CN102994620B - AKT1 gene mutation detection specificity primer and liquid chip thereof - Google Patents
AKT1 gene mutation detection specificity primer and liquid chip thereof Download PDFInfo
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Abstract
The invention discloses an AKT1 gene mutation detection specificity primer and a liquid chip thereof. The liquid chip mainly comprises: an ASPE primer composed of a 5'-terminal tag sequence and 3'-terminal specificity primer sequences focused on target gene mutation sites, wherein the specificity primer sequences comprise SEQ ID NO.5 and SEQ ID NO.6 focused on an E17K site, and/or SEQ ID NO.7 and SEQ ID NO.8 focused on an E49K site; a microsphere coated by an anti-tag sequence; and an amplimer. The consistency between the detection result of the detection liquid chip provided by the invention and the detection result of a sequencing method is high to 100%, and the wild-type and mutant parallel detection of a plurality of mutation sites is realized.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete a kind of AKT1 detection in Gene Mutation Auele Specific Primer and the liquid-phase chip of relating to.
Background technology
AKT is human homology's gene of viral oncogene v-akt, encoding serine Serineprotein kinase, in the relevant signal path of PI3K, play an important role, affect existence, propagation and the invasion and attack of cell, participate in comprising the cell processes of apoptosis and glucose metabolism.
Study and show at present, in lung cancer and mammary cancer, AKT gene activation and chemotherapeutics resistance significant correlation, AKT1 genetic expression raises and is considered to the reason of lung carcinoma cell to cis-platinum resistance; And in the research of breast cancer cell, AKT activates and significantly strengthens its resistance to chemotherapeutics.In addition, in surpassing 90% nonsmall-cell lung cancer, found the sustained activation of AKT gene and caused PI3K inhibitor and radiocurable resistance.
At present, the relevant sudden change of curative effect of AKT1 gene mainly contains E17K and E49K, E17K (49G > A) sudden change is that the L-glutamic acid of the codon coding of the 17th sports Methionin, this sudden change has changed the susceptibility to kinase inhibitor, and the L-glutamic acid that E49K (G145A) is the 49th bit codon coding sports Methionin, AKT1 transgenation has become the focus of drug efficacy study.
At present, the method that AKT1 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 AKT1 gene mutation detection liquid-phase chip, and this liquid-phase chip can be used for wild-type and the saltant type of individual event or two kinds of common genotype E17K of parallel detection AKT1 gene and E49K.
The technical scheme that realizes above-mentioned purpose is as follows:
An AKT1 gene mutation detection liquid-phase chip, includes:
(A). the wild-type designing respectively for the different mutational sites of AKT1 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.5 and the SEQ ID NO.6 in E17K site; And/or for SEQ ID NO.7 and the SEQ ID NO.8 in E49K site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.4;
(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.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 target sequence detection, that there is corresponding mutational site.
Preferably, described amplimer is SEQ ID NO.13 and the SEQ ID NO.14 for E17K, E49K site.
Preferably, described ASPE primer is: for the sequence being comprised of SEQ ID NO.1 and SEQ ID NO.5 in E17K site and the sequence being comprised of SEQ ID NO.2 and SEQ ID NO.6; And/or for the sequence being formed by SEQ ID NO.3 and SEQID NO.7 in E49K site and the sequence being 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 AKT1 detection in Gene Mutation.
The technical scheme that realizes above-mentioned purpose is as follows:
For the Auele Specific Primer of AKT1 detection in Gene Mutation, it is: for SEQ ID NO.5 and the SEQ IDNO.6 in E17K site; And/or for SEQ ID NO.7 and the SEQ ID NO.8 in E49K 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 AKT1 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, two kinds of mutational sites are detected and can be completed the amplification of 1 objective sequence 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 1AKT1 gene mutation detection liquid-phase chip, mainly includes:
One, ASPE primer
Wild-type and saltant type for two kinds of common genotype E17K of AKT1 gene and E49K, 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 1AKT1 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 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 two kinds of common genotype E17K of AKT1 gene and E49K, design of amplification primers, to (in Table 3), amplifies 1 target sequence that contains two mutational sites.
