism site target sequence of A107C, G323C, A210G and A75G.
Preferably, described amplimer is: for the SEQ ID NO.25 of A107C and/or G323C and SEQ ID NO.26, for the SEQ ID NO.27 of A210G and SEQ ID NO.28, for more than a pair of in the SEQ ID NO.29 of A75G and SEQ ID NO.30.
preferably, described ASPE primer is: the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.9 for A107C reaches the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.10, the sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.11 for G323C reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.12, the sequence that is comprised of SEQ ID NO.5 and SEQ IDNO.13 for A210G reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.14, more than a pair of in the sequence that forms for the sequence that is formed 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 is to provide a kind of specific primer sequence for the detection of CDKAL1 gene pleiomorphism.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind of specific primer sequence that detects for the CDKAL1 gene pleiomorphism, it is selected from: for the SEQ IDNO.9 of A107C and SEQ ID NO.10, for the SEQ ID NO.11 of G323C and SEQ ID NO.12, for the SEQ IDNO.13 of A210G and SEQ ID NO.14, for more than a pair of in the SEQ ID NO.15 of A75G and 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, and realistic especially application needs.In very a large amount of Auele Specific Primers, through lot of experiments, 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 basically there is not cross reaction between designed probe and anti-tag sequence, choosing and tag sequence label and the specifically combination of ASPE primer of tag sequence label, anti-tag sequence label, can avoid cross reaction, realize the parallel detection in a plurality of SNP site.
2. the present invention by the design experiences of contriver's long-term accumulation and a large amount of experimental implementation, has 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, 4 kinds of pleomorphism sites detect and can complete 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 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 CDKAL1 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 1CDKAL1 gene pleiomorphism detects liquid-phase chip, mainly includes:
One, ASPE primer
Wild-type and mutant for CDKAL1 gene four kinds of common genotype A107C, G323C, A210G and A75G 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 1CDKAL1 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 8 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
8 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 CDKAL1 gene four kinds of common genotype A107C, G323C, A210G and A75G, design of amplification primers amplifies respectively three target sequences that contain pleomorphism site to (seeing Table 3), and wherein A107C and G323C are positioned at the 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 respectively the stock solution of 100pmol/mL with 10mmol/L Tris Buffer.
Embodiment 2 uses embodiment 1CDKAL1 gene test liquid-phase chip to the detection of sample
The formula of described various solution is as follows:
MES damping fluid (pH5.0) formula (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 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 three pairs of primers, one step of multiplex PCR amplifies the three objective sequences that contain respectively CDKAL1 gene four kinds of common genotype A107C, G323C, A210G and A75G, wherein A107C and G323C are positioned at the same amplified production, the product size is respectively 605bp, 589bp and 282bp, and primer sequence (SEQ ID NO.25-30) 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.25-30 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 corresponding 16 kinds of microballoons of every group selection
5Individual/ml);
2. get respectively the microballoon of every kind of numbering of 1ul in the Eppendorf tube of 1.5ml;
3. microballoon is in 〉=centrifugal the 1-2min of 10000g;
4. supernatant discarded, microballoon is resuspended in 2 * Tm hybridization buffer of 100ul, 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 CDKAL1 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 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 and can detect exactly CDKAL1 gene polymorphism sites type, and 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 detection of the liquid-phase chip of the ASPE primer that embodiment 3 is different to the CDKAL1 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 CDKAL1 Gene A 107C and G323C site mutation, respectively for 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 is selected from SEQ ID NO.1-SEQ ID NO.8, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing that is coated on microballoon is selected from SEQ ID NO.17-SEQ ID NO.24.Specific design is as shown in following table (table 7).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 7 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 8 pattern detection result and Polymorphism Analysis
Table 9 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 1 and test group 5.Other different tag sequences and specific primer sequence collocation, with coming to the same thing of embodiment 2 and the present embodiment, concrete data are omitted.
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
Detect liquid-phase chip as example take CDKAL1 gene polymorphism sites A210G and A75G site mutation, take the complementary sequence forward or backwards of place, mutational site target sequence as template, respectively for 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 present invention 1, as shown in table 10.Wherein,
Interior base is pleomorphism site.
