CN103031369B - CDKN2A gene mutation detection specific primer and liquid chip - Google Patents
CDKN2A gene mutation detection specific primer and liquid chip Download PDFInfo
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Abstract
The invention discloses a CDKN2A gene mutation detection specific primer and a liquid chip. The liquid chip mainly comprises ASPE primers consisting of tag sequences at a 5' end and specific primer sequences aiming at target gene mutation sites at a 3' end, microspheres coated with anti-tag sequences and an amplification primer, wherein the specific primer sequences are as follows: SEQ ID NO.19 and SEQ ID NO.20 which aim at a G128T site, SEQ ID NO.21 and SEQ ID NO.22 which aim at a C143T site, SEQ ID NO.23 and SEQ ID NO.24 which aim at a T281A site, SEQ ID NO.25 and more than one of SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28 which aim at a G322A/C/T site, SEQ ID NO.29 and SEQ ID NO.30 which aim at a G329A site, SEQ ID NO.31 and SEQ ID NO.32 which aim at a G330A site, SEQ ID NO.33 and SEQ ID NO.34 which aims at a C341T site, and SEQ ID NO.35 and SEQ ID NO.36 which aim at a G358T site. The detection liquid chip provided by the invention has the advantages that the matching rate between the detection result and a sequencing method is up to 100%, and the wild-type and mutant parallel detection of multiple mutant sites can be realized.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete a kind of CDKN2A detection in Gene Mutation Auele Specific Primer and the liquid-phase chip of relating to.
Background technology
Cell cycle dependant kinase suppressor gene (cyclin-dependent kinase inhibitor, CDKN2A), be a kind of important cancer suppressor gene, belong to cell cycle dependent kinase enzyme inhibition factor gene family, there is the effect that regulates Cell apoptosis and proliferation.
People CDKN2A is positioned chromosome 9p 21, the two kinds of different cancer suppressor proteins of encoding, and the one, cell cycle dependant kinase arrestin p16 INK4a, by exons 1 α, 2 and 3 codings; Another is its variable frame gene (alterative reading frame, ARF) p14ARF product, by exons 1 β, 2 and 3 codings, therefore, CDKN2A gene claims again INK4a-ARF gene, cycle arrestin p16INK4a and the p14ARF of cancer suppressor gene CDKN2A coding have important cell cycle regulating effect, and the variation of CDKN2A gene is the common phenomenon in tumour generation and progression.
At present, the method that CDKN2A transgenation is carried out to determination and analysis seldom, mainly contains direct sequencing and PCR-RFLP analytical method, and wherein the most frequently used method has PCR-RFLP analytical method.PCR-RFLP method is the change of the restriction enzyme enzyme recognition site that causes based on transgenation, as lost or generation novel site in site, by a certain specific fragment of pcr amplification, use again digestion with restriction enzyme amplified production, the size of electrophoresis observation fragment, the transgenation that this method changes for detection of restriction enzyme site, can directly judge genotype, but this method can not be for 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 meet the needs of practical application.
Summary of the invention
One of object of the present invention is to provide CDKN2A gene mutation detection liquid-phase chip, and this liquid-phase chip can be used for separately or wild-type and the saltant type of parallel detection CDKN2A gene 10 kinds of common genotype G128T, C143T, T281A, G322A/C/T, G329A, G330A, C341T and G358T.
The technical scheme that realizes above-mentioned purpose is as follows:
A CDKN2A gene mutation detection liquid-phase chip, includes:
(A). the wild-type designing respectively for the different mutational sites of CDKN2A gene and the ASPE primer of saltant type: every kind of ASPE primer holds the specific primer sequence for goal gene mutational site to form by the tag sequence and 3 ' of 5 ' end, and described specific primer sequence is: for SEQ ID NO.19 and the SEQ ID NO.20 in G128T site; SEQ ID NO.21 and SEQ ID NO.22 for C143T site; SEQ ID NO.23 and SEQ ID NO.24 for T281A site; For the SEQ ID NO.25 in G322A/C/T site and more than one in SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28; SEQ ID NO.29 and SEQ ID NO.30 for G329A site; SEQ ID NO.31 and SEQ ID NO.32 for G330A site; SEQ ID NO.33 and SEQ ID NO.34 for C341T site; And/or for SEQ ID NO.35 and the SEQ ID NO.36 in G358T site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.18;
(B). there is anti-tag sequence microballoon coated, that there is different colours coding, in the middle of described anti-tag sequence is connected with microballoon, be also provided with spacerarm sequence; Described anti-tag sequence is selected from 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). for amplifying the primer that needs target sequence detection, that there is corresponding mutational site.
