CN103031368B - FGFR1 gene mutation detection specific primer and liquid chip - Google Patents
FGFR1 gene mutation detection specific primer and liquid chip Download PDFInfo
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Abstract
The invention discloses an FGFR1 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.5 and SEQ ID NO.6 which aim at a C374T site, and/or SEQ ID NO.7 and SEQ ID NO.8 which aim at a C754A 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 FGFR1 detection in Gene Mutation Auele Specific Primer and the liquid-phase chip of relating to.
Background technology
Fibroblast growth factor (fibroblast growth factors, FGFs) be the polypeptide class family of one group of structural similitude, and play a role by the fibroblast growth factor acceptor (fibroblast growth factor receptors, FGFRs) of cross-film high-affinity.FGFR belongs to receptor type protein tyrosine kinase, and this family mainly comprises FGFR1, FGFR2, FGFR3 and tetra-members of FGFR4.FGFR participates in regulating multiple processes such as cell proliferation, apoptosis, migration, new vessel generation.Because effect is extensive, FGFR and other RTK are subject to strict regulation and control under normal circumstances.In tumour, as in mammary cancer, bladder cancer, prostate cancer etc., FGFR activated mutant or ligand/receptor are crossed to express and are caused its sustained activation, not only closely related with the generation of tumour, development, poor prognosis etc., and also play a significant role in the process such as invasion and attack and transfer of tumor angiogenesis, tumour.Fibroblast growth factor acceptor 1 (Fibroblast Growth Factor Receptor 1, FGFR1) in wound healing, vascularization, process of tissue reparation, play a significant role, the present invention is directed to wild-type and the saltant type in two sites such as C374T and C754A of FGFR1 gene, providing can be separately or the liquid-phase chip of parallel detection.
At present, the method for FGFR1 transgenation being carried out to determination and analysis is little, 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 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 meet the needs of practical application.
Summary of the invention
One of object of the present invention is to provide FGFR1 gene mutation detection liquid-phase chip, and this liquid-phase chip can be used for separately or wild-type and the saltant type of two kinds of common genotype C374T of parallel detection FGFR1 gene and C754A.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind of FGFR1 gene mutation detection liquid-phase chip, includes:
(A). the wild-type designing respectively for the different mutational sites of FGFR1 gene and the ASPE primer of saltant type: every kind of ASPE primer is made up of for the specific primer sequence in goal gene mutational site tag sequence and the 3 ' end of 5 ' end, and described specific primer sequence is: for SEQ ID NO.5 and the SEQ ID NO.6 in C374T site; And/or for SEQ IDNO.7 and the SEQ ID NO.8 in C754A site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.4;
(B). there is microballoon that anti-tag sequence is coated with, 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.9～SEQ ID NO.12, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). for amplifying the primer that needs the target sequence detecting, there is corresponding mutational site.
Preferably, described amplimer is SEQ ID NO.13 and the SEQ ID NO.14 for C374T site; And/or for SEQ ID NO.15 and the SEQ ID NO.16 in C754A site.
Preferably, described ASPE primer is: for the sequence being made up of SEQ ID NO.1 and SEQ ID NO.5 in C374T site and the sequence that is made up of SEQ ID NO.2 and SEQ ID NO.6; And/or for the sequence being formed by SEQ ID NO.3 and SEQ ID NO.7 in C754A site and the sequence that formed by SEQ ID NO.4 and SEQ ID NO.8.
Another object of the present invention is to provide the Auele Specific Primer for FGFR1 detection in Gene Mutation.
The technical scheme that realizes above-mentioned purpose is as follows:
For the Auele Specific Primer of FGFR1 detection in Gene Mutation, it is: for SEQ ID NO.5 and the SEQ IDNO.6 in C374T site; And/or for SEQ ID NO.7 and the SEQ ID NO.8 in C754A 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 FGFR1 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 multiple 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, the also sudden change situation in the multiple mutational sites of parallel detection simultaneously, detects effect consistent.
3. detection method step of the present invention is simple, the amplification that can complete by a step PCR 2 target sequences that contain mutational site is detected in 2 kinds of mutational sites, the many uncertain factors that exist in the complex operations processes such as repeated multiple times PCR are avoided, thereby can greatly improve Detection accuracy, embody 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 1FGFR1 gene mutation detection liquid-phase chip, mainly includes:
One, ASPE primer
For wild-type and the saltant type of two kinds of common genotype C374T of FGFR1 gene and C754A, design respectively specific primer sequence.ASPE primer is made up 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 1FGFR1 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 4 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
4 kinds of microballoons selecting, purchased from Luminex company of the U.S., 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 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 (purchased from Luminex company) of individual above-mentioned numbering 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/L EDTA, 2-8 DEG C keeps in Dark Place.
Three, amplify the primer of the target sequence that contains mutational site
For two kinds of common genotype C374T of FGFR1 gene and C754A, design of amplification primers, to (in table 3), amplifies 2 target sequences that contain two mutational sites.
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/L Tris Buffer.
Embodiment 2 uses the detection to sample of FGFR1 gene mutation detection liquid-phase chip described in embodiment 1
The formula of described various solution is as follows:
MES damping fluid (pH5.0) formula (250ml) of 50mM:
2 × Tm hybridization buffer
| Reagent | 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 |
After filtration, be stored in 4 DEG C.
ExoSAP-IT test kit is purchased from USB company of the U.S..
