CN103031364A - FGFR3 gene mutation detection specific primer and liquid chip - Google Patents
FGFR3 gene mutation detection specific primer and liquid chip Download PDFInfo
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Abstract
The invention discloses an FGFR3 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.22 and SEQ ID NO.23, SEQ ID NO.24 and SEQ ID NO.25, SEQ ID NO.26 and SEQ ID NO.27, SEQ ID NO.28 and SEQ ID NO.29, SEQ ID NO.30 and SEQ ID NO.31, SEQ ID NO.32 and SEQ ID NO.33, SEQ ID NO.34 and SEQ ID NO.35, SEQ ID NO.36 and more than one of SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39 and SEQ ID NO.40, and/or SEQ ID NO.41 and SEQ ID NO.42. 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 FGFR3 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 the fibroblast growth factor acceptor (fibroblast growth factor receptors, FGFRs) that passes through the cross-film high-affinity plays a role.Fibroblast growth factor receptor3 (Fibroblast growth factor receptors 3, FGFR3) be a kind of tyrosine kinase receptor, it plays an important role in generation, promotion angiogenesis, wound healing and the tumour of cell mitogen form.FGFR3 belongs to transmembrane protein, when the receptor extracellular section after part FGFs is combined, acceptor forms homology or heterodimer, makes the tyrosine-kinase enzyme activation of cell inner segment, causes a series of cascade reactions in cell, finally causes the synthetic and cell fission of DNA.Mitogen signal transduction pathway and cell growth that FGFR3 can start the Ras-MAPK mediation simultaneously play inhibiting STAT signal transduction pathway; when normal development; cell is grown under the effect of two bars systems and is broken up; during the FGFR3 sudden change; the dysfunction activation of acceptor, thus normal growth and the growth organized destroyed.There are some researches show, the FGFR3 transgenation is relevant with the formation of tumour.
At present, the method that determination and analysis is carried out in the FGFR3 transgenation seldom mainly contains direct sequencing and PCR-RFLP analytical method, and wherein the most frequently used method has the PCR-RFLP analytical method.The PCR-RFLP method is based on the change of the restriction enzyme enzyme recognition site that transgenation causes, as losing or produce novel site in the site, by a certain specific fragment of pcr amplification, use again the digestion with restriction enzyme amplified production, the size of electrophoresis observation fragment, this method can directly be judged 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 provides the FGFR3 gene mutation detection liquid-phase chip, and this liquid-phase chip can be used for separately or wild-type and the mutant of parallel detection FGFR3 gene 12 kinds of common genotype R248C, G370C, S371C, Y373C, G380R, A391E, D641N, K650Q/E/T/M, G697C.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind of FGFR3 gene mutation detection liquid-phase chip includes:
(A). the wild-type that designs respectively for the different mutational sites of FGFR3 gene and the ASPE primer of mutant: every kind of ASPE primer is comprised of the tag sequence of 5 ' end and 3 ' the end specific primer sequence for the goal gene mutational site, and described specific primer sequence is: for SEQ ID NO.22 and the SEQ ID NO.23 in R248C site; SEQ ID NO.24 and SEQ ID NO.25 for the G370C site; SEQ ID NO.26 and SEQ ID NO.27 for the S371C site; SEQID NO.28 and SEQ ID NO.29 for the Y373C site; SEQ ID NO.30 and SEQ ID NO.31 for the G380R site; SEQ ID NO.32 and SEQ ID NO.33 for the A391E site; SEQ ID NO.34 and SEQ ID NO.35 for the D641N site; For the SEQ ID NO.36 in K650Q/E/T/M site and be selected among SEQ ID NO.37, SEQ ID NO.38, SEQ IDNO.39, the SEQ ID NO.40 more than one; And/or for SEQ ID NO.41 and the SEQ ID NO.42 in G697C site, described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.21;
(B). anti-tag sequence microballoon coated, that have the different colours coding is arranged, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon; Described anti-tag sequence is selected from SEQ ID NO.43～SEQ ID NO.63, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). be used for amplifying the primer that needs target sequence detection, that have corresponding mutational site.