Preferably, described amplimer is SEQ ID NO.55 and the SEQ ID NO.56 for G128T and/or C143T site; And/or for SEQ ID NO.57 and the SEQID NO.58 in T281A, G322A/C/T, G329A, G330A, C341T, G358T site.
Preferably, described ASPE primer is: for the sequence being comprised of SEQ ID NO.1 and SEQ ID NO.19 in G128T site and the sequence being comprised of SEQ ID NO.2 and SEQ ID NO.20; For the sequence being formed by SEQ ID NO.3 and SEQID NO.21 in C143T site and the sequence being formed by SEQ ID NO.4 and SEQ ID NO.22; For the sequence being formed by SEQ ID NO.5 and SEQ ID NO.23 in T281A site and the sequence being formed by SEQ ID NO.6 and SEQ ID NO.24; For the sequence being formed by SEQ ID NO.7 and SEQ ID NO.25 in G322A/C/T site, the sequence being formed by SEQ ID NO.8 and SEQ IDNO.26, the sequence being formed by SEQ ID NO.9 and SEQ ID NO.27 and/or the sequence that formed by SEQ ID NO.10 and SEQID NO.28; For the sequence being formed by SEQ ID NO.11 and SEQ ID NO.29 in G329A site and the sequence being formed by SEQ ID NO.12 and SEQ ID NO.30; For the sequence being formed by SEQ ID NO.13 and SEQ IDNO.31 in G330A site and the sequence being formed by SEQ ID NO.14 and SEQ ID NO.32; For the sequence being formed by SEQID NO.15 and SEQ ID NO.33 in C341T site and the sequence being formed by SEQ ID NO.16 and SEQ ID NO.34; And/or for the sequence being formed by SEQ ID NO.17 and SEQ ID NO.35 in G358T site and the sequence being formed by SEQ ID NO.18 and SEQID NO.36.
Another object of the present invention is to provide the Auele Specific Primer for CDKN2A detection in Gene Mutation.
The technical scheme that realizes above-mentioned purpose is as follows:
For the Auele Specific Primer of CDKN2A detection in Gene Mutation, it is: for SEQ ID NO.19 and the SEQ IDNO.20 in G128T site; SEQ ID NO.21 and SEQ ID NO.22 for C143T site; SEQ ID NO.23 and SEQ ID NO.24 for T281A site; For the SEQ ID NO.25 in G322A/C/T site and be selected from SEQ ID NO.26, SEQ IDNO.27 and SEQ ID NO.28 in more than one; SEQ ID NO.29 and SEQ ID NO.30 for G329A site; SEQ ID NO.31 and SEQ ID NO.32 for G330A site; SEQ ID NO.33 and SEQ IDNO.34 for C341T site; And/or for SEQ ID NO.35 and the SEQ ID NO.36 in G358T site.
Major advantage of the present invention is:
1. the identical rate of detection method provided by the present invention and sequencing is up to 100%.And detect the needed time well below conventional sequencing technologies, realistic especially application needs.Prepared CDKN2A gene mutation detection liquid-phase chip has extraordinary signal-noise ratio, and between designed probe and anti-tag sequence, substantially there is not cross reaction, 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 mutational sites.
2. the present invention, by the design experiences of the long-term accumulation of contriver 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 design can the sensitive mutational site of identifying specifically target detect, accurately distinguishes the genotype of various types; In same reaction system, between different Auele Specific Primers, substantially there is not cross reaction between Auele Specific Primer and the pcr amplification product of non-target detect, detection specificity is good, and cross reacting rate is lower than 3%; Except detecting Single locus sudden change situation, also the sudden change situation in a plurality of mutational sites of parallel detection simultaneously, detects effect consistent.
3. detection method step of the present invention is simple, 10 kinds of mutational sites are detected and can be completed the amplification of 2 target sequences that contain mutational site by a step PCR, the many uncertain factors that exist 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 poor defect of repeatability 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 detecting improves greatly, thereby the sensitivity detecting is further enhanced, and signal to noise ratio strengthens, and detected result more accurately and reliably.