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 the 2 objective sequences that contain respectively two kinds of common genotype C374T of FGFR1 gene and C754A, product size is respectively 322bp and 403bp, and primer sequence (SEQ ID NO.13-16) 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.13-16 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 DEG C of 3min; 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 40s, 30 circulations; 72 DEG C of 10min; 4 DEG C 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 DEG C, hatch 15min for 80 DEG C, the enzyme that deactivation is unnecessary.Enzyme is cut product after treatment and 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 multiple 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 DEG C of 2min; 94 DEG C of 30s, 54 DEG C of 1min, 72 DEG C of 2min, 30 circulations; 4 DEG C save backup.
Five, hybridization
According to design ASPE primer, the corresponding 4 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. encase 96 orifice plates with lucifuge with masking foil, 95 DEG C of 60s, 37 DEG C 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 DEG C, on Luminex instrument, detect.
Six, result detects and data analysis
Reaction after product detects by Luminex serial analysis instrument.Be greater than 100 as cut-off values taking saltant type fluorescent value (MFI), in the time that the MFI value of saltant type detection is greater than 100, judges that this sample exists this mutation type, otherwise judge that this sample is as corresponding wild-type.
Use present method to detect the FGFR1 transgenation of great amount of samples, detect with liquid-phase chip result and compare with sequencing, calculate the identical rate of method detected result provided by the present invention.Present method detects 20 increments FGFR1 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.Visible FGFR1 gene mutation detection liquid-phase chip provided by the present invention can detect the mutation type of FGFR1 gene exactly, and result is reliable and stable.
One of table 4 pattern detection result (MFI)
Table 5 sample FGFR1 gene mutation type analytical results
| Catalogue number(Cat.No.) | Liquid-phase chip detected result | Sequencing result |
| 1 | Wild-type | Wild-type |
| 2 | C754A sudden change | C754A sudden change |
| 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 | C374T sudden change | C374T sudden change |
| 9 | Wild-type | Wild-type |
| 10 | Wild-type | Wild-type |
| 11 | Wild-type | Wild-type |
| 12 | C374T sudden change | C374T sudden change |
| 13 | Wild-type | Wild-type |
| 14 | Wild-type | Wild-type |
| 15 | Wild-type | Wild-type |
| 16 | C754A sudden change | C754A sudden change |
| 17 | Wild-type | Wild-type |
| 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 FGFR1 gene mutation site
One, the design (selection of tag sequence and Anti-tag sequence) that prepared by liquid-phase chip
Detect liquid-phase chip as example taking FGFR1 gene C 754A site mutation, respectively for the wild-type of C754A 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-SEQID NO.4, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing being coated on microballoon is selected from SEQ ID NO.9-SEQ ID NO.12.Specific design is as shown in following table (table 6).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 6 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 7 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 2.Other different tag sequences and specific primer sequence collocation, with coming to the same thing of embodiment 2 and the present embodiment, concrete data are omitted.
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 4FGFR1 detection in Gene Mutation specific primer sequence
One, the design (selection of wild-type and saltant type specific primer sequence) that prepared by liquid-phase chip
Detect liquid-phase chip as example taking the mutational site of FGFR1 gene C 374T, taking the complementary sequence forward or backwards of this place, mutational site target sequence as template, respectively for the wild-type of C374T 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 8.Wherein, in frame, base is mutational site.
Table 8 specific primer sequence
Detect liquid-phase chip as example taking the mutational site of FGFR1 gene C 374T, select different specific primer sequences for C374T, 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 9).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 9 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 10 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 3.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 (4)
1. a FGFR1 gene mutation detection liquid-phase chip, is characterized in that, includes:
(A). the wild-type designing respectively for the different mutational sites of FGFR1 gene and the ASPE primer of saltant type: every kind of ASPE primer is made up of for the specific primer sequence in goal gene mutational site tag sequence and the 3 ' end of 5 ' end, and described specific primer sequence is: for SEQIDNO.5 and the SEQIDNO.6 in C374T site; And/or for SEQID NO.7 and the SEQIDNO.8 in C754A site; Described tag sequence is selected from SEQIDNO.1～SEQIDNO.4;
(B). there is microballoon that anti-tag sequence is coated with, 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 SEQIDNO.9～SEQIDNO.12, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). for amplifying the primer that needs the target sequence detecting, have corresponding mutational site, described amplimer is: for SEQIDNO.13 and the SEQIDNO.14 in C374T site; And/or for SEQIDNO.15 and the SEQ IDNO.16 in C754A site.
2. FGFR1 gene mutation detection liquid-phase chip according to claim 1, is characterized in that, described ASPE primer is: for the sequence being made up of SEQIDNO.1 and SEQIDNO.5 in C374T site and the sequence that is made up of SEQIDNO.2 and SEQIDNO.6; And/or the sequence forming for the sequence being formed by SEQIDNO.3 and SEQIDNO.7 in C754A site and by SEQ IDNO.4 and SEQIDNO.8.
3. FGFR1 gene mutation detection liquid-phase chip according to claim 1, is characterized in that,
(A) described ASPE primer is: for the sequence being made up of SEQIDNO.1 and SEQIDNO.5 in C374T site and the sequence that is made up of SEQIDNO.2 and SEQIDNO.6; With the sequence being formed by SEQIDNO.3 and SEQID NO.7 for C754A site and the sequence being formed by SEQIDNO.4 and SEQIDNO.8;
(B) there is microballoon that anti-tag sequence is coated with, 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 SEQIDNO.9～SEQIDNO.12, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C) described amplimer is: for SEQIDNO.13 and the SEQIDNO.14 in C374T site; With SEQIDNO.15 and the SEQIDNO.16 for C754A site.
4. according to the FGFR1 gene mutation detection liquid-phase chip described in claim 1-3 any one, it is characterized in that, described spacerarm is 5-10 T.
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