Preferably, described amplimer is: for SEQ ID NO.64 and the SEQ ID NO.65 in R248C site; SEQ ID NO.66 and SEQ ID NO.67 for G370C, S371C, Y373C, G380R, A391E site; SEQ ID NO.68 and SEQ ID NO.69 for D641N, K650Q/E/T/M site; And/or for SEQ ID NO.70 and the SEQ ID NO.71 in G697C site.
Preferably, described ASPE primer is: the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.22 for the R248C site reaches the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.23; The sequence that is comprised of SEQ ID NO.3 and SEQ IDNO.24 for the G370C site reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.25; The sequence that is comprised of SEQ IDNO.5 and SEQ ID NO.26 for the S371C site reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.27; The sequence that is comprised of SEQ ID NO.7 and SEQ ID NO.28 for the Y373C site reaches the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.29; The sequence that is comprised of SEQ ID NO.9 and SEQ ID NO.30 for the G380R site reaches the sequence that is comprised of SEQ ID NO.10 and SEQID NO.31; The sequence that is comprised of SEQ ID NO.11 and SEQ ID NO.32 for the A391E site reaches the sequence that is comprised of SEQID NO.12 and SEQ ID NO.33; The sequence that is comprised of SEQ ID NO.13 and SEQ ID NO.34 for the D641N site reaches the sequence that is comprised of SEQ ID NO.14 and SEQ ID NO.35; For the sequence that is formed by SEQ IDNO.15 and SEQ ID NO.36 in K650Q/E/T/M site and be selected from the sequence that formed by SEQ ID NO.16 and SEQ ID NO.37, in the sequence that formed by SEQ ID NO.17 and SEQ ID NO.38, the sequence that is formed by SEQ ID NO.18 and SEQ ID NO.39, the sequence that formed by SEQ ID NO.19 and SEQ ID NO.40 more than one; And/or for the sequence that is formed by SEQ IDNO.20 and SEQ ID NO.41 in G697C site and the sequence that is formed by SEQ ID NO.21 and SEQ ID NO.42.
Another object of the present invention provides the Auele Specific Primer for the FGFR3 detection in Gene Mutation.
The technical scheme that realizes above-mentioned purpose is as follows:
Described specific primer sequence is: for SEQ ID NO.22 and the SEQ ID NO.23 in R248C site; SEQ ID NO.24 and SEQ ID NO.25 for the G370C site; SEQ ID NO.26 and SEQ ID NO.27 for the S371C site; SEQ ID NO.28 and SEQ ID NO.29 for the Y373C site; SEQ ID NO.30 and SEQ IDNO.31 for the G380R site; SEQ ID NO.32 and SEQ ID NO.33 for the A391E site; SEQ ID NO.34 and SEQ ID NO.35 for the D641N site; For the SEQ ID NO.36 in K650Q/E/T/M site and be selected among SEQ ID NO.37, SEQ IDNO.38, SEQ ID NO.39, the SEQ ID NO.40 more than one; And/or for SEQ ID NO.41 and the SEQ ID NO.42 in G697C site.
Major advantage of the present invention is:
1. the identical rate of the detected result of FGFR3 gene mutation detection liquid-phase chip provided by the present invention and sequencing is up to 100%, and the needed time, realistic especially application needed well below sequencing technologies commonly used.Prepared FGFR3 gene mutation detection liquid-phase chip has extraordinary signal-noise ratio, and basically there is not cross reaction between designed probe and the 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 mutational sites.
2. 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 the 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 sudden change situation in a plurality of mutational sites of simultaneously parallel detection detects effect consistent.
3. detection method step of the present invention is simple, 12 kinds of mutational sites are detected and can be finished four amplifications that contain the target sequence in mutational site by a step 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, so that prepared microballoon can be applicable to different test items, has very strong expansion.The fluorescent signal value that detects improves greatly, thereby so that the sensitivity that detects is further enhanced, signal to noise ratio strengthens, and detected result more accurately and reliably.