Embodiment
Embodiment 1CDKN2A gene mutation detection liquid-phase chip, mainly includes:
One, ASPE primer
Wild-type and saltant type for CDKN2A gene 10 kinds of common genotype G128T, C143T, T281A, G322A/C/T, G329A, G330A, C341T and G358T, design respectively specific primer sequence.ASPE primer is comprised of " tag sequence+specific primer sequence ".ASPE primer sequence is as shown in the table:
The ASPE primer sequence (tag sequence+specific primer sequence) of table 1CDKN2A gene
Every ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence for anti-tag sequence on corresponding microballoon, and 3 ' end is saltant type or the special primer fragment (as shown in Table 1 above) of wild-type.All ASPE primers are synthesized 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 tag sequence, reduce to greatest extent between the anti-tag sequence of each microballoon and secondary structure that tag and ASPE Auele Specific Primer fragment may form, on 18 kinds of microballoons numberings of selection and microballoon, corresponding anti-tag sequence is as shown in table 2:
Corresponding anti-tag sequence on table 2 microballoon numbering and microballoon
18 kinds of microballoons selecting, purchased from U.S. Luminex company, are coated in anti-tag sequence on microballoon.Between anti-tag sequence and microballoon, be connected with the spacerarm sequence of 5-10 T, before each anti-tag sequence, add the spacerarm sequence of the preceding paragraph 5-10 T, anti-tag sequence is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.By synthetic anti-tag sequence sterilizing ddH
2o is made into the stock solution of 100nmol/ml.Described spacerarm is for for by anti-tag and microsphere surface is spaced apart or anti-tag is placed in to 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 efficiency 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), 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 coated process of microballoon is as follows:
Get respectively 5 * 10
6the carboxylated microballoon of individual above-mentioned numbering (purchased 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) (purchased from the Pierce Chemical company) working fluid of preparation 10ng/ml.Toward the EDC working fluid that adds 2.5ul in microballoon suspension, constant-temperature incubation 30 minutes, then add the EDC working fluid of 2.5ul, then constant-temperature incubation 30 minutes.After reaction finishes, the Tween-20 with 0.02% washs once, then washs once with 0.1% SDS liquid.The Tris-EDTA solution [10mmol/L Tris (pH8.0)] that the microballoon that is coated with anti-tag sequence after washing is resuspended in to 100ul, in 1mmol/LEDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains mutational site
For CDKN2A gene 10 kinds of common genotype G128T, C143T, T281A, G322A/C/T, G329A, G330A, C341T and G358T, design of amplification primers, to (in Table 3), amplifies 2 target sequences that contain mutational site.
Table 3 amplifies the primer of the target sequence with mutational site
All primers are synthesized 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/LTris Buffer.
The detection of CDKN2A gene mutation detection liquid-phase chip described in embodiment 2 utilization embodiment 1 to sample
The formula of described various solution is as follows:
MES damping fluid (pH5.0) formula (250ml) of 50mM:
2 * Tm hybridization buffer
After filtration, be stored in 4 ℃.
ExoSAP-IT test kit is purchased from U.S. USB company.
Biotin labeled dCTP is purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
Methods involving with reference to < < molecular cloning > > about DNA extraction, obtains DNA to be detected.
Two, the pcr amplification of testing sample
Design 2 pairs of primers, multiplex PCR one step amplifies 2 target sequences that contain respectively CDKN2A gene 10 kinds of common genotype G128T, C143T, T281A, G322A/C/T, G329A, G330A, C341T and G358T, product size is respectively 285bp and 248bp, and primer sequence (SEQ ID NO.55-58) is shown in shown in above-mentioned table 3.
First prepare multiple PCR primer working fluid: respectively get respectively the primer stock solution 100ul of SEQ ID NO.55-58 in 1.5ml Eppendorf tube, mix and be multiple PCR primer working fluid.Multi-PRC reaction system is as follows:
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;
Hatch 15min for 2.37 ℃, 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, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on reacted product band.
First the ASPE primer working fluid that preparation mixes: get respectively the corresponding wild-type of gene to be detected and saltant type ASPE primer stock solution 10ul in 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
According to design ASPE primer, the corresponding 18 kinds of coated microballoons of every group selection (as described in Example 1), every kind of microballoon concentration is 2.5 * 10
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, and vortex mixes;
5. get the above-mentioned microballoon suspension of 25ul in the 96 corresponding holes of hole filter plate, 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. with masking foil, encase 96 orifice plates with lucifuge, 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. are resuspended in microballoon in 1 * Tm hybridization buffer of 75ul, and adding 15ul concentration is the SA-PE (SA-PE) of 10ug/ml;
Hatch 15min for 12.37 ℃, on Luminex instrument, detect.