Table 3 amplifies the primer of the target sequence with mutational site
| SEQ NO | Mutational site | Type | Amplimer (5 '-3 ') |
| 13 | E17K and E49K | Forward | GCTGTGGTGGGGCGAGTCTG |
| 14 | Reverse | TCCAGCCAACCCCCCAAATC |
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 AKT1 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
| Reagent | Source | Final concentration | The consumption of every 250ml |
| 1MTris-HCl，pH8.0 | Sigma T3038 | 0.2M | 50ml |
| 5M NaCl | Sigma S5150 | 0.4M | 20ml |
| Triton X-100 | Sigma T8787 | 0.16％ | 0.4ml |
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 1 pair of primer, multiplex PCR one step amplifies the 1 objective sequence that contains respectively two kinds of common genotype E17K of AKT1 gene and E49K, and product size is 384bp, and primer sequence (SEQ ID NO.13-14) 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.13-14 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 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. 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 AKT1 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 AKT1 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.Visible AKT1 gene mutation detection liquid-phase chip provided by the present invention can detect the mutation type of AKT1 gene exactly, and result is reliable and stable.
One of table 4 pattern detection result (MFI)
Table 5 sample AKT1 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 | E17K sudden change | E17K sudden change |
| 4 | Wild-type | Wild-type |
| 5 | Wild-type | Wild-type |
| 6 | Wild-type | Wild-type |
| 7 | Wild-type | Wild-type |
| 8 | Wild-type | Wild-type |
| 9 | Wild-type | Wild-type |
| 10 | Wild-type | Wild-type |
| 11 | Wild-type | Wild-type |
| 12 | Wild-type | Wild-type |
| 13 | E49K sudden change | E49K sudden change |
| 14 | Wild-type | Wild-type |
| 15 | Wild-type | Wild-type |
| 16 | E17K sudden change | E17K 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 AKT1 gene mutation site
One, the design that prepared by liquid-phase chip (selection of tag sequence and Anti-tag sequence)
It is example that the AKT1 gene E17K site mutation of take detects liquid-phase chip, respectively for the wild-type of E17K 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 IDNO.4, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing being coated on microballoon is selected from SEQ ID NO.9-SEQID 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 like 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
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 concrete data are omitted.And 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.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 4AKT1 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 AKT1 gene E49K, 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 E49K 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,
interior base is mutational site.
Table 8 specific primer sequence
Take the mutational site of AKT1 gene E49K, to detect liquid-phase chip be example, for E49K, 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 9).Synthetic, the anti-tag sequence of ASPE primer are coated with microballoon, amplimer, detection method like 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 (6)
1. an AKT1 gene mutation detection liquid-phase chip, is characterized in that, includes:
(A). the wild-type designing respectively for the different mutational sites of AKT1 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.5 and the SEQ ID NO.6 in E17K site; And/or for SEQ ID NO.7 and the SEQ ID NO.8 in E49K site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.4;
(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.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 target sequence detection, that there is corresponding mutational site.
2. AKT1 gene mutation detection liquid-phase chip according to claim 1, is characterized in that, described amplimer is: for SEQ ID NO.13 and the SEQ ID NO.14 in E17K, E49K site.
3. AKT1 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.5 in E17K site and the sequence being comprised 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 E49K site and the sequence being formed by SEQ IDNO.4 and SEQ ID NO.8.
4. AKT1 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.5 in E17K site and the sequence being comprised of SEQID NO.2 and SEQ ID NO.6; With the sequence being formed by SEQ ID NO.3 and SEQ ID NO.7 for E49K site and the sequence being formed by SEQ ID NO.4 and SEQ ID NO.8;
(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.9～SEQ ID NO.12, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C) described amplimer is: for SEQ ID NO.13 and the SEQ ID NO.14 in E17K, E49K site.
5. according to the AKT1 gene mutation detection liquid-phase chip described in claim 1-4 any one, it is characterized in that, described spacerarm is 5-10 T.
6. for the Auele Specific Primer of AKT1 detection in Gene Mutation, it is characterized in that, described specific primer sequence is: for SEQ ID NO.5 and the SEQ ID NO.6 in E17K site; And/or for SEQ ID NO.7 and the SEQ ID NO.8 in E49K site.
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| CN201110269791.4A CN102994620B (en) | 2011-09-13 | 2011-09-13 | AKT1 gene mutation detection specificity primer and liquid chip thereof |
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| CN201110269791.4A CN102994620B (en) | 2011-09-13 | 2011-09-13 | AKT1 gene mutation detection specificity primer and liquid chip thereof |
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