Embodiment
Embodiment 1
The described FGFR3 gene mutation detection liquid-phase chip of the present embodiment mainly includes:
One, ASPE primer
Wild-type and mutant for FGFR3 gene 12 kinds of common genotype R248C, G370C, S371C, Y373C, G380R, A391E, D641N, K650Q/E/T/M, G697C 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 1FGFR3 gene
Every the ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence for anti-tag sequence on the 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, corresponding anti-tag sequence is as shown in table 2 on 21 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
21 kinds of microballoons selecting are coated in the anti-tag sequence on the microballoon available from U.S. Luminex company.Be connected with the spacerarm sequence of 5-10 T between anti-tag sequence and the 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 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 such 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 adds the EDC working fluid of 2.5ul again, and constant-temperature incubation is 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/L Tris (pH8.0)] of 100ul, among the 1mmol/L EDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains the mutational site
For FGFR3 gene 12 kinds of common genotype R248C, G370C, S371C, Y373C, G380R, A391E, D641N, K650Q/E/T/M, G697C, design of amplification primers amplifies 2 target sequences that contain 6 mutational sites to (seeing Table 3).
Table 3 amplifies the primer of the target sequence with mutational 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/LTrisBuffer.
Embodiment 2 uses embodiment 1 described FGFR3 gene mutation detection liquid-phase chip to the detection of sample
The prescription of described various solution is as follows:
MES damping fluid (pH5.0) prescription (250ml) of 50mM:
2 * Tm hybridization buffer
| Reagent | The source | Final concentration | The consumption of every 250ml |
| 1MTris-HCl，pH8.0 | SigmaT3038 | 0.2M | 50ml |
| 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 about DNA extraction, obtain DNA to be detected.
Two, the pcr amplification of testing sample
Design 4 pairs of primers, one step of multiplex PCR amplifies 4 target sequences that contain respectively FGFR3 gene 12 kinds of common genotype R248C, G370C, S371C, Y373C, G380R, A391E, D641N, K650Q/E/T/M and G697C, the product size is respectively 415bp, 376bp, 329bp and 454bp, and primer sequence (SEQ ID NO.64-71) is seen shown in the 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.64-71 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 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 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 and 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 the design the ASPE primer, the corresponding 21 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, 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. 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 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 the SA-PE (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 detects by Luminex serial analysis instrument.Greater than 100 as the cut-off value, the MFI value that detects when mutant is judged that there is this mutation type in this sample, otherwise is judged that this sample is corresponding wild-type greater than 100 the time take mutant fluorescent value (MFI).
Use present method to detect the FGFR3 transgenation of great amount of samples, compare with the liquid-phase chip result with the sequencing detection, calculate the identical rate of method detected result provided by the present invention.Present method detects 20 increments FGFR3 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.As seen FGFR3 gene mutation detection liquid-phase chip provided by the present invention can detect the mutation type of FGFR3 gene exactly, and the result is reliable and stable.