Six, result detects and data analysis
Reaction after product detects by Luminex serial analysis instrument.The saltant type fluorescent value (MFI) of take is greater than 100 as cut-off value, when the MFI value of saltant type detection is greater than 100, judges that this sample exists this mutation type, otherwise judges that this sample is as corresponding wild-type.
Use present method to detect the CDKN2A transgenation of great amount of samples, with sequencing, detect with liquid-phase chip result and compare, calculate the identical rate of method detected result provided by the present invention.Present method detects 20 increments CDKN2A genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.Visible CDKN2A gene mutation detection liquid-phase chip provided by the present invention can detect the mutation type of CDKN2A gene exactly, and result is reliable and stable.
One of table 4 pattern detection result (MFI)
Two of table 5 pattern detection result (MFI)
Three of table 6 pattern detection result (MFI)
Table 7 sample CDKN2A gene mutation type analytical results
| Catalogue number(Cat.No.) | Liquid-phase chip detected result | Sequencing result |
| 1 | Wild-type | Wild-type |
| 2 | Wild-type | Wild-type |
| 3 | G128T sudden change | G128T sudden change |
| 4 | Wild-type | Wild-type |
| 5 | G329A sudden change | G329A sudden change |
| 6 | G330A sudden change | G330A sudden change |
| 7 | Wild-type | Wild-type |
| 8 | G322C sudden change | G322C sudden change |
| 9 | C143T sudden change | C143T sudden change |
| 10 | Wild-type | Wild-type |
| 11 | G358T sudden change | G358T sudden change |
| 12 | Wild-type | Wild-type |
| 13 | Wild-type | Wild-type |
| 14 | Wild-type | Wild-type |
| 15 | Wild-type | Wild-type |
| 16 | Wild-type | Wild-type |
| 17 | G329A sudden change | G329A sudden change |
| 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 CDKN2A gene mutation site
One, the design that prepared by liquid-phase chip (selection of tag sequence and Anti-tag sequence)
Take CDKN2A gene G128T and T281A site mutation, to detect liquid-phase chip be example, respectively for the wild-type of G128T and T281A and the specific primer sequence of saltant type design ASPE primer 3 ' end, the tag sequence of ASPE primer 5 ' end is selected from SEQ ID NO.1-SEQ ID NO.18, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing being coated on microballoon is selected from SEQ ID NO.37-SEQ ID NO.54.Specific design is as shown in following table (table 8).Synthetic, the anti-tag sequence of ASPE primer are coated with microballoon, amplimer, detection method etc. as described in embodiment 1 and embodiment 2.
Design prepared by table 8 liquid-phase chip
Two, sample detection
The liquid-phase chip that adopts above-mentioned design to prepare, detects sample 21-40 by testing process described in embodiment 2 and method, and detected result is as follows:
Table 9 pattern detection result and gene mutation analysis
Table 10 pattern detection result and gene mutation analysis
From experimental result, ASPE primer uses different Tag sequences, and its result is still reliable and stable, but ASPE primer is while selecting in embodiment 1 collocation of tag sequence and specific primer sequence, effect better (signal to noise ratio is better), 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.
Other is for the liquid-phase chip in different mutational sites, and ASPE primer uses different Tag sequences, and its result is still reliable and stable, and while selecting in embodiment 1 collocation of tag sequence and specific primer sequence, effect is better, and concrete data are omitted.
The selection of embodiment 4CDKN2A detection in Gene Mutation specific primer sequence
One, the design that prepared by liquid-phase chip (selection of wild-type and saltant type specific primer sequence)
Take the mutational site of CDKN2A gene C 143T and G329A, to detect liquid-phase chip be example, the complementary sequence forward or backwards of this place, mutational site target sequence of take is template, respectively for the wild-type of C143T and G329A and the specific primer sequence of saltant type design ASPE primer 3 ' end, comprise preferred specific primer sequence and 2 alternative specific primer sequences in the embodiment of the present invention 1, as shown in table 11.Wherein,
interior base is mutational site.