Table 10 specific primer sequence
Detect liquid-phase chip as example take CDKAL1 gene polymorphism sites A210G and A75G site mutation respectively, select different specific primer sequences for A210G and A75G, 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 11).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 11 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 12 pattern detection result and Polymorphism Analysis
Table 13 pattern detection result and Polymorphism Analysis
Can find out from the present embodiment, when the ASPE primer was selected in embodiment 1 collocation of specific primer sequence and tag sequence, effect better (signal to noise ratio is better) was 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.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 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 CDKAL1 gene pleiomorphism detects liquid-phase chip, it is characterized in that, mainly includes:
A. the wild-type and the mutant specificity ASPE primer that design respectively for the different pleomorphism sites of CDKAL1 gene, every kind of ASPE primer is comprised of the tag sequence of 5 ' end and the specific primer sequence for the goal gene pleomorphism site of 3 ' end, described specific primer sequence is: for SEQ ID NO.11 and the SEQ ID NO.12 of G323C, and be selected from for the SEQ ID NO.9 of A107C and SEQ ID NO.10, for the SEQ ID NO.13 of A210G and SEQ ID NO.14, for more than a pair of in the SEQ ID NO.15 of A75G and SEQ ID NO.16; Described tag sequence is selected from the sequence in SEQ ID NO.1～SEQ ID NO.8;
B. be coated with respectively the microballoon of special anti-tag sequence, described anti-tag sequence can be correspondingly with A in selected tag sequence complementary pairing, described anti-tag sequence is selected from the sequence in 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 the primer that amplification has the above pleomorphism site target sequence of A107C, G323C, A210G and A75G, described amplimer is: for SEQ ID NO.25 and the SEQ ID NO.26 of A107C and/or G323C, and be selected from for the SEQ ID NO.27 of A210G and SEQ ID NO.28, for more than a pair of in the SEQ ID NO.29 of A75G and SEQ ID NO.30.
2. CDKAL1 gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that, described ASPE primer is: the sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.11 for G323C reaches the sequence that is comprised of SEQ ID NO.4 and SEQ IDNO.12, and the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.9 that is selected from for A107C reaches the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.10, the sequence that is comprised of SEQ ID NO.5 and SEQ ID NO.13 for A210G reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.14, more than a pair of in the sequence that forms for the sequence that is formed by SEQ ID NO.7 and SEQ IDNO.15 of A75G and by SEQ ID NO.8 and SEQ ID NO.16.
3. CDKAL1 gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that, mainly includes:
A. specificity ASPE primer: for the sequence that is formed by SEQ ID NO.1 and SEQ ID NO.9 of A107C and the sequence that is formed by SEQID NO.2 and SEQ ID NO.10, the sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.11 for G323C reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.12, the sequence that is comprised of SEQ ID NO.5 and SEQ IDNO.13 for A210G reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.14, reach with the sequence that is formed by SEQ IDNO.7 and SEQ ID NO.15 for A75G the sequence that is formed by SEQ ID NO.8 and SEQ ID NO.16,
B. be coated with respectively the microballoon of special anti-tag sequence, described anti-tag sequence can be correspondingly with A in selected tag sequence complementary pairing, described anti-tag sequence is selected from the sequence in 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: for the SEQ ID NO.25 of A107C and G323C and SEQ ID NO.26, for the SEQ ID NO.27 of A210G and SEQ ID NO.28 with for SEQ ID NO.29 and the SEQ ID NO.30 of A75G.
4. according to claim 1-3 described CDKAL1 gene pleiomorphisms of any one detect liquid-phase chip, it is characterized in that, described spacerarm sequence is 5-10 T.
5. be used for the Auele Specific Primer that the CDKAL1 gene pleiomorphism detects, it is characterized in that, described specific primer sequence is: for SEQ ID NO.11 and the SEQ ID NO.12 of G323C and be selected from for the SEQ ID NO.9 of A107C and SEQ ID NO.10, for the SEQ ID NO.13 of A210G and SEQ ID NO.14, for more than a pair of in the SEQ ID NO.15 of A75G and SEQ ID NO.16.
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| 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 |
| CN115651984B (en) * | 2022-10-20 | 2023-04-14 | 山东大学 | Application of biomarker in evaluating tumor taxus chemotherapy drug resistance |
-
2011
- 2011-06-09 CN CN 201110153876 patent/CN102191337B/en not_active Expired - Fee Related
Non-Patent Citations (8)
| Title |
|---|
| A variant in CDKAL1 influences insulin response and risk of type 2 diabetes;Steinthorsdottir V等;《NATURE GENETICS》;20070426;第39卷(第6期);第770-775页 * |
| 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;Rong R等;《DIABETES》;20090228;第58卷;第478-488页 * |
| Common variants of the novel type 2 diabetes genes CDKAL1 and HHEX/IDE are associated with decreased pancreatic β-cell function;Pascoe L等;《DIABETES》;20071231;第56卷;第3101-3104页 * |
| GWAS在2型糖尿病相关基因研究中的应用;杨雨晗等;《中国生物化学与分子生物学报》;20100731;第26卷(第7期);第617-721页 * |
| 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》.2007,第56卷 |
| 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》.2009,第58卷 |
| Steinthorsdottir V等.A variant in CDKAL1 influences insulin response and risk of type 2 diabetes.《NATURE GENETICS》.2007,第39卷(第6期), |
| 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》.2010,第26卷(第7期), |
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