One of table 4 pattern detection result (MFI)
Table 5 pattern detection result's (MFI) two
Table 6 pattern detection result's (MFI) three
Table 7 sample FGFR3 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 | The Y373C sudden change | The Y373C sudden change |
| 4 | Wild-type | Wild-type |
| 5 | The K650E sudden change | The K650E sudden change |
| 6 | The G380R sudden change | The G380R sudden change |
| 7 | Wild-type | Wild-type |
| 8 | The R248C sudden change | The R248C sudden change |
| 9 | Wild-type | Wild-type |
| 10 | Wild-type | Wild-type |
| 11 | The G380R sudden change | The G380R sudden change |
| 12 | Wild-type | Wild-type |
| 13 | Wild-type | Wild-type |
| 14 | Wild-type | Wild-type |
| 15 | The K650M sudden change | The K650M sudden change |
| 16 | Wild-type | Wild-type |
| 17 | S371C sudden change, D641N sudden change | S371C sudden change, D641N sudden change |
| 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 FGFR3 gene mutation site
One, the design (selection of tag sequence and Anti-tag sequence) of liquid-phase chip preparation
Detect liquid-phase chip as example take FGFR3 gene R248C and S371C site mutation, respectively for the wild-type of R248C and S371C 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.21, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing that is coated on the microballoon is selected from SEQ ID NO.43-SEQ ID NO.63.Specific design is shown in following table (table 8).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 8 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned design preparation, by embodiment 2 described testing processes and method sample 21-40 is detected, detected result is as follows:
Table 9 pattern detection result and gene mutation analysis
Table 10 pattern detection result and gene mutation analysis
| Sequence number. | The S371C-w detected value | The S371C-m detected value |
| Group4 | Group5 | Group6 | Group4 | Group5 | Group6 | |
| Negative control | 18 | 26 | 28 | 16 | 27 | 16 |
| 21 | 3722 | 4304 | 3048 | 82 | 70 | 87 |
| 22 | 2525 | 3983 | 2982 | 77 | 62 | 77 |
| 23 | 3395 | 4062 | 2856 | 81 | 64 | 90 |
| 24 | 3053 | 4045 | 2705 | 84 | 72 | 96 |
| 25 | 3061 | 4058 | 3099 | 77 | 71 | 89 |
| 26 | 3111 | 4210 | 3562 | 578 | 874 | 576 |
| 27 | 3956 | 4862 | 3391 | 85 | 66 | 83 |
| 28 | 3607 | 4929 | 3500 | 70 | 57 | 68 |
| 29 | 3018 | 3924 | 3208 | 81 | 72 | 88 |
| 30 | 2939 | 3889 | 3025 | 55 | 54 | 64 |
| 31 | 3521 | 4207 | 3605 | 61 | 50 | 54 |
| 32 | 3626 | 4643 | 3705 | 46 | 39 | 43 |
| 33 | 4097 | 4978 | 3832 | 50 | 34 | 56 |
| 34 | 4022 | 4564 | 3943 | 75 | 53 | 57 |
| 35 | 2613 | 3670 | 2536 | 48 | 41 | 66 |
| 36 | 2877 | 4009 | 2982 | 68 | 67 | 93 |
| 37 | 3388 | 4408 | 3641 | 72 | 63 | 76 |
| 38 | 3657 | 4518 | 3653 | 34 | 31 | 34 |
| 39 | 3350 | 4035 | 2763 | 43 | 38 | 44 |
| 40 | 3821 | 4883 | 4051 | 74 | 63 | 69 |
From experimental result as can be known, the ASPE primer uses different Tag sequences, and its result is still reliable and stable, but the ASPE primer is selected when the tag sequence is arranged in pairs or groups with specific primer sequence among the embodiment 1, 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.
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 selects when the tag sequence is with the specific primer sequence collocation among the embodiment 1, and effect is better, concrete data omission.
The selection of embodiment 4FGFR3 detection in Gene Mutation specific 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 the mutational site of FGFR3 gene Y373C and A391E, take the forward or backwards complementary sequence of this place, mutational site target sequence as template, respectively for the wild-type of Y373C and A391E and the specific primer sequence of mutant design ASPE primer 3 ' end, comprise preferred specific primer sequence and 2 alternative specific primer sequences in the embodiment of the invention 1, as shown in table 11.Wherein,
Interior base is the mutational site.
Table 11 specific primer sequence
Detect liquid-phase chip as example take the mutational site of FGFR3 gene Y373C and A391E, select different specific primer sequences for Y373C and A391E, the tag sequence of ASPE primer 5 ' end then is fixed as the best effect sequence among the embodiment 1, and select with it corresponding anti-tag sequence, specific design is shown in following table (table 12).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 12 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned design preparation, by embodiment 2 described testing processes and method sample 41-60 is detected, detected result is as follows:
Table 13 pattern detection result and gene mutation analysis
Table 14 pattern detection result and gene mutation analysis
By the present embodiment as seen, when the ASPE primer was selected among the 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.Other derives from different specific primer sequences and the collocation of tag sequence of the forward or backwards complementary sequence of place, target detect site sequence, with coming to the same thing of embodiment 2 and the present embodiment, namely still be that the specific primer sequence described in the embodiment 1 is better from different tag sequence arranging effects, concrete data are omitted.