Table 11 specific primer sequence
Take the mutational site of CDKN2A gene C 143T and G329A, to detect liquid-phase chip be example, for C143T and G329A, select different specific primer sequences, the tag sequence of ASPE primer 5 ' end is fixed as the best effect sequence in embodiment 1, and select the anti-tag sequence of answering in contrast, specific design is as shown in following table (table 12).Synthetic, the anti-tag sequence of ASPE primer are coated with microballoon, amplimer, detection method etc. as described in embodiment 1 and embodiment 2.
Two of design prepared by table 12 liquid-phase chip
Two, sample detection
The liquid-phase chip that adopts above-mentioned design to prepare, detects sample 41-60 by testing process described in embodiment 2 and method, and detected result is as follows:
Table 13 pattern detection result and gene mutation analysis
Table 14 pattern detection result and gene mutation analysis
From the present embodiment, when ASPE primer is selected in embodiment 1 collocation of specific primer sequence and tag sequence, effect better (signal to noise ratio is better), referring to the present embodiment test group 7 and test group 10.Other derives from different specific primer sequences and the collocation of tag sequence of the complementary sequence forward or backwards of place, target detect site sequence, with coming to the same thing of embodiment 2 and the present embodiment, be still that the specific primer sequence described in embodiment 1 is better from different tag sequence arranging effects, concrete data are omitted.
Other multiple specific primer sequence for different mutational sites and the collocation of tag sequence, with coming to the same thing of embodiment 2 and the present embodiment, the selected Auele Specific Primer of embodiment 1, has better signal to noise ratio, detect effect also better, concrete data are omitted.
Be more than for the illustrating of possible embodiments of the present invention, but this embodiment is not in order to limit the scope of the claims of the present invention, allly do not depart 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 CDKN2A gene mutation detection liquid-phase chip, is characterized in that, includes:
(A). the wild-type designing respectively for the different mutational sites of CDKN2A gene and the ASPE primer of saltant type: every kind of ASPE primer holds the specific primer sequence for goal gene mutational site to form by the tag sequence and 3 ' of 5 ' end, and described specific primer sequence is: for SEQ ID NO.19 and the SEQ ID NO.20 in G128T site; SEQ ID NO.21 and SEQ ID NO.22 for C143T site; SEQ ID NO.23 and SEQ ID NO.24 for T281A site; For the SEQ ID NO.25 in G322A/C/T site and be selected from SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28 in more than one; SEQ ID NO.29 and SEQ ID NO.30 for G329A site; SEQ ID NO.31 and SEQ ID NO.32 for G330A site; SEQ ID NO.33 and SEQ ID NO.34 for C341T site; And/or for SEQ ID NO.35 and the SEQ ID NO.36 in G358T site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.18;
(B). there is anti-tag sequence microballoon coated, that there is different colours coding, in the middle of described anti-tag sequence is connected with microballoon, be also provided with spacerarm sequence; Described anti-tag sequence is selected from 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). for amplifying the primer that needs target sequence detection, that have corresponding mutational site, described amplimer is: for SEQ ID NO.55 and the SEQ ID NO.56 in G128T and/or C143T site; And/or for SEQ ID NO.57 and the SEQ ID NO.58 in T281A, G322A/C/T, G329A, G330A, C341T, G358T site.
2. CDKN2A gene mutation detection liquid-phase chip according to claim 1, it is characterized in that, described ASPE primer is: for the sequence being comprised of SEQ ID NO.1 and SEQ ID NO.19 in G128T site and the sequence being comprised of SEQ ID NO.2 and SEQ ID NO.20; For the sequence being formed by SEQ ID NO.3 and SEQ ID NO.21 in C143T site and the sequence being formed by SEQ ID NO.4 and SEQ ID NO.22; For the sequence being formed by SEQ ID NO.5 and SEQ ID NO.23 in T281A site and the sequence being formed by SEQ ID NO.6 and SEQ ID NO.24; For the sequence being formed by SEQ ID NO.7 and SEQ ID NO.25 in G322A/C/T site and select free SEQ ID NO.8 and the sequence that SEQ ID NO.26 forms, the sequence being formed by SEQ ID NO.9 and SEQ ID NO.27, the sequence that formed by SEQ ID NO.10 and SEQ ID NO.28 in more than one; For the sequence being formed by SEQ ID NO.11 and SEQ ID NO.29 in G329A site and the sequence being formed by SEQ ID NO.12 and SEQ ID NO.30; For the sequence being formed by SEQ ID NO.13 and SEQ ID NO.31 in G330A site and the sequence being formed by SEQ ID NO.14 and SEQ ID NO.32; For the sequence being formed by SEQ ID NO.15 and SEQ ID NO.33 in C341T site and the sequence being formed by SEQ ID NO.16 and SEQ ID NO.34; And/or for the sequence being formed by SEQ ID NO.17 and SEQ ID NO.35 in G358T site and the sequence being formed by SEQ ID NO.18 and SEQ ID NO.36.
3. CDKN2A gene mutation detection liquid-phase chip according to claim 1, is characterized in that:
(A) described ASPE primer is: for the sequence being comprised of SEQ ID NO.1 and SEQ ID NO.19 in G128T site and the sequence being comprised of SEQ ID NO.2 and SEQ ID NO.20; For the sequence being formed by SEQ ID NO.3 and SEQ ID NO.21 in C143T site and the sequence being formed by SEQ ID NO.4 and SEQ ID NO.22; For the sequence being formed by SEQ ID NO.5 and SEQ ID NO.23 in T281A site and the sequence being formed by SEQ ID NO.6 and SEQ ID NO.24; For the sequence being formed by SEQ ID NO.7 and SEQ ID NO.25 in G322A/C/T site, the sequence being formed by SEQ ID NO.8 and SEQ ID NO.26, the sequence being formed by SEQ ID NO.9 and SEQ ID NO.27 and the sequence that formed by SEQ ID NO.10 and SEQ ID NO.28; For the sequence being formed by SEQ ID NO.11 and SEQ ID NO.29 in G329A site and the sequence being formed by SEQ ID NO.12 and SEQ ID NO.30; For the sequence being formed by SEQ ID NO.13 and SEQ ID NO.31 in G330A site and the sequence being formed by SEQ ID NO.14 and SEQ ID NO.32; For the sequence being formed by SEQ ID NO.15 and SEQ ID NO.33 in C341T site and the sequence being formed by SEQ ID NO.16 and SEQ ID NO.34; With the sequence being formed by SEQ ID NO.17 and SEQ ID NO.35 for G358T site and the sequence being formed by SEQ ID NO.18 and SEQ ID NO.36;
(B) there is anti-tag sequence microballoon coated, that there is different colours coding, in the middle of described anti-tag sequence is connected with microballoon, be also provided with spacerarm sequence; Described anti-tag sequence is selected from SEQ ID NO.37～SEQ ID NO.54, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C) described amplimer is: for SEQ ID NO.55 and the SEQ ID NO.56 in G128T and/or C143T site; With SEQ ID NO.57 and the SEQ ID NO.58 for T281A, G322A/C/T, G329A, G330A, C341T, G358T site.
4. according to the CDKN2A gene mutation detection liquid-phase chip described in claim 1-3 any one, it is characterized in that, described spacerarm is 5-10 T.
5. for the Auele Specific Primer of CDKN2A detection in Gene Mutation, it is characterized in that, described specific primer sequence is: for SEQ ID NO.19 and the SEQ ID NO.20 in G128T site; SEQ ID NO.21 and SEQ ID NO.22 for C143T site; SEQ ID NO.23 and SEQ ID NO.24 for T281A site; For the SEQ ID NO.25 in G322A/C/T site and be selected from SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28 in more than one; SEQ ID NO.29 and SEQ ID NO.30 for G329A site; SEQ ID NO.31 and SEQ ID NO.32 for G330A site; SEQ ID NO.33 and SEQ ID NO.34 for C341T site; And/or for SEQ ID NO.35 and the SEQ ID NO.36 in G358T site.
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Non-Patent Citations (3)
| Title |
|---|
| CDKN2A基因变异与肿瘤;王静等;《中国优生与遗传杂志》;20071231;第15卷(第7期);114-116页 * |
| Maria Concetta Fargnoli，et al.CDKN2a/p16INK4a Mutations and lack of p19ARF Involvement in Familial melanoma Kindreds.《The Journal of investigative dermatology》.1998,第111卷(第6期),1202-1206页. * |
| 王静等.CDKN2A基因变异与肿瘤.《中国优生与遗传杂志》.2007,第15卷(第7期),114-116页. * |
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