Other is for multiple specific primer sequence and the collocation of tag sequence in different mutational sites, with coming to the same thing of embodiment 2 and the present embodiment, namely embodiment 1 selected Auele Specific Primer has better signal to noise ratio, detect effect also better, concrete data are omitted.
More than be for the specifying of possible embodiments of the present invention, but this embodiment limits claim 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 claim of the present invention.
Claims (6)
1. a FGFR3 gene mutation detection liquid-phase chip is characterized in that, includes:
(A). the wild-type that designs respectively for the different mutational sites of FGFR3 gene and the ASPE primer of mutant: every kind of ASPE primer is comprised of the tag sequence of 5 ' end and 3 ' the end specific primer sequence for the goal gene mutational site, and described specific primer sequence is: for SEQ ID NO.22 and the SEQ ID NO.23 in R248C site; SEQ ID NO.24 and SEQ ID NO.25 for the G370C site; SEQ ID NO.26 and SEQ ID NO.27 for the S371C site; SEQID NO.28 and SEQ ID NO.29 for the Y373C site; SEQ ID NO.30 and SEQ ID NO.31 for the G380R site; SEQ ID NO.32 and SEQ ID NO.33 for the A391E site; SEQ ID NO.34 and SEQ ID NO.35 for the D641N site; For the SEQ ID NO.36 in K650Q/E/T/M site and be selected among SEQ ID NO.37, SEQ ID NO.38, SEQ IDNO.39, the SEQ ID NO.40 more than one; And/or for SEQ ID NO.41 and the SEQ ID NO.42 in G697C site, described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.21;
(B). anti-tag sequence microballoon coated, that have the different colours coding is arranged, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon; Described anti-tag sequence is selected from SEQ ID NO.43～SEQ ID NO.63, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). be used for amplifying the primer that needs target sequence detection, that have corresponding mutational site.
2. FGFR3 gene mutation detection liquid-phase chip according to claim 1 is characterized in that, described amplimer is: for SEQ ID NO.64 and the SEQ ID NO.65 in R248C site; SEQ ID NO.66 and SEQ ID NO.67 for G370C, S371C, Y373C, G380R, A391E site; SEQ ID NO.68 and SEQ ID NO.69 for D641N, K650Q/E/T/M site; And/or for SEQ ID NO.70 and the SEQ ID NO.71 in G697C site.
3. FGFR3 gene mutation detection liquid-phase chip according to claim 1, it is characterized in that, described ASPE primer is: the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.22 for the R248C site reaches the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.23; The sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.24 for the G370C site reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.25; The sequence that is comprised of SEQ ID NO.5 and SEQ ID NO.26 for the S371C site reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.27; The sequence that is comprised of SEQ ID NO.7 and SEQ IDNO.28 for the Y373C site reaches the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.29; The sequence that is comprised of SEQ IDNO.9 and SEQ ID NO.30 for the G380R site reaches the sequence that is comprised of SEQ ID NO.10 and SEQ ID NO.31; The sequence that is comprised of SEQ ID NO.11 and SEQ ID NO.32 for the A391E site reaches the sequence that is comprised of SEQ ID NO.12 and SEQ ID NO.33; The sequence that is comprised of SEQ ID NO.13 and SEQ ID NO.34 for the D641N site reaches the sequence that is comprised of SEQ ID NO.14 and SEQ ID NO.35; For the sequence that is formed by SEQ ID NO.15 and SEQ ID NO.36 in K650Q/E/T/M site and be selected from the sequence that formed by SEQ ID NO.16 and SEQ ID NO.37, in the sequence that formed by SEQ ID NO.17 and SEQ IDNO.38, the sequence that is formed by SEQ ID NO.18 and SEQ ID NO.39, the sequence that formed by SEQ ID NO.19 and SEQ IDNO.40 more than one; And/or for the sequence that is formed by SEQ ID NO.20 and SEQ ID NO.41 in G697C site and the sequence that is formed by SEQ ID NO.21 and SEQ ID NO.42.
4. FGFR3 gene mutation detection liquid-phase chip according to claim 1 is characterized in that,
(A). described ASPE primer is: the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.22 for the R248C site reaches the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.23; The sequence that is comprised of SEQ ID NO.3 and SEQ IDNO.24 for the G370C site reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.25; The sequence that is comprised of SEQID NO.5 and SEQ ID NO.26 for the S371C site reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.27; The sequence that is comprised of SEQ ID NO.7 and SEQ ID NO.28 for the Y373C site reaches the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.29; The sequence that is comprised of SEQ ID NO.9 and SEQ ID NO.30 for the G380R site reaches the sequence that is comprised of SEQ IDNO.10 and SEQ ID NO.31; The sequence that is comprised of SEQ ID NO.11 and SEQ ID NO.32 for the A391E site reaches the sequence that is comprised of SEQ ID NO.12 and SEQ ID NO.33; The sequence that is comprised of SEQ ID NO.13 and SEQ ID NO.34 for the D641N site reaches the sequence that is comprised of SEQ ID NO.14 and SEQ ID NO.35; The sequence that forms for the sequence that is formed by SEQ ID NO.15 and SEQ ID NO.36 in K650Q/E/T/M site and the sequence that is formed by SEQ ID NO.16 and SEQ IDNO.37, by SEQ ID NO.17 and SEQ ID NO.38, the sequence that is formed by SEQ ID NO.18 and SEQ IDNO.39 and the sequence that is formed by SEQ ID NO.19 and SEQ ID NO.40; Reach the sequence that is formed by SEQ ID NO.21 and SEQ ID NO.42 with the sequence that is formed by SEQ ID NO.20 and SEQ ID NO.41 for the G697C site;
(B). anti-tag sequence microballoon coated, that have the different colours coding is arranged, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon; Described anti-tag sequence is selected from SEQ ID NO.43～SEQ ID NO.63, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). described amplimer is: for SEQ ID NO.64 and the SEQ ID NO.65 in R248C site; SEQ ID NO.66 and SEQ ID NO.67 for G370C, S371C, Y373C, G380R, A391E site; SEQ ID NO.68 and SEQ ID NO.69 for D641N, K650Q/E/T/M site; With SEQ ID NO.70 and the SEQ IDNO.71 for the G697C site.
5. each described FGFR3 gene mutation detection liquid-phase chip is characterized in that according to claim 1-4, and described spacerarm is 5-10 T.
6. be used for the Auele Specific Primer of FGFR3 detection in Gene Mutation, it is characterized in that, described specific primer sequence is: for SEQ ID NO.22 and the SEQ ID NO.23 in R248C site; SEQ ID NO.24 and SEQ ID NO.25 for the G370C site; SEQ ID NO.26 and SEQ ID NO.27 for the S371C site; SEQ ID NO.28 and SEQ IDNO.29 for the Y373C site; SEQ ID NO.30 and SEQ ID NO.31 for the G380R site; SEQ ID NO.32 and SEQ ID NO.33 for the A391E site; SEQ ID NO.34 and SEQ ID NO.35 for the D641N site; For the SEQ ID NO.36 in K650Q/E/T/M site and be selected among SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, the SEQ ID NO.40 more than one; And/or for SEQ ID NO.41 and the SEQ ID NO.42 in G697C site.
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| CN103789416A (en) * | 2014-01-04 | 2014-05-14 | 杭州艾迪康医学检验中心有限公司 | Method and oligonucleotide for detecting FGFR3 gene G380R site mutation |
| CN103789416B (en) * | 2014-01-04 | 2015-09-23 | 杭州艾迪康医学检验中心有限公司 | Detect method and the oligonucleotide of FGFR3 G380R site mutation |
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